1567Fort Leeders of NJFort Lee High School Fort Lee, NJ 07024High SchoolTeam not yet accepted001http://2014HS.igem.org/Team:Fort_Leeders_of_NJhttp://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=195520140High School1285METUHS-AnkaraMETU Development Foundation High School Ankara, Cankaya, Turkey www.odtugvo.k12.tr/ankaraHigh SchoolCO to CO2 Converter & CO Monitoring SystemCarbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We?ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill-switch design based on the lac-operon. The kill-switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device.1531BBa_K1285999http://2014HS.igem.org/Team:METUHS-AnkaraBBa_K1285000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164820140High SchoolLog in   Team:METUHS-Ankara/index.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense Project Briefly, our design helps monitoring the level of carbonmonoxide in a medium. Our system will be able to convert carbonmonoxide to a less harmful gas, carbondioxyde by metabolic pathways. We call it "CO-Sense".   View our project page     Team We participate from METU Highschool located  at Ankara, Turkey.           Find out more about our team   Tweets by @METUHSiGEM Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/index.htmlproject.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense Project Description Design Results Human Practices Lab Notebook Carbon monoxide (CO) is a colorless and odorless gas which can be very poisonous and dangerous. Since it is produced from commonly used household devices and industry, in the case of any leakage it can cause severe poisonings and deaths. Many people die in Turkey because of mishandled ascot and blast heaters. According to statistics 10,154 people, meaning 14 out of every 100,000 people, were intoxicated.   The aim of our project is to prevent these cases. In order to achieve this, we plan to develop a biological device which will include a detection and a conversion system. Firstly, our detection mechanism is based on light dependent sensors. These sensors gather data from the bacteria, in turn triggering an alarm system. Secondly, in the event of CO presence, our conversion system will be activated in order to convert carbon monoxide into carbon dioxide (CO2). To accomplish this transformation, we are using an enzyme called Carbon Monoxide Dehydrogenase (CODH). Finally, as a safety measure we will include a kill switch mechanism that aims to inactivate the system.   CO to CO2 Converter & CO Monitoring System   Carbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We’ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill-switch design based on the lac-operon. The kill-switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device. constitutive promoter Kill switch   we use an mRNA interferase, MazF, which cleaves mRNA’s at specific sequences. In our kill switch, we used anti-sense RNA principle as a template. According to this principle, MazF, which is constantly produced via a constitutive promoter, is got inactivated by Anti-MazF construct. In order to trigger this mechanism, we used IPTG, a harmless molecule for the bee which at the same time does not appear in the honey too. When IPTG is present in the environment, LacI is inhibited by IPTG and therefore promoter gets activated. With the activated promoter of it, Anti-MazF is produced and inactive MazF. As long as IPTG exists, MazF gets inactivated continuously; therefore, the bacteria maintain their lives. On the other hand, if bacteria exists in a IPTG-free environment, Anti-MazF producing stops, which leads to MazF producing and bacteria get killed by MazF. (Circuit image courtesy of METU iGEM Team 2013) In this page, you can find out the computational modelling for our design and finished system.   Carbon monoxide   With our research we were able to find out that 1500 ppm of CO gas means death in an hour. To make calculations we need to convert this value into something we can work with easily, such as moles.   1500 ppm = 0.15%   If we take a 30 meter squared room with 3 meter ceiling height, the overall volume of the room is 90m^3 which equates to 90000 liters.   By using Ideal Gas Law, we can say that the amount of moles of CO present in 135 Liters is around about 4 moles. So what we have learned? In a big size room, there should be at least 4 moles of CO molecules for it to be considered as deadly.   CODH Activity   Even though we were not able to find detailed parameters for this particular enzyme, we were able to work out a very simple formula to calculate the amount of CO we will be able to convert each second.   r = CODH activity rate p = plasmid copy number b = amount of bacteria s = synthesis rate [CO]=amount of CO (moles) A = fraction of CO converted   *Lets call r.p.b.s: "c" since we can assume that it will stay constant. *Our calculations show that "c = 31777209688000" or "3.2 x 10^14"   When we divide c by the Avagadro number we can find out what fraction of moles of CO we will be able to convert each time frame. Even though "c" seems like a huge number, the Avagadro number is much bigger.   3.2 x 10^14 / 6.02 x 10^23  =  0.5 x 10^-9   This mans that we are able to convert roughly 0.000000001 of the CO available each second.   Here is a little script we wrote with Python programming language with modules "matplotlib" and "numPy":   from pylab import *   timer = 0 f = 0.000000001 s = 7200 #seconds co = 4 # moles co2 =0 # moles values = [ ]   for i in range(s):     co2 = co2 + co*f     co = co - co2     if co <= 0:         break     values.append(co2)     timer += 1   x = arange(0,timer,1) plot(x,values)   xlabel('time (s)') ylabel('CO2 (moles)') title('CO2 - Time') grid(True) show()     Please note that, even though we are subtracting the amount of CO converted from the initial CO value, the graph still does not have a visible curve to it. It is obvious that even though we are able to convert a huge number of CO molecules each given second, there are way to much CO then our system can convert.   By increasing the surface are of the single-layer e-coli colonies, for example take a 10 x 10 grid of the same exact system, we can achieve 100 times more efficient converters which can shave of two 0's from the results.   Down below you can see the photos of the colonies we were able to get and the electronic circuit design.     Trying out the color sensor. Red pen on the sensor light up red. Blue ruler on top, blue light. Final images of the design. Today, improving technology and ideas lead students to be the constructors of better future. That’s why as METU DF High School iGEM 2014 team we made researches and experiments to widen the perspective of what synthetic biology can achieve. We know that synthetic biology is the way of constructing core components which can be modeled by merging them with the fundamentals of biology. We assume that by the efficient use of synthetic biology we can lighten the way through future and save many lives.   Therefore, this year we worked hard to put forth an experiment that can lead the way to our better future. As METU HS iGEM team, we believe that synthetic biology is a tool that can turn the world to a better place. And iGEM is an opportunity for us to show people that synthetic biology is helpful for the humankind. That's why we did many events to introduce people synthetic biology. We have given a bake sale which included bacteria & DNA muffins and x-ray crystallography cake.                                     The muffins got retweeted by the iGEM HQ!       Furthermore we interviewed some students in our school to see how much they know about synthetic biology world. We decorated our school with DNA's and posters of our team on DNA day. Also as METU HS iGEM team we have attended to a Synthetic Biology Day to expand our knowledge. It is important for the next generations to be familiar with synthetic biology, in order to achieve this we gave a presentation to 7 and 8th graders. We have informed them about iGEM and biology. And our aim is to present iGEM to everyone to convince people that synthetic biology is useful. So we have told people about our team and iGEM at the opening of an ODTU DF school in Denizli. In order to maintain our goal we believed that a sale of work will be a nice introduction of iGEM and synthetic biology. We cooked cupcakes in order to sale and we designed them as bacteria and everything about synthetic biology. Secondly, we also designed an enormous DNA shaped gumdrop. The most critical part was we recommended iGEM and talk with them about the project we were doing. Our main purpose was to make people know what synthetic biology is and create awareness about these topics. To conclude we have tried and we are still trying to spread synthetic biology in order to acknowledge human. As METU DF High School iGEM 2014 team we work hard to settle the importance of synthetic biology by referring various projects.   We believe that awareness and education is the key of action, acceptance and understanding. Our project contains lots of valuable experiments that can prove the useful remedies of the efficient use of synthetic biology.   You can find every photo and video we referred to at the fun page by following this link or find it at the extras page!   Competent Cell • Take 1 µL cell (3 µL cell for us ) 100 ml LB 37ºC 2h (OD 0.600 200 rpm) • Divide 100 ml into 2 falcons & centrifuge for 10 minutes 5000 rpm +4ºC • Discard supernatant • Add 10 µL 0.1 M (molarity) CaCl2 • Dissolve pellet by gently shaking • Incubate on ice 10 min. • Centrifuge 5000 rpm for 5 min. +4ºC • Discard supernatant • Put 2 ml CaCl2 and dissolve pellet • Put in ice for 5 min. & keep it -80ºC 80% glycerol   Transformation • Thaw competent cells on ice • Mix ligation mix (100 µL cells 5 µL plasmids) • Incubate cells on ice 30 min. • Heat shock at 42ºC 90 sec. • Incubate cells on ice 5 min. • Add 900 µL LB • Incubate at 37ºC for 80 min. • Centrifuge at 3000 rpm for 10 min. • Discard supernatant • Resuspend the pellet in 100 µL LB • Spread cells on LB Agar with antibiotic K34F - DT Chl K34B - RFP Chl K25J - GFP Chl     Transformation again! Selin Hoca Transformation   Pet28a Pet28a+gene RFP+ DT   We cannot insert the plasmid!! Something’s wrong with the competent cells!!   Comp 1 - Comp 2  -     RFP + DT    V   Pet28a Comp 3  -    Amp, Kan        Kan     The second transformation. Competent cell, why didn’t they work? 1) Lack of CaCl2 2) CaCl2 wasn’t cold enough 3) Non homogeneous cells (The closest) 4) OD was not enough   Transformation, why didn’t it work? 1) Heat shock 2) Cell died in centrifuge 3) Cell died whilst spreading • Heat • Mechanic force   Plasmid Isolation   .Gel Electrophoresis 10 µL                                           .Stock 15 µL  Gel Extraction     A1         K1          RFP Needed the plasmids! Plasmid Isolation - 2 µL cell pellet, lyse down the cells take plamids!                                                                                                               16.05.2014 Transformation                 pCooM       with changes of 50 µL cell               pCooF          + 10 µL  plasmid           Digestion NEB Buffer   5 µL BSA              0.5 µL Enzyme 1     0.5 µL Enzyme 2     0.5 µL µL Plasmid       1000 ng/µL   To complete to 50 µL, add ddH2O/dH2O Keep it in water bath for 2.5 hours at 37ºC   LIGATION 10 x T4 ligase buffer                      2 µL 6:1 M ratio to invert to vector Add dH2O T4 ligase                                         1 µL     Incubate at room temtperature  1 hours and 15 min. at 65ºC for enzyme activation Alternative: +4ºC over night   Calculatory Insert Amount   Insert mass in ng = 6 x  insert length in 6 µL / Vector length 6 g...   ...x vector mass in mg     -Gel -Gel Extraction   Vector - 2500 bp Insert  - 500 bp 1300 - to prepare the agarose gel 1.95 g of agarose (nearly 2%)     -Gel Results -PCR -Gel   Why didn’t it result correct? There was no DNA Problem with gel Part we don’t want in the gel Lack of the amount TAE? Agarose 6x loading dye Plasmid  -  Digestion, Ligation EtBr DNA degredation                                 Conditions Initial denaturation   -   94ºC     -30 sec Cycles                          94ºC      10-30 sec (30-35)                        45-65ºC (Tm= 58 ºC) 15-60 sec                     2000+1800 bp (32-33)                        65ºC   50 sec/kb (⁓2 min) Final extension          65ºC  10 min Hold                            +4ºC   8F 9R    /   10F 11R - primers used! Nutrient agar plate inoculation RFP                     GFP IPTG + IPTG -     Nutrient agar plate inoculation IPTG + and – Inoculation on broth media IPTG x2 MP x4     ​​• IPTG induction in Broth Media (1)• Miniprep pCooM, pCooF (1)                 Holin- Antiholin• CODH from BCG PCR (2)• Kill Switch Transformation (3)                  MazF- AntimazF• Digestion & Ligation (?)           BCG -primer(Rhodospirillum rubrum  primer)            Transformation of kill switch (with changes of LB - 150 µL at last step) Gel Extraction Genomic DNA Isolation Chl & Tet Inoculation     Empty: MazF, AntimazF (streak plates) (4 inoculations) Genomic DNA Isolation • Suspend colonies in 120 µL TEN Buffer                                                         40 µM Tris, 1 µM EDTA, 150 µM NaCl • Incubate at 100ºC for 10 min • Centrifuge at 13.000 rpm • Take supernatant store at +4ºC Gel Extraction • Slice the gel • Add 1:1 volume binding buffer • Incubate 50-60ºC for 10 min. • Transfer up to 800 µL of the solubilized gel solution to the purification column • Cent. 1 min, discard flow-through • 100 µL binding buffer cent. 1 min, discard flow-through • 700 µL wash buffer, cent. 2 min discard flow-through • Column to eppendorf 50 µL Elution buffer dH2O, cent. 1 min. • Discard column store it at -20ºC     1) PCR 2) Gel Electrophoresis - Genomic DNA + Gel Extraction   -PCR                                                                                             L    Uncut 2    2.2   2.1   Uncut 4   4   BA   BK    L   2T   4T    4L                                    = Gel Electrophoresis   Gel Extraction Nutrient Agar plate inoculation LB at the last step of transformation is 150 µL Ligation: Same with 13.06.2014 but overnight +4ºC incubation  prepared at 19.45 17.06.2014 - at 11:45, will be transformed.     17.06.14 - Transformation                     100 - 10                     50   - 5 Transformation - heat shock 75’’                               100   LB at the last step Selin inoculation - amount of LB was more than needed. 0.5 eppendorf - it was opened before centrifuge (contamination)     4F   9R - primers                                             1- 2 GE               3- 2.2 PCR - 2. Sample                                              2- 2.1   LIGATION T4 ligase                                1 µL T4 ligase buffer                    1 µL Vector                                   4     6     2     5     3 Insert                                    4     2     6     3     5                                              1:1   3:1  1:3  5:3   3:5 16ºC     30’ RT        30’ RT        30’ +4ºC    Overnight                                                                                       Transformation: Öykü: 1st: (3) 2 GE 2h A (3:1)  -  2nd: 2 GE A 301 Can (Öykü): 1st: (4) 2 GE A 2h (1:3) - 2nd: 2 GE A 301 Almira: 1st: (2) 2GE A 2h 1:1 (3:5) - 2nd: 2 GE A 301 – (5) 2GE A 301 Martin: 1st: (1) 2GE A 2h 5:3 (1:1) - 2nd: 2 GE A 301         Transformation - +4 ONC PCR - 2 - 2’ PCR Purification Digestion Gel Gel Extraction Ligation 16ºC   1h                RT     2h                +4     ONC Gel Gel Extraction Transformation       23.04.2014 28.04.2014 29.04.2014 01.05.2014 16.05.2014 23.05.2014 25.05.2014 30.05.2014 09.06.2014 10.06.2014 11.06.2014 12.06.2014 13.06.2014 16.06.2014 17.06.2014 18.06.2014 19.06.2014 20.06.2014 Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/projecreference.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense http://www.lifetechnologies.com/tr/en/home/life-science/cell-analysis/antibodies-and-secondary-detection/primary-antibodies/gfp-antibodies.html   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC35403/   Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum Jongyun Heo, Cale M. Halbleib*, and Paul W. Ludden† Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, WI 53706 Communicated by R. H. Burris, University of Wisconsin, Madison, WI, May 9, 2001 (received for review February 13, 2001)   http://2012.igem.org/Team:METU/KillSwitchOverview http://2012.igem.org/Team:METU/CODH         Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/referencattributions.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense First and foremost, we would like to thank to METU iGEM Team for showing extreme support, sharing their knowledge and experience whenever we needed it. They let us use their equipment and labs, supervised us all the time! So huge credit goes to METU iGEM Team.   Find out more about METU iGEM following this link. Huge thanks to these people. Below their name, you can see why there were so important to us!   Okan Tezcan   -Guidance and lab work   Selin Su Yirmibeşoğlu   -Protocols and lab work   İlkem Kumru   -Wiki design and modelling   Ayça Çırçır   -Lessons about molecular biology   Melike Dönertaş   -Mathematical modelling and chracterization   Wiki design and mathematical model programming, the events listed in the human practices, lab work was done by the METU HS iGEM Team with the help of listed people given. There were no 3rd parties involved in the system design, modelling and wiki design. All content used is work of METU HS iGEM Team unless stated opposite. Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/attributionFrom 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense Project Briefly, our design helps monitoring the level of carbonmonoxide in a medium. Our system will be able to convert carbonmonoxide to a less harmful gas, carbondioxyde by metabolic pathways. We call it "CO-Sense".   View our project page     Team We participate from METU Highschool located  at Ankara, Turkey.           Find out more about our team   Tweets by @METUHSiGEM Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankarhttp://fisofowi.netii.net/images/metuhslogo_alpha-u2751.pngteam.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense Who are we? Teachers & Advisors Safety Questions We participate from METU Development Foundation Private High School located at the METU campus, Ankara, Turkey. This is our school's first iGEM experience and we are very thankful to everyone who gave support. Under this field, you can find the students and the instructors name, their photo and a brief info about them.  Hey, I'm Almira and I'm a third grader in the METU High School. Along with my science studies I also play cello professionally in the Presidential Symphony Orchestra and I study as a private student in the Music Pedagogy department of Gazi University. When it comes to my personal interests a little, I've been drawing and painting since I was very little and I also write all kinds of fictional stories. After I graduate from METU H.S, my plan is to move to Sweden to study university. Since I've always been a creative person in my own ways I'm interested in creating, designing and developing all kinds of things, which makes me interested in genetics and biology so much, because it's a study and an effort to understand the very first creation on this earth. And the reason I volunteered to join IGEM is, I thought it's a very good opportunity for me to dig a little deeper into it beforehand and get a little experience. My name is Batuhan. I'm 16 years old and I go to METU High School. I like working as a team and I'm interested in highly biology. I have a lot of hobbies that I don't regularly do, however I play basketball and I have been doing Wing Tsun, which is known as 'Kung Fu', for years. I like discussing and bringing out new, original idea and being part of projects, and that's why I joined the iGEM team. Hi I’m Bengisu Birer and I am a 10th grade student at METU High School. I noticed my interest in molecular biology last year. That’s why I decided to be a part of the IGEM team.  With the help of IGEM I learnt lots of thing about bacteria which I can’t learn at school. IGEM has many benefits like helping us to think more deeply and faster. Thanks to IGEM, I have improved myself in synthetic biology. Also, it helps evolve your creativity and this affects other classes, too. Hi everyone, I’m Can Soygür. I’m 16 now and a 10th grade student in METU Development Foundation High School. At school, I've found out I have a special interest in biology, particularly in molecular biology. Therefore, I joined our school’s iGEM HS team to improve my biology and have fun with my friends. In my free times I often chat with my friends or play PC games, mostly first person shooter. I also spend time doing my new interest: archery. By the way, my favourite hobby is reading a lot, especially humorous magazines, political books and some interesting novels. My name is Cem Anıl and I am a senior in METU Development Foundation High School. I have a great passion for science and this is the primary reason why I'm here in the IGEM High School Team. I am planning to pursue an academic career in computational neuroscience in the future. Though I find each branch of science extremely intriguing, the areas that attract my interest most are physics, neuroscience and molecular biology. My biggest intention is to contribute to the research aiming to reach artificial intelligence. I am also extremely involved in music. I play the piano, compose classical music and jazz pieces and perform in several school and regional concerts. I take great pleasure from discussing science, philosophy, arts, mathematics, music and cinema with people. As for sports, I used to play tennis a lot a few years ago and participated in numerous tournaments; now I only continue playing tennis as a hobby. I am grateful to be a part of this great science organization which gave me the chance to acquire a lot of theoretical and practical knowledge in synthetic biology. I am Deniz Siso and I'm a senior at METU Development Foundation High School. This is our first iGEM experience as a team. I joined the team because I really want to do something good for the world and the people such as solving environmental problems. Also, I want to study biological sciences in college. This also encourages me to participate in iGEM which will enrich my biological knowledge and give me lab experience. In addition, I learn to work as a team thanks to iGEM and I believe that this will be very useful for me in the future, especially in college. I am very happy and excited to be a part of this amazing team. Hi, I am Ece Özmen, a 10th grade student at METU High Scool. iGEM is all new to our school and to us, this is the first year of the team. iGEM helps us discover the world of synthetic biology and it is a huge opportunity for our future careers. Besides learning about synthetic biology, we also learn how to work as a team. Also I believe that to enjoy every single moment of this competition is very important. This is what I tell people when they ask the question "What is iGEM?". Hey there!  I am Umut, and a 11th grade student at METU High School. I have had a lot of fields of interest in my life but I have never been a very great student. My grades are just a tad bit over average. To me, academic success is its own thing. You should be able to move along and still be able to work on your interests. In my free times I write some code (Java, Python, C/C++) and make some fun applications and/or games. I also really enjoy solving the Rubik's Cube, playing jazz piano improvisation, listening to technology podcasts, discussing quantum physics with my father and creating origami models. The iGEM club helped me get over my laziness and made me remember how much I enjoyed learning in the first place. Hey! My name is Öykü Deniz Demiralay. I am a junior in METU HS. I love biology learning about new stuff, studying about it makes me happy and peaceful. I love working, being busy gets my mind off my problems. Also producing new things and being able to help people is what I mostly like about working. I believe that this will be the opportunity to take me to the upper levels in the area, in this case biology, I am working on. iGEM has so many appealing sides. I loved the experiments since I was a little child, iGEM gave me the opportunity to improve my skills. I hope for the best for all candidates. My name is Safa Taflan. I am a tenth grader in Metu High School. As my favorite subject is Biology, I reallt wanted to take part in iGEM. I believe that iGEM will help us improve our lab skills and make us more interested in biology. As a student who has grown up with science fiction movies about biology, it is exhilarating to even take part in iGEM. I wish everyone good luck and have fun! Hi, I am Selin and I am an eleventh grade student. It is our first year in iGEM as a team and I am pretty excited about the idea of genetically engineered machines. Examples of what could be done by synthetic biology seemed so crazy that I immediately decided on joining the newly forming iGEM team in our school. I learned about so much that I didn’t even know existed. I also deeply love chemistry and electronics. I play volleyball and have practice almost every day. Volleyball has become a big part of my life. Hi, I'm Umay Eren Ertekin. I'm 15 and currently at 10th grade in METU High School. Science has always been one of my biggest interests, most of all chemistry and biology- so iGEM has been an amazing opportunity for me to learn about and get some practice with synthetic biology. I'm extremely glad I joined :) . I hope to continue doing what I love and have a scientific career in the future. I also really enjoy reading, writing, learning new things and playing the piano. My favourite genres in books are fantasy and science fiction, though I have many favourite books of other types as well, and my favourite sport is badminton. Ofcourse we were not alone while doing all this. We owe a huge thanks to the ones who helped us reach to this point. They thoughtu s the fundamentals, helped us be more organized and kept things with in track. Okan Tezcan (Msc graduate in cancer biology, Middle East Technical University):   Hello, I am the instructor of METU High School IGEM team and also one of the biology teachers in METU High School. After receiving my master degree, I have been sharing my academic experiences with the team members. Together with my team members, I am working on a biosynthetic project for showing the contribution to this field.   Students notes: We can not express our love to this person with word theirselves. Even though he was a little rough on us at times, he is very caring and his advices helped us reach to this point! Side Selin Su Yirmibeşoğlu (Team advisor, wetlab specialist)   Hi, I'm Selin. I graduated from METU Biology department. I was a team member in METU 2012 iGEM team and team leader in METU 2013 iGEM team. This year, I am an advisor of METU HS team. METU HS team is unbelieveable. All members of the team are curious, I am really proud of all members of this wonderful and mischievous team! I really enjoyed making things with them based on synthetic biology and also I am really proud of being a part of directing young minds to the science :)   Student notes: Without the help of Selin, we would be nowhere near where we are now. She helped us with the lab work, protocols and general microbiology knowledge. Thank you for being awesome and helpful! İlkem Kumru (IT Master, tech whiz)   Without his guidance, the wiki would look like 80's jam! Huge shoutout goes to İlkem for showing us how a team wiki should look and function like. He is that kind of person who is out scene, but has a big role in making things work seamless. Ayça Çırçır (The professor)   We don't know what we would do without her wisdom. At the start of the year, we took lessons from her to gain basic knowledge and expertise about synthetic biology, molecular biology, biotechnology and lab equipment. We always knew that there was someone out there who we can reach to anytime, and it was for sure that she had an answer to our problems. Kudos to her! Melike Dönertaş (math-head, modelling)   The mathematical modelling of the system would not be possible without her. She helped us find the parameters we need and made deep reasearch about the enzyme activity. A huge thank you goes to Melike. 1.Would any of your project ideas raise safety issues in terms of researcher, public or environmental safety?   Our project works with carbonmonoxide thus we as the METU HS team had to be very careful with the amount of CO and CO2 we are dealing with while the testing. Other than the dangerous gases, there are no checimally dangerous substance used.   Also, by designing a kill-switch system, we made sure that our bacteria with modified genes can never escape were we want them to be in and cause contamination.   2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?   We have followed all the safety procedures during the isolation process from the cells we had and we successfully isolated the DNA without any problems which might have raised safety issues.   3.Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country?   In METU we have a biosafety and ethical research center which monitors the safety of our projects. The members of METU Biology department are also members of National Biosafety Coordinating Committee. We are able to consult to them whenever it is necessary. All the procedures that are used in the lab are also approved procedures that are used by these people mentioned above.   4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?   To us, if the goal is to provide secure bio-systems, a set of standarts and protocols should be considered. For example, integrating an automated cell-lysis system every genetic part that ship, would make sure that the bacteria are not going anywhere. Humans are very intelligent machines, but we are not perfect. If we can load the safety system into the bacteria, it will be the cell itself that deals with it, which has a lower possibility to fail compared to complexer systems like the human brain.     5. What kind of precoutions did your team took in the lab?   In the lab, we always cleared our hands and the tables with alcohol. Teammates with long hair made sure that their hair was not in the way. Also, we executed every protocol that involved contact with air, near two sets of flame beams. This way we made sure that there was no contamination to the environenment or into our experiment setup.       Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/teaextras.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense We would like to thank everbody who contrabuted our work one way or other. Follow this link to see the full attributions page! It was not always serious business around here. We had lots and lots of fun whilst working on our project! You can visit here to discover photos and videos and more about the events and casual lab day work! To view the sources of any kind of information we used, please follow this link. Attributions Fun Stuff Reference Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/extracontact-us.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense Contact METU HS iGEM   E-mail address: metuhsigem@gmail.com   ODTÜ Lisesi, Çankaya, Ankara 06800 Turkey You can also follow us on instagram @METUHSIGEM   Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/contact-ufun.html From 2014hs.igem.org METU HS iGEM 2014 Home Project Team Extras Attributions Fun Reference Contact Us CO-sense We even  dressed up! School decoration Such muffins! Very yummy! Sometimes you need a break.. Hard work pays of. Best team photo ever! Getting ready for the movie! Just found DNA's 3D configuration.. It is about the pencil 30 minutes to build, 4 seconds to sell School decoration! (even more) ONLY 6 LEFT Now 4.. Crank up the price tag! Serving to our teachers ! Non stop selling action! Speechless. Wetlab hard work.. Operation code: OLDIFY Modelling is always fun! A little walk down the road, after long lab work! It is getting crowded over here! Even the entrance! (even more decoration) It is everywhere :O Let iGEM surround you! Doing a presentation about iGEM and CO-sense Can you see the strawberry DNA? Why not Waiting for PCR has never been this awesome And...The cleaning process! |<< < > 1 - 28 To the left, you can see images from everyday life of the wetlaber, photos from the fair and presentation to the 8th graders. We have a ton more pictures on our social media pages.   Find us on Facebook, Twitter and Instagram at @METUHSIGEM pages! CO, WE ARE COMING!   COs are in air, everywhere! Killing every person, silently… Hey iGEM, we stay silent to this? We came, to save humanity! What sort of foe this is, sneaky… Grudges our lives, stingy! Even takes away the ones most lovely, It won’t anymore put saddles on our backs! Hey CO, don’t you pity to children? Leaking from all stoves, heaters Poisoning the sleeping ones slyly By inhibiting their enzymes. Enough of it, what’s that suffer? We’re finding an aid against you! With bacteria, we’ll convert all of you One by one to CO2. Into every house you try to get in Our bacteria will come in. Even if you go in to one bravely, They’ll warn every person, with colors! Are over the days you take lives! Stay in past these all weak states! Everlasting is the created masterpiece! At last, we got rid of your sorrow.     iGEM   It calls people to MIT, Go, it says; to synthetic biology… Ever a nice project goes there, Medals is what it gets: Bronze, silver, gold…   Retrieved from "http://2014hs.igem.org/Team:METUHS-Ankara/fu1292Lethbridge CanadaChinook High School, Lethbridge, Alberta, Canada Winston Churchill High School, Lethbridge, Alberta, Canada Catholic Central High School, Lethbridge, Alberta, Canada Lethbridge Collegiate Institution, Lethbridge, Alberta, CanadaHigh SchoolA Synthetic Biology Approach to Preventing Increasing Antibiotic ResistanceIn April 2014, the World Health Organization released a report about the increasing danger of antibiotic resistance stating ?A post-antibiotic era ? in which common infections and minor injuries can kill ... is a very real possibility for the 21st century.? People continue to misuse antibiotics by not finishing their prescription, using them to combat inappropriate pathogens like viruses, and using them liberally in agriculture. When this happens, antibiotics can enter the water table. This selective pressure causes organisms in the water to adapt and become resistant to the antibiotics. Reducing the amount of antibiotics in the water supply will help to reduce this selective pressure. To remediate antibiotics in the environment we plan to generate a strain of E. coli that will export an enzyme known as Beta-lactamase to degrade Beta-lactam antibiotics. Ultimately, this will help reduce the selective pressure causing the evolution for antibiotic resistance.1661BBa_K1292999http://2014HS.igem.org/Team:Lethbridge_CanadaBBa_K1292000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165520140http://2014hs.igem.org/files/presentation/Lethbridge_Canada.pdfhttp://2014hs.igem.org/files/poster/Lethbridge_Canada.pdfHigh SchoolLog in   Team:Lethbridge Canada/art From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Human Practices Art Outreach Science can seem daunting to the general public, especially when the science in question involves the microscopic. Not being able to visualize the idea can make understanding the concept very difficult. Accordingly, our team collaborated with the New Media Department at the University of Lethbridge under the guidance of Dr. Will Smith. We are not artists, but we attempted to communicate some of the ideas surrounding synthetic biology through physical media. We are slated to appear in a synthetic biology gallery in November. Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/artpril From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Notebook April Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/apriFrom 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Project Fighting Antibiotic Resistance Parts Our Tools Math Modelling our System Safety We can Dance Notebook Methods and Materials Water Treatment Human Practices Reaching Out to the Community App Part Searcher Team Sponsors Thanks to Our Supporters Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canadhttp://2014hs.igem.org/wiki/images/a/a5/LethHS2014_Igemlogo_whiteletters.pnghumanpractices From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Human Practices Overview This year, our human practices consisted of several projects concerning community outreach. We were able to give several presentations including to local high schools, city council, MP’s and MLA’s. Through these we spread awareness about iGEM and our project this year. Along with this, we visited the local wastewater treatment plant and have come into contact with the Oldman Watershed Council - a local non-profit organization. On the more creative side, we created an educational children’s book as an introduction to molecular biology, and we are planning to have a synthetic biology themed art exhibit displaying our teams art work. In terms of technology, we created a parts registry app available for all Apple devices. Education Fighting Antibiotic Resistance Government Fighting Antibiotic Resistance Water Treatment Fighting Antibiotic Resistance Children's Book Fighting Antibiotic Resistance Art Outreach Fighting Antibiotic Resistance Application Fighting Antibiotic Resistance Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/humanpracticeteam From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Team Students Brandon I am in grade 10 at Winston Churchill High School. I enjoy video games, watching movies, designing shirts, practising karate and attempt to code/script RPG games. When I’m not doing school work or anything thing for iGEM I am either in my basement watching movies/television while on my computer playing games or I am practising karate. I am a black belt in karate and have been practising for almost 9 years. I joined iGEM because I thought it would be a nice experience to see synthetic biology and learn about it. I was thinking about going into a career in science being in Chemistry, Biology or Biochemistry. Brandon “Girl whenever I am near you, I undergo anaerobic respiration because you take my breath away” Christina My name is Christina Kong and I am a Grade 12 student who goes to Catholic Central High School. I became interested in iGEM because it is revolves around science and learning which I find enjoyable. I particularly enjoy the lab work portion of iGEM because it's a very hands-on aspect of the competition and I am more of a kinesthetic learner as well. I enjoy math as it is consistent and logical; there's little room for the grey answers, and you can always find a definite solution to the problem. My favorite activities would be playing badminton and exercising. Christina You have nicer legs than an Isosceles right triangle! Dinula I am a grade 10 student at Winston Churchill High School. I enjoy programming, playing the guitar, and watching movies. When I’m not stuck indoors, I like going running and biking. I joined iGEM because the feats of synthetic biology fascinated me and I wanted to learn more about the processes involved. I additionally wanted to develop my computer programming skills by working on the wiki and software aspect of the competition. Dinula I wish I were adenine, because then I could get paired with U Dylan ​Hi, I'm Dylan Sutherland. I go to Chinook High School and have many interests. Of these interests, I am most interested in video games, movies, and reading. I'm not really a sports guy but I enjoy watching and playing rugby. I've been part of the high school iGEM team for two years now and find the science behind it fascinating. In school, I tend to focus mainly on biology and physics. I am a grade twelve student and next year I am going to the University of Lethbridge. Dylan Is it getting hot in here? Or is it just our bond that is forming? Hope My name is Hope. I am 17 years old and am enrolled in my final year at Chinook High School. I will be attending the University of Lethbridge this fall where I will major in Biochemistry and minor in French. In my spare time I love to run, crochet and read. I am also an avid Harry Potter fan. Hope Alcohol and calculus don't mix. Never drink and derive. Krista My name is Krista Fjordbotten, and I am a grade 12 student at Chinook High School. I joined high school iGEM because I felt it would help me gain knowledge about the field of synthetic biology and lab work in general, as I have great interest in the sciences. Next year I plan to attend the University of Lethbridge doing an undergraduate degree in Neuroscience. Outside of iGEM, I enjoy reading, hiking and boating. Krista I wish I were adenine because then I could get paired with U. McKailyn My name is McKailyn Ives, I am currently in grade eleven at Winston Churchill High School. Prior to joining the iGEM team I had little knowledge of synthetic biology but this experience has taught me a lot and I know it will benefit me in my future. I enjoy being apart of a program that sets goals to improve the world and community for the better. In my spare time I like to play basketball and rugby. I hope to make a career out of Humanitarian Aid when I'm older because I love to help people. McKailyn Once I made a Chemistry joke... but there was no reaction. Peter Hi! My name is Peter Quo! Pleasure to meet you, I am a connoisseur of living and thus I like all aspects of life. Favorite pastimes range from archery to horology. I joined iGEM because I wanted to learn synthetic biology and wanted the growing science fields to be apart of my future. Also meeting like minded people is always a plus. Peter Are your eyes an event horizon? ... because they suck me right in. Ronja I’m Ronja Kothe, a Grade 10 Student of Winston Churchill High School in Lethbridge. I play classical guitar and clarinet. My favourite subjects in school are math and science. I am very interested in biology and I thought that iGEM would provide me with an exciting new opportunity to learn more about synthetic biology in a hands-on environment. It has been totally worth it! My favourite part is working in the lab. After High School, I would like to tackle the sciences; right now virology seems like an interesting career path for me. On a side note, I moved to Canada from Germany 8 years ago; “Auf Wiedersehn!” Ronja “You're like an exothermic reaction, you spread your hotness everywhere!” Roya My name is Roya Akbary and I am a grade 11 student at Winston Churchill High School. I have always had a passion for science especially biology! I enjoy iGEM because I love being a part of a team of people who have similar interests as me. After high school, I would love to pursue a career in health care. I am currently volunteering at the hospital which I am really enjoying. I also play the piano, the French horn, and am a freelance shower singer. Additionally, I like acting in school plays and watching movies especially musicals. Roya Are you made of copper and tellurium? 'Cause you're CuTe! Sally My name is Sally Xu and I am in Grade 11 at Winston Churchill High School. I really enjoy chemistry and biology in school which is why I joined the iGEM program. I believed it would be a great experience to work in labs and synthesize a project that our team came up with. Another aspect that I am interested in is computer programming. I plan to take a major part in the coding of our iGEM wiki. Sally Is your sine pi/2? Because baby, you’re the 1. Sunny My name is Sunny Sun and I am a grade 11 student at Winston Churchill High School. In my free time I like to read, play the piano, sketch, paint and spend time with my family. I became a member of this iGem team initially because of my interest in the sciences, however this experience has provided me with valuable knowledge and great hands-on activities far exceeding basic textbook materials. My favourite part of iGem is working in the labs among peers and conducting real experiments that hold real life applications. In the future, I hope to pursue a career in medical research and further broaden my knowledge of science. Sunny Have you heard the one about the sick chemist? If you can't helium, and you can't curium, you'll probably have to barium. Tiffany D Heyos mayos! I’m Tiffany Dang, I am a grade eleven student at Winston Churchill High School. I love to be active and play sports –rugby, badminton, tennis, and rock climbing. If I’m not outside you can find me with my nose in a book or surfing the internet looking for something new to learn. I joined iGEM because I found the field intriguing as it has many applications to everyday life. I also love working in a laboratory setting outside of school and trying to find a solutions to problems we encounter. After high school? I plan to do something in the field of science where I can work in a lab and help people simultaneously –a biomedical engineer. Tiffany D You're so hot, you denature my proteins! ;) Tiffany T My name is Tiffany Trinh. I am a grade 11 student at Winston Churchill High School. I joined iGEM because, science is one of my favourite subjects and I am really interested in learning new things. I’ve learned a lot from iGEM like the use of equipment, methods and operations inside the laboratory, and what synthetic biology is all about. Utilizing the knowledge that I learned through iGEM, I hope to continue my studies in the field of science. Some of my hobbies are playing badminton, playing piano and making crafts. One day I hope to look back and see iGEM as the stepping stone to my career of choice. Tiffany T I'm more attracted to you then F is attracted to an electron. Team Advisors Chris Hey! My name is Chris and I like to do things! Have you ever wondered what your eyes do when you're asleep? Weird right? Yeah, weird. Any ways, I do science and philosophy. Additionally, I model on the side while I work in a lab at the University of Lethbridge. Stay in school! Chris No pick up line here folks. Move along. I don't have the arm strength to lift all of you. Elaine Hi, I am a biochemistry major at the University of Lethbridge with the hope of pursuing a career in veterinary medicine in the future. As a member of the high school iGEM division for the past three years, I have obviously developed a passion for synthetic biology. I find this field of study very versatile and as a result, very fascinating. iGEM has given me the opportunity as a high school student to work with many professionals in various fields. Now returning as an adviser this year, I get to see the students learn just as I did, and it is really nice to be able to share with them what I have learned. Elaine Once I made a Chemistry joke... but there was no reaction. Kieran Hello, my name is Kieran McCormack. I participated at the 2013 High School competition as a student and this year I am taking on a new role as an Adviser for the team. I currently finished my first year of Biochemistry at the University of Lethbridge, which allows for a nice contrast between the normal science world and the more defined synthetic biology world. I enjoy doing new activities and have found great joy from being an adviser on the team; whether that be through new friendships, communications or spread of knowledge. I have enjoyed learning about myself throughout this year, and hopefully you can continue to do so into the future. Kieran What do you call an educated tube? A graduated cylinder. Wesley My name is Wesley Mosimann, I am going into my second year of university as a dual major in biochemistry and physics. My passion is science, but I also enjoy reading, rugby, and teaching. This is my 3rd year in iGEM, but my first year as an advisor, and it certainly is different from the other side. Wesley like the ideal vacuum, you are the only thing in my universe. Yoyo Hello! I am a student at the University of Lethbridge currently finishing first year in the Chemistry program. As a previous high school student I was enthralled by iGEM mostly by the interesting projects past teams have created (I’m looking at you, BactoBlood). Back now as an advisor; I want to be able to give other High School students the same opportunity I was given, that is, to be able to work in a university lab. I hope to one day pursue a career in the medical field, though the specifics are yet to be determined. In my spare time I enjoy video games, watching TV, and exercise, and of course spending time with fiends. Yoyo I was gonna write a Chemistry joke, but all the good ones argon. Team Mentors HJ Originally from Germany I moved to Canada in 2005 to start a research group on the structure and function of the bacterial protein synthesis machinery, a cellular process targeted by over 50% of the known antibiotics. I am intrigued by the molecular design and function of this essential bio-nanomachine. I try to unravel the underlying design principles in order to enable the rational design and engineering of novel bio-nanomachines. I am essentially asking the question if such novel bio-machines can be constructed from simple and fundamental principles or are these assemblies just to complex. Well and that’s why it was extremely easy to rope me into doing iGEM. HJ What did one sister chromatid say to the other? Stop copying me! Justin I'm Justin. I think biology is way more fun when looking at it though the eyes of an engineer, so synthetic biology is a perfect fit for me. I am very excited to be working full-time for the Lethbridge iGEM team for the second year in a row. When not in the lab I downhill/freeride mountain bike, ride motocross, play guitar, and enjoy walks on the beach in San Francisco. Justin Why can't you trust an atom? Because they make up everything. Zak Hey, I'm Zak, and I am an iGEMmer. I thought I was almost free of my fascination with genetic technologies when I transferred to the neuroscience program at the U of L in 2011 from biology at the U of R, but I lapsed right back when I learned that iGEM existed. I thought I had refocused when I began doing behavioural experiments with rats, but immediately searched out a M.Sc. project that has me engineering mouse brain cells. I also love camping, fishing, and hiking. If I have to be out of the lab, I'd like to be way out there. Zak What's a sleeping brain's favourite rock band? R.E.M. Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/tearesults From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Project Colony Forming Unit Assay CFU Assay In order to determine the number of viable cells in a liquid culture, we used a Colony Forming Unit (CFU) assay. This simple assay involves the collection and serial dilution of samples of a liquid culture. These dilutions are then plated and colonies are counted. To determine the number of CFUs per milliliter, you use this formula: CFU/mL = (# of colonies*dilution factor)/volume plated in mL’s We used this assay to generate a standard curve representing the effect of ampicillin concentration on CFU/mL of liquid E. coli cultures with no antibiotic resistance. Here is the curve we have generated: We plan to use this curve to determine the concentration of ampicillin in media that we treat with our project. Dialysis In order to remediate antibiotics in liquids without releasing cells into the liquid, we used a 1000KD molecular weight cut off (MWCO) dialysis tube. By placing our beta-lactamase producing cells in this tube and placing this tube in an ampicillin containing liquid, we hope to release the beta-lactamase enzyme into the liquid and degrade the ampicillin. We ran this test with 10ml (OD600 = 4.628) of cells containing an ampicillin resistant plasmid encoding beta-lactamase in the dialysis tube. We took samples of the ampicillin-containing media every hour and used those samples for another CFU assay. It is clear that the concentration of ampicillin in the media is decreasing, but there is still work to be done to be sure that it is the dialysis treatment and not heat or pH having this effect. We will have those results come the Jamboree! Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/resultwatertreatment From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Water Treatment Waste Water Treatment Plant Visit On June 13, 2014, members of our iGEM team visited Lethbridge’s local waste water treatment plant in order to get an idea of our project could be implemented into a waste water treatment plant. Before visiting the waste water treatment plant, we had envisioned our E.coli to be trapped in size 1,000 Dalton dialysis tubes that would only allow the media in which the cells are suspended in, cell debris from broken cells as well as our enzyme, beta-lactamase, to escape. We were planning on having another 1,000,000 Dalton dialysis tube included in our construct through which the media could diffuse through by continuously pumping fresh water into our construct. This would decrease the concentration of the media on the opposite side of the dialysis tube and thus allow for constant diffusion of the media. We wanted to eliminate the risk of genetically modified DNA finding its way into the water system by inserting filters similar to filters found in miniprep columns that would capture the DNA. This would enable only the enzymes to be excreted into the water system. Tour of the Wastewater Treatment Plant The waste water treatment plant already uses bacteria found in a Human’s gut ecosystem for the cleaning process in order to eliminate nitrogen and phosphorus in the water. Excessive nitrogen/phosphorus in the water can stimulate algae growth, which results in a decrease of dissolved oxygen for other aquatic species. The bacteria in the cleaning process were first introduced into an anaerobic environment, which caused them to take up nitrogen/phosphorus. Next, they were put into an aerobic environment in which the nitrogen/phosphorus inside of their cells was excreted back out into the water. The last step of the process was to move the cells back to an anaerobic environment in which they would take up even more nitrogen/phosphorus than they previously had in their system. Through these steps, they cleaned up the water from excessive nitrogen/phosphorus. In addition to the bioreactor, the waste water treatment plant also used Ultraviolet radiation (UV light) to treat its water, as a final preventative measure. In this final step, UV light was shone underneath the stream of water that rushes out of the waste water treatment plant. The UV light degrades any DNA/proteins, including viruses, to ensure that no microorganisms can replicate the left-overs, after everything enters the water system. In addition, UV light is much safer and cheaper to implement than adding chemicals such as chlorine into the water to disinfect it. Lethbridge Wastewater Treatment PlantHow our bioreactor would work After having toured the waste water treatment plant, we now believe our initial complex construct is no longer necessary; however, instead we can implement our construct into a waste water treatment plant as a large bioreactor that contains our E.coli cells. Since the waste water treatment plant already uses bacteria in its cleaning process, there are enough nutrients in the waste water for our E.coli to survive and thus we do not need to include any LB media in our construct. In addition, adding our E.coli directly into the waste water system would no longer be an issue regarding the release of genetically modified organisms because the E.coli would be contained within the bioreactor and all additional DNA and proteins would be degraded under the UV light before the water is released into the environment. Being able to add our E.coli cells directly into the water also allows us to export beta-lactamase to E.coli's periplasmic space, rather than exporting beta-lactamase directly out of the cell. This means that only the E.coli cells (that contain our beta-lactamase) and the antibiotics would need to come in contact with eachother in order for our construct to start degrading antibiotics, instead of having to force the small beta-lactamase enzymes and antibiotics together. Back (Left to Right): Krista Fjordbotten, Dylan Sutherland, Zak Stinson, Yoyo Yao, Brandon Hall Front (Left to Right): Sunny Sun, Tiffany Trinh, Doug Kaupp, Wesley Mosimann, Dinula De Silva Overall, the tour of Lethbridge's waste water treatment plant was very useful as it helped us to envision the industrial applications of our project. Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/watertreatmebook From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Human Practices Children's Book Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/boomay From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Notebook May Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/maparts From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Project Parts This year, our construct consists of attaching different signal sequences to red flourescent proteins. In addition to E.coli exporting beta-lactamase to the periplasmic space, our project also consists of characterising three different signal sequences: K331007 (Beta-lactamase Bla Signal Sequence) that will export the protein to the periplasmic space of the cell K331008 (Outer Membrane Protein OmpA) exporting the protein to the outer membrane K331009 (Heat Stable Toxin I) which will excrete the protein out of the cell membrane K331007 relates specifically to our project as it is the signal sequence already present in beta-lactamase. K331009 also relates as it will export our other antibiotic degrading enzymes, such as erythromycin esterase A, out of the cell membrane. We are characterizing these three signal sequences as part of our project this year in order to better understand their functions. Although the Parts Registry outlines where the different signal sequences move the protein to in the cell, it is hard to visually see the protein. In addition we aim to characterize these signal sequences with confocal microscopy to see how effectively they work under different environmental conditions. Our construct also uses J04500 (IPTG inducible promoter with RBS) as our promoter as it is a well characterized promoter, and was used by the 2012 Lethbridge High School iGEM team for their insulin secretion construct. To test our construct in the lab, we have used RFP as our gene of interest with a double terminator in order to visually see the export of our protein. When K331007 (beta-lactamase Bla signal sequencing) and K331008 (outer membrane protein OmpA) are used, the protein will be located in the periplasmic membrane and outer membrane of the cell respectively. K331009 (heat stable toxin I) however when used will export the protein out of the cell. Using our dialysis tubing test in the lab, if our construct has K331009 we will see RFP being exported into the surrounding media signalling our construct is successful. From there in our final construct we can replace RFP as our gene of interest with beta-lactamase and erythromycin esterase A. Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/partpp From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Human Practices App The parts registry website is an awesome tool for finding information about parts we use in the lab. We consistently used it in the lab to retrieve information about the parts such as the length of sequence and part type. In the lab, we did not have easy access to a full desktop computer; therefore we used our mobile devices to search up parts about information. However, we found it to be a big nuisance using the parts registry on our mobile devices. But to be honest, the parts registry does not look aesthetically pleasing on mobile devices. Furthermore, you need to constantly zoom in and out and pan around to access the information. To make it even more difficult, in the lab we usually had our hands full and we could only use one hand to navigate the website on our mobile devices. Pretty soon, this got incredibly frustrating and we came with the idea to create a mobile app for the parts registry. When we were designing the app, we kept in to keep the app’s user interface as simple and clean as possible, while still keeping the app aesthetic. This will ensure easy access to the information and the best user experience as the user will be able to find the information they need, in a nice, efficient manner. We plan on adding cool features to this app including a service that allows you to print and email part information, download parts for offline usage, as well as virtually displaying the on a DNA ladder. We plan on making this app available for free on the Apple app store. The app will only be available on iPhones, iPod Touches, and iPads as we ran into problems creating the app on other platforms. But that is not to say that other platforms won’t be supported in the future. Screenshots: The App in Action.Download App from iTunes Appstore Download our app source filesApp is released under the Copyleft license (For more information, go to https://www.gnu.org/copyleft/copyleft.html) Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/apsponsors From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Team Sponsors We'd like to thank all of the sponsors for their amazing and continued support. Their generous contributions have made our team and project possible. Alberta Innovates Technology Futures has been one of our most consistent and enthusiastic sponsors. Thanks to their support, our team has had the opportunity to attend three fully-funded workshops, including an introductory synthetic biology workshop, a workshop focused on presentation skills and public speaking, as well as a preparatory workshop featuring actual iGEM judges and specialists in the field. AITF has also provided the funding behind this program. Furthermore, we'd like to extend our thanks to the University of Lethbridge for graciously providing laboratory space and equipment with which to conduct our experiments. Thank you to Alberta RNA Research and Training Institute for providing the team with wet lab equipment and general lab supplies.You are the foundations to our project and we would like to extend our overwhelming gratitude for all of the help you have provided us this year! Team Attributions We would also like to extend our endless gratitude to the following people: Mayor Chris Spearman and Councillors Jeff Carlson, Jeff Coffman, Blaine Hyggen, Liz Iwaskiw, Joe Mauro, Bridget Mearns, Rob Miyashiro and Councillor Ryan Parker for their time and support in our endeavours; Our MLA Greg Weadick and MP Jim Hillyer for their time and attention; Aleta Neufeld, Bev Burke, Karen Carney, Colleen Valin and Gloria Roth for putting us in contact with the aforementioned members of our municipal, provincial, and federal government respectively and organizing our meetings; Doug Kaupp and Duane Paul Guzzi of the Water and Wastewater Treatment Plants from the City of Lethbridge for providing an insight look at how wastewater is treated and advice on our project; Alan Wu and Iain George for their effort in putting us into contact with experts in water treatment at the aquatic level and the municipality of Pine Creek respectively; Dr. Will Smith for allowing us the space, advice and help for our Art Outreach Project; Adam Christiansen for all his advice and for answering all of our Wiki related questions; The staff of the Lethbridge School District No. 51 and Holy Spirit Roman Catholic Separate School Division No. 4; And all of Wieden lab and Kothe lab for generously aiding us in troubleshooting problems along the way. Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/sponsormarch From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Notebook March Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/marcsafety From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Notebook Safety Safety questions created by iGEM are designed not only for high school students but all members of the iGEM competition including collegiate and entrepreneurial to remind everyone of the importance of safety even in the competition. 1. Would any of your project ideas raise safety issues in terms of researcher safety, public safety or environmental safety? For the Lethbridge High School iGEM team we work in a laboratory here at the University of Lethbridge with Containment Level One - which “requires no special design features beyond those suitable for a well-designed and functional laboratory. Containment is achieved through the use of practises normally employed in a basic microbiology laboratory” -Public Health Agency of Canada. In addition safety in the laboratory setting is a priority concern for us. Before entering a lab setting we are required to wear proper lab attire including a lab coat, glasses and gloves. Long pants, closed toed shoes are required and long hair must be tied back. In every lab we work in at the University of Lethbridge a fire extinguisher, emergency shower, eye wash station, and first aid kit is readily available. We were all informed of where they are located in the labs we work in during our safety training. Although there are some hazardous chemicals we are exposed to or work with such as ethidium bromide, we work under the constant supervision of one of our advisors who assist us whenever there is a potential risk. Before entering in the lab as well we were given WHMIS as well as lab training on the basics of working in a university grade lab. For our project this year we are working with a non-pathogenic strain of E.coli known as DH5which according to the World Health Organization as rated as a Risk Level One -a microorganism unlikely to cause human or animal disease. In the lab we are mimicking our water treatment system on a small scale by the use of dialysis tubing with pores 1 000 000 daltons big. The pores are large enough to allow the export of just beta-lactamase into the surrounding media but not large enough to allow the E.coli to move across the concentration gradient. If our system was commercialized a similar membrane would be used which would allow just the beta-lactamase to move across the concentration gradient. However we have talked to our local waste water treatment plant on advice for the case where the E.coli is able to move across the membrane. They have advised us that at the end of the process of treating both waste and drinking water, UV light is used to kill any remaining debris and organisms in the water before the use in the public. Also to address the issue of debris due to cell death, the membrane used in our system would have pores small enough to prevent the debris from escaping to the surrounding media. However we have also designed a safety mechanism if this measure is not sufficient in which we would have a separate pipe leading from our treatment tank composed of a material similar to an EZ-10 column. The filter in EZ-10 columns are positively charged trapping the cell debris, and in our system would apply the same way to manage all cell debris. The debris then would be contained and sent to be treated as biological waste. We have also discussed the safety behind our project with our local waste water treatment plant as well to gain professional and sound advice. 2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? Our constructs this year are very similar with the exception of the signal sequences in which we have used Bba_K331007, Bba_K331008 and Bba_K331009. However our genes of interest beta-lactmase and erythromycin do not raise any safety concerns in a lab setting. If all protocols are followed using the BioBrick in the lab the part is safe to use. 3. Is there a local biosafety group, committee, or review board at your institution? At the University of Lethbridge to address biosafety Risk and Safety Services is the committee responsible. The goal of the committee is to “... educate, mentor, foster and grow an environment to all members of the University of Lethbridge community where each accepts an individual and shared responsibility in growing a culture that is rich in the assessment, management and control of risk and safety.” -U of L Risk and Safety Services. Their focus is not solely on students but everyone using the campus. For Lethbridge High School iGEM they are aware of our project and support our work provided we follow the Laboratory Safety Guidelines outlined by Health Canada: Public Health Agency of Canada. 4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? Nature.com released a paper which introduces a potential safety issue in the engineering of synthetic bases. Biochemists have developed synthetic bases similar in structure to the four common base pairs of adenine, guanine, cytosine and thymine which have been shown in laboratory results to build DNA that can interact with enzymes. The synthesis of the bases is based on the idea that “ … Rather than hydrogen bonding to keep the two strands of DNA together, these strands nestle up against each other through hydrophobic interactions” -John Timmer. As a result it could be possible to put these synthetic bases into media with growing cells, allow their movement across the cell’s membrane and be incorporated into the cell’s metabolism. For iGEM, the innovation of synthetic bases has potential to be incorporated into project ideas if the theory is successful. Although the innovation may help to reduce safety issues by targeting needed bases for cells, the incorporation of this element poses a risk through malignant use. In addition to the risk of new Biobricks containing synthetic bases being reused by other teams without knowledge to the effects on their systems. To address the issue we propose that if synthetic bases be incorporated into the competition, that knowledge be accessible to all teams and to standardize the practise by a panel of professionals on this field. In addition the creation of synthetic cells does not have a given protocol, and so poses danger in the method and chemicals that could be used. By providing a set protocol on the synthesis of synthetic bases by experts and the biochemists involved it will allow others to work safely with given instructions. Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/safetmath From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Project Math To find the amount of ampicillin inside of a system (relative to a starting concentration) we must be able to calculate how fast ampicillin is removed from the system. We used a closed environment (dialysis experiment) and placed ampicillin resistant bacteria into spiked media and took measures every hour of the ampicillin concentration (relative to the CFU assay) and plotted a graph of the ampicillin concentration versus time. This allows us to find how much ampicillin we will lose over a period of time. This assumes beta-lactamase to be a black box, but we would like to elucidate its role in the degradation of ampicillin, so we started by modeling the growth of beta-lactamase over time with the intent of finding the rate at which beta lactamase catalyses ampicillin. AMPICILLIN We currently have a graph of our colony forming units versus time spent in the dialysis experiment, but our baseline to compare # of colony forming units to concentration of ampicillin is skewed due to errors in experimentation. We will be able to provide a graph of ampicillin concentration vs. time at the jamboree, but for now we only have colony forming units vs. time. Graph of CFU’s vs timeFrom this, you can see that the amount of ampicillin decreases with increasing time. This is a result of more Beta-lactamase being produced by the cells, which degrades our antibiotic.In the future we would like to expand this to different values of E0 and plot concentration vs time instead of CFUs. With this information we could define a rate constant for the degradation of ampicillin relevant to initial volumes of ampicillin and e.coli by defining functions that relate [A] to time. We can see how these relate relative to E0 and form an equation looking something like this: BETA LACTAMASENUMBER OF E. COLI To find the total amount of Beta-lactamase produced by each cell, the initial number of bacteria must be determined: FORWARD (SYNTHESIS) RATE OF BETA LACTAMASE:The following expression calculates the rate that Beta-lactamase is produced for a single cell by determining the product of the Amino Acids produced per second and the inverse of Amino Acids per Beta lactamase with the following expression: Credits go to the 2009 Beijing iGEM team for calculating the average translation speed of e. coli. This determines the amount of Beta-lactamase produced per second by each individual bacteria.TOTAL AMOUNT OF BETA LACTAMASE PRODUCED:Now we have to factor in the amount of e.coli we have at any given point in time. Assuming we are in doubling phase the whole time, we can (very closely) approximate the doubling time of e.coli and solve for the number of e.coli in our system at any time, and therefore, the REVERSE (DEGRADATION) RATE OF BETA LACTAMASE:To determine the amount of Beta-lactamase that is being lost over time, two variables are used in the following expression: OVERALL BETA LACTAMASE PRESENT:We combine both expressions for the Forward (synthesis) rate and the Reverse (degradation) rate for Beta-lactamase: By including the half-life of Beta-lactamase we come across an implicit differential equation in the final expression and this practically unsolvable by hand. We tried using the mathematical software Maple to solve it; unfortunately we couldn’t make it work in the end and are left to make some approximations.We will assume that if we experiment for less than an hour we can ignore the half life of beta lactamase and get the following equation again. However, if we have larger periods of time we cannot reliably calculate the amount of beta lactamase we have. In the future, we would like to reliably calculate the number of beta lactamase in the system at any point in time, and then calculate the rate at which beta lactamase will degrade ampicillin as a function of time, hopefully giving similar values to the ampicillin rate constant. REFERENCES:http://2009.igem.org/Team:PKU_Beijing/Modeling/Parameters Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/matjune From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Notebook June Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/junproject From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Project The Problem The World Health Organization (WHO) recently published a report on the dramatic increase of antibiotic resistances in bacteria around the world, stating that “A post-antibiotic era – in which common infections and minor injuries can kill – far from being an apocalyptic fantasy, is instead a very real possibility for the 21st Century”(1). Antibiotic resistances have serious medicinal and financial consequences. According to the Centres for Disease Control, approximately 70% of all people with bacterial infections in hospitals in the Unites(2) or 1.4 million people get bacterial infections that are resistant to at least one prevalently used antibiotic in hospitals in the United States every year. Furthermore, the National Academy of Science estimates an increase of $4 billion in healthcare costs every year in the United States associated with antibiotic resistant bacteria(2). How do these antibiotic resistances develop? When bacteria are constantly exposed to high levels of antibiotics, they try to develop a defence mechanism against these antibiotics by taking in extracellular parts and even mutating their own DNA to produce an “anti antibacterial protein,” that disrupts the function of the antibiotics by either binding to it or degrading it. Those bacteria that are successful in warding off the antibiotics replicate through natural selection and are said to have an antibiotic resistance. Lately the concentration of antibiotics in the water system, that would cause bacteria to develop antibiotic resistances, has been rising. One of the main ways antibiotics enter the water system is from run-off from feedlots. Antibiotics are fed to 83% of cattle feedlots and 84% of sheep farms in the United States in order to promote growth(3). Additionally, an estimated 75% of all antibiotics fed to animals are not fully digested and will eventually enter the environment(2). A class of antibiotics that are commonly found in the water is penicillin. Penicillin is distinguished by its beta-lactam ring, which is a cyclic, 4-part amide ring attached to a carbonyl group, that prevents cell wall formation and thus kills the bacteria(4). In response to penicillin, the bacteria will produce the enzyme beta-lactamase, which degrades the beta-lactam ring in penicillin using hydrolysis (5). 1. WHO report on antimicrobial resistance2. Antibiotics and the Food Animal Industry3. Preventing Antibiotic Resistance Act of 20134. Lactam5. Siddheshwar, S.S et al (2013). Need and Scope of Development of β-lactums Project The Water Treatment Idea The Lethbridge HS iGEM team is attempting to find a solution to tackle the global issue of increased antibiotic resistances in bacteria by focusing on reducing the levels of antibiotics, primarily penicillin, in the water system. To do this, we are having E.coli excrete beta-lactamase into its periplasmic space. Once the penicillin and the E.coli meet, and the pencillin enters into the E.coli's periplasmic space, the beta-lactamase will degrade the penicillin. This way the overall concentration of antibiotics in the water will start to decrease. Furthermore, we would like to characterize these signal sequences with other antibacterial resistance genes in order to have E.coli excrete other proteins, such as erythromycin esterase A, that would target a different class of antibiotics and therefore make our construct more effective. We hope to be able to apply our construct in a water treatment plant in the form of a bioreactor in order to decrease the levels of penicillin in the water system and limit the amount of time the bacteria will have to adapt to the stressed, antibiotic-rich environment. Project Future Directions There are many things we could continue with as an iGEM team. This includes additional correspondence with the water treatment plant, as it would be useful in solidifying how we could implement our project on a large scale, and how useful it would be in a real world. The characterization of our signal sequences with other enzymes would also be interesting because if our construct works with other “anti-antibiotics” we would be able to implement systems like ours in areas where antibiotics are prevalent in high concentrations. For our math model, we only showed one graph for our results of ampicillin concentration vs time spent in the system (where E0 = 3.7x1010); if we produced more graphs where E0 varied, we could define a rate constant for ampicillin degradation relative to the starting number of E.coli.It would look something like this:\ This would lead to more accurate values of how much ampicillin you have at a point in time. We also hope to continue to reach out to our community and make them aware of iGEM in general as well as what we are doing. One of our main goals for the future is to display our art pieces in an art exhibition for the city in November, even though that is outside of our season for this year. We feel that the support of our community is essential to our success as a team and the more people that we can involve, the more opinions we will gain, thus leading to a more informed and stronger project. Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/projegovernment From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Government City Hall Presentations As a part of the outreach branch of our Human Practices, we decided to make a presentation to our Municipal Government. We contacted the city council and were able to receive a slot on their agenda for June 9th, 2014 at one of their weekly meetings. We wanted to use this opportunity to spread awareness to local leaders about iGEM, our team and our project. We were allowed a 10 minute presentation and a following Q and A session. In our presentation, we defined synthetic biology and the use of E. coli in iGEM. We also described the iGEM competition and Lethbridge’s past participation in Jamborees. Then, we were able to bring forward the issue of antibiotic resistance and share what we have accomplished with our project this year. Presentations at Lethbridge City Council Top (Left to Right): Mayor Chris Spearman, Chris Isaac Bottom (Left to Right): Tiffany Dang, Roya Akbary, Christina KongThe city council officials were very interested in iGEM and our project. They were very impressed with what we are doing at a high school level. Many questions were raised including concerns with the use of E. coli and whether or not there are ways of breaking down antibiotics without using bacteria. City council members were well aware of the issue of antibiotic resistance and were interested to hear our teams proposed solution. After the Q and A, the mayor and city council members thanked us for our presentation and wished us good luck and success at the Jamboree! They requested that we send them our finished Wiki and that we stay in touch to let them know what we accomplish at the Jamboree. Presenting to city council was a very exciting experience as a scientist and as a citizen! We are so grateful to have had this opportunity to communicate with our city leaders and officials and to have received such positive feedback! Government Greg Weadick, Member of Legislative Assembly In addition to meeting with our Member of Parliament as a means to inform our federal government about the iGEM program, we have also contacted our Members of Legislative Assembly Ms. Bridget Pastoor and Mr. Greg Weadick as representatives of our provincial government. Our project this year deals with an issue affecting Albertan citizens, as we are heavily involved in the agriculture and natural gas industry –both, which heavily use water. For us we wanted an outside perspective on the ethics of our project and to learn possible ways it could affect Albertans. We also wanted to inform our provincial government about how high school students in the province were getting involved with science –specifically synthetic biology –through the iGEM program. We have been fortunate enough as well to have the opportunity to meet with Mr. Greg Weadick during our season. However our meeting will not occur until after Wikifreeze on June 23. We hope to inform Mr. Weadick more about the iGEM program, its applications and our own general comments about our experiences. As well as to hear his opinion on our project and its practicality. Government Jim Hillyer, Member of Parliment On May 23, 2014 representatives from our team met with Lethbridge's MP Jim Hillyer. We wanted to bring awareness about what iGEM is and what it has to offer High School students. We discussed our antibiotic resistances project and we were able to teach Jim Hillyer more about the problem with antibiotics in the water. We got great feedback about our project and the iGEM program itself. This experience helped us get an outside perspective on the ethics of our project. Our own federal government is also beginning to accept synthetic biology and it's application. Jim Hillyer is very excited and intrigued about what we do and is definitely cheering the Lethbridge Canada team on!(Left to Right) Chris Isaac, Jim Hillyer, Tiffany Dang, Dinula De Silva, Tiffany Trinh, Elaine Bird Back to TopRetrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/governmenotebook From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Safety Fighting Antibiotic Resistance March Fighting Antibiotic Resistance April Fighting Antibiotic Resistance May Fighting Antibiotic Resistance June Fighting Antibiotic Resistance Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/notebooEducation From 2014hs.igem.org Lethbridge High School Project ProblemWater Treatment IdeaPartsMathResults Notebook SafetyMarchAprilMayJune Human Practices EducationGovernmentWater TreatmentChildren's BookArt OutreachApplication Team StudentsAdvisorsMentorsSponsors Education High School Presentations This year as a form of outreach we decided to visit local high schools and talk to them about synthetic biology. We visited LCI (Lethbridge Collegiate Institute), Chinook High School, CCH (Catholic Central High), and Winston Churchill High School over the span of two weeks - going into all biology and chemistry classes in grades nine through eleven. Presentations at Winston Churchill High School. We informed the students and staff about synthetic biology,our project and the basic components that allow it to work like enzymes, E. coli, and our lab equipment. In addition to presenting, we let the students and staff take part in an experiment where they extracted DNA from a strawberry using basic household ingredients, including: strawberries, meat tenderizer, rubbing alcohol, laundry detergent, glass jars, measuring cups, and strainers. The aim of these presentations were to engage and expand the young minds of our community. Without a doubt, these presentations did so and gained many students' interest to join iGEM. Education Exploration Expo Exploration Expo was a single day event hosted by the University of Lethbridge, aimed at showcasing the science department of the University through interactive displays, research presentations, and performances. This event connects the University to a broader community and has allowed us to reach out to individuals of all ages and backgrounds. Exploration Expo was held on June 7th 2014 and our team set up a booth showcasing synthetic biology. As it was aimed at promoting education and understanding among all, we prepared a simple experiment involving everyday materials that was suitable to be conducted in such an environment. The experiment was extracting DNA from strawberries using household detergent and alcohol (95% ethanol); the option of extracting self DNA was also provided. Volunteer Group at the Exploration Expo A crude extract of DNA can be obtained from a strawberry homogenate. Strawberries are blended in a mixture of salt and water and are then added to a beaker. Meat tenderizer and detergent are then added to break up the cells. The free DNA in the solution can be precipitated with rubbing alcohol. The resulting DNA is visible to the naked eye and can be physically manipulated. This activity will give participants a hands-on experience with DNA. Procedure: 1. Blend 100ml strawberries in 200ml of saltwater solution.2. Filter homogenate into beaker3. Add 30ml detergent and a pinch of meat tenderizer 4. Stir gently, and double the volume with ethyl alcohol5. Allow students to manipulate DNA with tooth picks Names of some individuals who participated in our booth This interactive event allowed many people to come and inquire about synthetic biology and iGEM in particular. It was a great way for young kids to learn about science through a fascinating experiment to which many of them were captivated by. We were also able to reach out to members in the community who usually have no connection with the science field, and it was great to educate them with an informative demonstration. In addition, our team was given the opportunity to speak formally in front of a crowd of people about iGEM and our project this year. This event was the perfect opportunity for us to reach out and interact with the whole community and inform them of who we are and what we do. Education The Oldman Watershed Council The Oldman Watershed Council is a local non-profit organization in Lethbridge. They aim to find solutions to environmental challenges that impact us all. They are particularly concerned with water quality. Seeing that our project deals with solving the issue of the presence of antibiotics in water systems, we decided to contact this organization as a part of our Human Practices sector. We wanted to know if they had any concerns about antibiotics in water, if they were aware of the issue, and if they found our system idea viable. We received positive feedback and the Council offered to promote our team and project on their social media sites. Additionally, they offered us an opportunity to write for their blog. We will use this opportunity to create a series of three installments of blog posts outlining our project, our success at the jamboree, and our future plans and goals regarding our project. This will provide a great vessel for raising awareness about the rising issue of antibiotic resistance and how we are trying to use synthetic biology to solve it. Back to Top Retrieved from "http://2014hs.igem.org/Team:Lethbridge_Canada/Educatio1253TAS TaipeiTaipei American School Taipei, Taiwan www.tas.edu.twHigh SchoolE. mortality: Extending Cell Life by Regulating Telomere LengthTelomeres are repeating sequences of TTAGGG nucleotides at the ends of somatic cell chromosomes. These sequences protect cellular genomes from harmful effects associated with chromosome shortening during cell replication. Due to the finite length of telomeres, telomere shortening is known to be a primary contributor to cellular senescence and cell death. To allow human somatic cells to replicate indefinitely, we have engineered a biological circuit with three primary components to regulate telomere length. (1) Expression of the reverse transcriptase enzyme telomerase to extend telomeres (2) Regulation of the amount of telomerase expressed using an oscillatory mechanism (3) Operation of a safety device to prevent possible cancerogenesis. Through the cooperation of these three mechanisms, and the tools of synthetic biology, lifespan extension is possible.1411BBa_K1253999http://2014HS.igem.org/Team:TAS_TaipeiBBa_K1253000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=160920140http://2014hs.igem.org/files/presentation/TAS_Taipei.pdfhttp://2014hs.igem.org/files/poster/TAS_Taipei.pdfHigh SchoolLog in   Team:TAS Taipei From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:TAS_Taipei1304Ravenwood RaptorsRavenwood 1724 Wilson Pike Brentwood, TN 37027 http://www.wcs.edu/rhs/High School801BBa_K1304999http://2014HS.igem.org/Team:Ravenwood_RaptorsBBa_K1304000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=167620140http://2014hs.igem.org/files/presentation/Ravenwood_Raptors.pdfhttp://2014hs.igem.org/files/poster/Ravenwood_Raptors.pdfHigh SchoolLog in   Team:Ravenwood Raptors From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:Ravenwood_Raptors1265Biotech HSBiotechnology High School 5000 Kozloski Road Freehold, NJ 07728High School1801BBa_K1265999http://2014HS.igem.org/Team:Biotech_HSBBa_K1265000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162620140High SchoolLog in   Team:Biotech HS/3A From 2014hs.igem.org Contents 1 3A Assembly Kit: Block 1 Team 1.1 Step #1: Growing1.2 Step #2: Miniprep1.3 Step #3: Restriction Digest1.4 Step #4: Ligation1.5 Step #5: Transformation1.6 Comments 3A Assembly Kit: Block 1 Team Step #1: Growing Step #2: Miniprep Step #3: Restriction Digest Step #4: Ligation Step #5: Transformation Comments Retrieved from "http://2014hs.igem.org/Team:Biotech_HS/3AFrom 2014hs.igem.org You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs)File:Biotech HS team.png Your team picture Team Biotech_HSOfficial Team Profile<iframe src="http://bthsigem2014.weebly.com/team-profile.html"frameborder="1" scrolling="auto" width="100%" height="600"></iframe>Contents1 Project2 Notebook3 Results/Conclusions4 Safety5 Attributions6 Human Practices7 Fun! ProjectWhat are you working on this semester? NotebookShow us how you spent your days. Results/ConclusionsWhat did you achieve over the course of your semester?3A Assembly Kit SafetyWhat safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? AttributionsWho worked on what? Lindsey Sniffen Description goes here Julia Fineberg Sarah Falotico Joyce Lee Caitlin Yung Cristina Panetta Troy Shafranek Brianna Berlin Sasha Elizar Human PracticesWhat impact does/will your project have on the public? Fun!What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:Biotech_H2014hs.igem.org/wiki/images/2/28/Biotech_HS_logo.png1276NC School Sci MathNorth Carolina School of Science and Math Durham, NC, USA www.ncssm.eduHigh School1401BBa_K1276999http://2014HS.igem.org/Team:NC_School_Sci_MathBBa_K1276000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163820140High SchoolLog in   Team:NC School Sci Math From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:NC_School_Sci_Math/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:NC School Sci Math logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:NC School Sci Math team.png Your team picture Team NC_School_Sci_Math Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project What are you working on this semester? Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:NC_School_Sci_Math1269FHS Frederick MDFrederick High School Frederick, MD USA http://education.fcps.org/fhs/High SchoolEngineering an anaerobic indicator to monitor the health of microbial fuel cellsWe?re seeking to create microbial fuel cells to experiment with genetic engineering and produce a clean source of energy and water. In order to fashion a more effective microbial fuel cell, we need to monitor the viability of bacteria within the fuel cell. Microbial fuel cells produce electricity and water when metal-breathing bacteria respire via an anode placed within an anaerobic compartment of the fuel cell. In order to monitor the health of these power-generating anaerobes, we designed an operon that will cause the microbes to fluoresce when growing in the oxygen-depleted anode chamber. Our operon consists of an oxygen-sensitive promoter, which activates gene expression in the absence of oxygen, and an open reading frame, which encodes a protein that fluoresces without the need for oxygen. This simple reporter system will allow us to determine when anaerobic conditions inside the fuel cell are optimal for bacteria growth and power generation.721BBa_K1269999http://2014HS.igem.org/Team:FHS_Frederick_MDBBa_K1269000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163020140http://2014hs.igem.org/files/presentation/FHS_Frederick_MD.pdfhttp://2014hs.igem.org/files/poster/FHS_Frederick_MD.pdfHigh SchoolLog in   Team:FHS Frederick MD/Basic Research From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Basic Research We hope that our team and others will can use our oxygen-sensitive reporter system to improve S. oneidensis growth and microbial fuel cell design. For example, researchers could use our fluorescent protein to study the length of time it takes from initial placement of the bacteria in the fuel cell until electrical energy is generated. Researchers could also use the gene to determine the optimal bacterial concentration that needs to be implemented in order to maximize energy output. With the glowing protein, researchers may be able to formulate the optimal growth media for the bacteria. Certain media may generate more electricity or more nutrients may need to be added in order to maximize performance. Farmers may also be able to look at what crops produce certain nutrients and then plan from there as to how to generate electricity from the fermentation of these plants. The gene could also be used to study methods for increasing electrical output on a larger scale and thus increasing fuel cell performance. Return to the iGEM 2014 HS Main Page Retrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/Basic_ResearchDescription From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS DescriptionThe Frederick High School Bioengineering team was formed in October 2012 to give students in the Frederick MD public school an opportunity to learn about molecular and synthetic biology by by working on after-school bioengineering projects. In the club's first year, members decided to explore the development and use of microbial fuel cells as a potential source of renewable energy. We began by building a simple fuel cell using the commercially available MudWatt kit and soil collected from a local pond. The MudWatt experiment lead us to consider the possibilities of building a more refined two-chamber microbial fuel cell in which we would use fluorescent bacteria to visually monitor the growth of bacteria on the cathode.Although we were not able to participate in last year's iGEM competition we're looking forward to attending the June 2014 Jamboree.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/DescriptioMaterials and Methods From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Materials and MethodsFollowing the direction of work completed by Drepper et al. 2007, the LOV fluorescent gene was modified for use in this experiment by altering one amino acid in the protein increasing the illumination given by the bacteria compared to the wild type gene. The increased fluorescence of the bacterial colonies makes the bacterial growth within the fuel cell easier to monitor. The NirB gene has already been synthesized by other iGEM teams (BBa_K763002) and was reproduced here only by adding a prefix and suffix nucleotide strain in order to use it in the 3A Assembly process. The LOV gene also received this same prefix and suffix sequence. Please see our project section for details on how the LOV and NirB genes were designed.Each engineered gene was synthesized as an IDT gBlock fragment and incorporated into the pSB1C3 construction plasmid by cutting each with restriction enzymes followed by ligation. After different plasmids containing one of the genes were made they were propagated in separate E. coli bacterial colonies. Each strain was cultivated in a liquid broth and centrifuged to concentrate a bacterial pellet. The DNA was then extracted from the bacteria and purified using QIAprep purification kit. Before continuing we had to confirm the incorporation of the plasmid into the bacterial DNA so a sample was run through gel electrophoresis. Finally, the plasmids were submitted for sequencing. The plasmid containing the LOV gene was successful with the proper DNA sequence present. However, the plasmid with the NirB gene failed to form properly so we were unable to complete the 3A assembly process to then combine those plasmids. This will become a future project for our group in order to continue fashioning a more effective microbial fuel cell.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/Materials_and_MethodNotebook From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Lab Notebook 20 Feb 2014: Received iGEM supplies 03 Apr 2014: Digested NirB and LOV inserts 10 Apr 2014: Ligated NirB and LOV genes into pSB1C3 14 Apr 2014: Transformed E. coli with LOV and NirB constructs 21 Apr 2014: Ligated NirB and LOV genes into pSB1C3 28 Apr 2014: Transformed E. coli with LOV and NirB constructs 01 May 2014: Prepared TSS buffer 05 May 2014: Ligatied the RFP plasmid 08 May 2014: Subcultured E. coli NEB10 Beta 08 May 2014: Tested transformation efficiency of competent cells 12 May 2014: Digested NirB, LOV, pSB1C3 DNA fragments with EcoRI and PstI 15 May 2014: Ligated NirB and LOV into pSB1C3 20 May 2014: Transformed E. coli with LOV and NirB constructs 29 May 2014: Cultured individual NirB and LOV colonies 02 Jun 2014: Purified NirB and LOV plasmids 03 Jun 2014: Visualized NirB and LOV plasmids on a gel 04 Jun 2014: Recultured NirB and LOV transformants 05 Jun 2014: Extracted plasmid DNA from bacterial transformants 06 Jun 2014: Observed plasmid DNA on a gel 09 Jun 2014: Submitted samples for sequencing 11 Jun 2014: Resubmitted DNA for sequencingReturn to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/NotebooLOV Domain From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS LOV DomainLOV stands for light oxygen voltage. It is a sensor protein that detects the presence of blue light (365 nm). In its wild type form it is used by higher plants, fungi, and bacteria. In higher plants LOV controls phototropism and chloroplasts relocation. In this form it absorbs blue light (365 nm) and in the wild state flavin mononucleotides (FMN) link to cysteine. This results in LOV not being able to efficiently emit green light (495 nm) due to FMN.We choose to modify LOV as our anaerobic environment and growth indicator. We choose LOV over green fluorescent proteins (GFP) due to the fact that GFPs are completely dependant on molecular oxygen to glow. However, due to FMN, LOV cannot release green light (495 nm). Thomas Drepper found a solution to this problem. Drepper and his fellow researchers realized the effects of FMN and found away to remove it. By eliminating the LOV domain's cysteine amino acid, FMN had nothing to bind to. Following Drepper's model we replaced the cysteine amino acid at position 62 of the Bacillus subtilis-derived LOV domain with an alanine:001 MASFQSFGIP GQLEVIKKAL DHVRVGVVIT DPALEDNPIV YVNQGFVQMT GYETEEILGK061 NARFLQGKHT DPAEVDNIRT ALQNKEPVTV QIQNYKKDGT MFWNELNIDP MEIEDKTYFV121 GIQNDITKQKThis is where our models departed. Instead of optimizing our gene for E.coli, we choose to optimize codon usage for S. oneidensis. We used the online Java Codon Adaptation Tool to create a DNA sequence which has been optimized for our use in the following ways: Optimized for S. oneidensis codon usage. Avoids use of the four 3A Assembly restriction enzymes: EcoRI, SpeI, XbaI, PstI. Avoids internal prokaryotic ribosome binding sites.When using these parameters, JCAT produces the following nucleic acid sequence:ATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTCAATTAGAAGTTATCAA 50AAAAGCTTTAGATCACGTTCGTGTTGGTGTTGTTATCACTGATCCAGCTT 100TAGAAGATAACCCAATCGTTTACGTTAACCAAGGTTTCGTTCAAATGACT 150GGTTACGAAACTGAAGAAATCTTAGGTAAAAACGCTCGTTTCTTACAAGG 200TAAACACACTGATCCAGCTGAAGTTGATAACATCCGTACTGCTTTACAAA 250ACAAAGAACCAGTTACTGTTCAAATCCAAAACTACAAAAAAGATGGTACT 300ATGTTCTGGAACGAATTAAACATCGATCCAATGGAAATCGAAGATAAAAC 350TTACTTCGTTGGTATCCAAAACGATATCACTAAACAAAAAGAATACGAAA 400AATTATTAGAAFinally, we added a TAA stop codon to the end of the sequence to ensure termination of the translation.We used the sequence alignment tool to confirm that this was in fact the correct gene. The result show an exact match.100.0% identity in 137 residues overlap; Score: 712.0; Gap frequency: 0.0%Intended 1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGKTranslated 1 MASFQSFGIPGQLEVIKKALDHVRVGVVITDPALEDNPIVYVNQGFVQMTGYETEEILGK ************************************************************Intended 61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFVTranslated 61 NARFLQGKHTDPAEVDNIRTALQNKEPVTVQIQNYKKDGTMFWNELNIDPMEIEDKTYFV ************************************************************Intended 121 GIQNDITKQKEYEKLLETranslated 121 GIQNDITKQKEYEKLLE *****************We used an E. coli codon usage application to see how well it will do in the bacteria. This analysis shows that there are multiple occurrences of the codon TTA (for leucine) which is poorly expressed in E. coli. The next most common Leucine codon in Shewanella is CTG, which is also well tolerated in E. coli. The following sequence replaces every instance of the Leucine TTA codon with CTG:atggcttctttccaatctttcggtatcccaggtcaactggaagttatcaaaaaagctctgM A S F Q S F G I P G Q L E V I K K A LgatcacgttcgtgttggtgttgttatcactgatccagctctggaagataacccaatcgttD H V R V G V V I T D P A L E D N P I VtacgttaaccaaggtttcgttcaaatgactggttacgaaactgaagaaatcctgggtaaaY V N Q G F V Q M T G Y E T E E I L G KaacgctcgtttcctgcaaggtaaacacactgatccagctgaagttgataacatccgtactN A R F L Q G K H T D P A E V D N I R TgctctgcaaaacaaagaaccagttactgttcaaatccaaaactacaaaaaagatggtactA L Q N K E P V T V Q I Q N Y K K D G TatgttctggaacgaactgaacatcgatccaatggaaatcgaagataaaacttacttcgttM F W N E L N I D P M E I E D K T Y F VggtatccaaaacgatatcactaaacaaaaagaatacgaaaaactgctggaataaWe also added the following terminal restriction sites to allow our LOV gene to be comparable with the 3A assembly process. Prefix5' GTTTCTTCGAATTCGCGGCCGCTTCTAGAG[part] 3'Suffix5' [part]TACTAGTAGCGGCCGCTGCAGGAAGAAAC 3'To check over our work we also ran the sequence through the [NEB Cutter] to ensure that the 3A assembly restriction sites are correctly located at the ends of the sequence and not elsewhere within the open reading frame. The results show that the restriction sites are present where required and not elsewhere.This is the BioBrick-formatted, Shewanella-optimized, gene we ordered from the sequencing company for insertion into the pSB1C3 plasmid:>LOVGTTTCTTCGA ATTCGCGGCC GCTTCTAGAG atggcttctt tccaatcttt cggtatccca ggtcaactgg aagttatcaa aaaagctctg gatcacgttc gtgttggtgt tgttatcact gatccagctc tggaagataa cccaatcgtt tacgttaacc aaggtttcgt tcaaatgact ggttacgaaa ctgaagaaat cctgggtaaa aacgctcgtt tcctgcaagg taaacacact gatccagctg aagttgataa catccgtact gctctgcaaa acaaagaacc agttactgtt caaatccaaa actacaaaaa agatggtact atgttctgga acgaactgaa catcgatcca atggaaatcg aagataaaac ttacttcgtt ggtatccaaa acgatatcac taaacaaaaa gaatacgaaa aactgctgga ataaTACTAG TAGCGGCCGC TGCAGGAAGA AACReturn to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/LOV_DomaiSponsors From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Ars Biotechnica is a nonprofit profit organization, which was established by our mentors, to help our high school and others build and maintain synthetic biology labs. Clinical RM helped our team purchase needed equipment and supplies when we were just starting up. The Battelle National Biodefense Institute helped cover our iGEM registration costs. New England Biolabs provided our team with many of the enzymes and reagents we needed to for this project.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/SponsorOutlook From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS OutlookThis year's experiments have resulted in minor setbacks and major successes. In our endeavors we have successfully combined the LOV fluorescent domain into our plasmid pSB1C3. The following years remain bright with possibilities. In years following this, future iGEMs members will succeed where we have failed, in which they will unite NirB and pSBLC3. Such completion would lead to the union of two plasmids through combining the promoter and LOV gene. Thus reaching our stride by placing our gene into Shewanella oneidensis to create our anaerobic/bacterial growth indicator. Future members will continue our work and research until a proficient fuel cell is created. Given our dedication and desire to succeed we hope to see our members bringing a functioning fuel cell into iGEM 2020.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/OutlooProject From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Retrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/ProjecElectrophoresis From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS ElectrophoresisAs seen in the gel, the first five bands, after the DNA marker well, show that the pSB1C3/NirB plasmid was transformed and showed an appropriate size banding pattern. The next five wells represented the pSB1C3/LOV plasmid. As seen in the gel, only two samples show bands. Further experiments will need to be completed to study why transformation do not occur fully.We sequenced those samples which showed strong bands on the gel.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/ElectrophoresiSafety From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS SafetyAll members were required to take a lab safety class developed and presented by one of our club members. To create a safe environment for ourselves, we went through basic safety and hazards overview for every lab. To ensure nothing was contaminated, we sterilized our work station using Q-18 disinfectant with a combination of bleach. We also used Lysol wipes on any surface in direct contact with lab materials. For personal safety, we would use Purell before and after taking off gloves. Eye protection was required in lab areas to protect eye health and safety. We also tested and confirmed that the eye wash station was completely functional before experiments were conducted. In addition, we wore lab coats that covered us from collar to knee. All hazardous materials were dealt with safely and with the environment in mind. Only iGEM-supplied E. coli were used. Loose hair was tied back. Proper supervision was a priority, with either Mark Trice or Dave Rozak present for all experiments.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/SafetPublic Awareness From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Public AwarenessThis year the Frederick High Bioengineering Club is not only on the fast track to Boston but also to recognition. Purely through word of mouth Maryland Senator Ben Cardin came to Frederick High to visit with the group. The team presented their research on biofuel cells from the previous years, explained in detail why they won their division at last year's Frederick County science fair, and described what they were currently working on. Thanks to Senator Cardin's interest in our budding iGEM team, we received some local news coverage. Given this opportunity, the team members took the time to also highlight the importance of STEM, expressing how STEM programs such as AP Biology, Chemistry, Environmental Science, and Calculus were great opportunities for students to get their feet wet and explore STEM. This media coverage provoked the local Career and Technology center at Frederick Community College to consider making their own iGEM teams with the aid of the non profit organization created by team mentors Mark Trice, Dave Rozak, and Gary Lopez. The Frederick News Posts covered Senator Cardin's time with the iGEMs group. Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/Public_AwarenesRenewable Energy From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Renewable EnergyThe renewable energy being produced by the fuel cell is the electricity. With the growing epidemic of energy scarcity, this production of a clean renewable source of energy with readily available use of nutrients is a unique answer to an age-old problem. The microbial fuel cell produces electricity by converting cellular energy released during metabolic reactions into electrical energy. This is due to bacteria growing on a negative electrode, as long as there is waste for the bacteria to consume electricity will continue to produce. An alternative fuel that is produce by microbial fuel cells is hydrogen in an anaerobic environment. The waste eating bacteria can also be in lakes to clean from organic pollutants benzene and toluene.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/Renewable_EnergHumanPractices From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Human PracticesAs renewable energy and alternative energy resources have become more and more important in the world we live in today, answers, not suggestions, must be found. The research we enacted will allow naturally-occurring bacteria in the soil to generate electricity, rather than traditional methods, such as coal. Through the genetic engineering of the bacteria, we will be able to enhance the bacteria's ability to deposit electrons on the electrodes to produce electricity.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/HumanPracticeNirB Promoter From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS NirB PromoterThe NirB gene is reliant on a fumarate and nitrate reductase (FNR), which allows the promoter to activate when there is no oxygen present, as well as facilitate regulation of transcription that is responsible for the growth under anaerobic conditions. When oxygen is not present, the 4Fe-4S complex helps join two FNR components. As this happens, it becomes a protein complex that attaches to the NirB promoter part of the DNA, which results in the production of the LOV protein. In an experiment by the 2012 Valencia Biocampus iGEM team (see BBa_K763002), sterile oil was glossed over the tube containing the bacteria. The bacteria were then able to use the oxygen until the levels were completely depleted, resulting in a fully anaerobic environment. Once fully anaerobic, the bacteria began to produce the team's fluorescent gene product, which was under the control of the NirB promoter.We will similarly use the oxygen-sensitive NirB promoter to regulate transcription of the fluorescent LOV protein that we are designing.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/NirB_PromoteSequencing From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS SequencingIn the last week of school, as classes were winding down, we sent our plasmid samples out for sequencing using the BioBrick standard forward sequencing primer (FV2). Our first shot at sequencing the samples yielded poor results because our template concentration was too high.We quickly resubmitted another set of samples and this time asked the company to adjust the template concentrations prior to sequencing. This yielded good results with mixed outcomes.The bad news: None of the five pSB1C3/NirB transformants showed signs of having integrated the NirB promoter. We are in the process of considering what went wrong and how to get better results next time.The good news: We were thrilled to discover that both of the pSB1C3/LOV plasmid samples had successfully incorporated our re-engineered copy of the LOV gene. While other teams have made BioBricks containing the NirB oxygen sensitive promoters (BBa_K763002), we believe we were the first to create a BioBrick containing an optimized version of the LOV domain, which should have better fluorescent properties than green fluorescent protein in anaerobic environments.The following sequence alignment shows consensus among the engineered LOV domain and the two cloned plasmid inserts (002-027-06 and 002-027-08).CLUSTAL 2.1 multiple sequence alignmentLOV --------------------------------------------GAATTC 1402-027-08-VF2_D11.ab1 TTCNGATAAAAAAAATCCTTAGCTTTCGCNANNGNNGATTTCTGGAATTC 10002-027-06-VF2_C11.ab1 TTCAGATAAAAAAAATCCTTAGCTTTCGCNNAGGANGATTTCTGGAATTC 99 ******LOV GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 6402-027-08-VF2_D11.ab1 GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 15002-027-06-VF2_C11.ab1 GCGGCCGCTTCTAGAGATGGCTTCTTTCCAATCTTTCGGTATCCCAGGTC 149 **************************************************LOV AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 11402-027-08-VF2_D11.ab1 AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 20002-027-06-VF2_C11.ab1 AACTGGAAGTTATCAAAAAAGCTCTGGATCACGTTCGTGTTGGTGTTGTT 199 **************************************************LOV ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 16402-027-08-VF2_D11.ab1 ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 25002-027-06-VF2_C11.ab1 ATCACTGATCCAGCTCTGGAAGATAACCCAATCGTTTACGTTAACCAAGG 249 **************************************************LOV TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 21402-027-08-VF2_D11.ab1 TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 30002-027-06-VF2_C11.ab1 TTTCGTTCAAATGACTGGTTACGAAACTGAAGAAATCCTGGGTAAAAACG 299 **************************************************LOV CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 26402-027-08-VF2_D11.ab1 CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 35002-027-06-VF2_C11.ab1 CTCGTTTCCTGCAAGGTAAACACACTGATCCAGCTGAAGTTGATAACATC 349 **************************************************LOV CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 31402-027-08-VF2_D11.ab1 CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 40002-027-06-VF2_C11.ab1 CGTACTGCTCTGCAAAACAAAGAACCAGTTACTGTTCAAATCCAAAACTA 399 **************************************************LOV CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 36402-027-08-VF2_D11.ab1 CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 45002-027-06-VF2_C11.ab1 CAAAAAAGATGGTACTATGTTCTGGAACGAACTGAACATCGATCCAATGG 449 **************************************************LOV AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 41402-027-08-VF2_D11.ab1 AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 50002-027-06-VF2_C11.ab1 AAATCGAAGATAAAACTTACTTCGTTGGTATCCAAAACGATATCACTAAA 499 **************************************************LOV CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 46402-027-08-VF2_D11.ab1 CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 55002-027-06-VF2_C11.ab1 CAAAAAGAATACGAAAAACTGCTGGAATAATACTAGTAGCGGCCGCTGCA 549 **************************************************LOV G---------GAAG---------------------------------AAA 47202-027-08-VF2_D11.ab1 GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAA 60002-027-06-VF2_C11.ab1 GTCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAA 599 * *** ***Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/SequencinTransformations From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS TransformationsIt may be hard to see in this photo, but there were 10-50 colonies on the chloramphenicol agar plates we inoculated with pSB1C3/NirB and pSB1C3/LOV transformed E. coli! We also ran a negative control and saw no sign of growth. After weeks of trying to transform commercially-produced LyoComp bacteria and homemade chemically competent E. coli, the latter approach finally achieved a high enough transformation efficiency for us to propagate and test multiple clones containing our synthetic NirB and LOV genes. In subsequent experiments, we subcultured five colonies from each plate, extracted the plasmid DNA, and analyzed it using gel electrophoresis and DNA sequencing.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/TransformationFrom 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS OverviewWe are interested in creating a microbial fuel cell that utilizes the facultative anaerobic bacteria, Shewanella oneidensis, to produce renewable energy and clean water. In order to optimize the growth conditions in the anaerobic chamber of the fuel cell, a fluorescent protein marker will be added so to visualize bacterial growth under different conditions. We plan to accomplish this using the NirB oxygen-sensitive promoter to induce expression of the glowing gene only when oxygen is scarce. Furthermore, we must engineer a fluorescent LOV domain, which is optimized for expression in S. oneidensis and capable of fluorescence under anaerobic conditions. This genetic construct will help us ensure that the bacteria are actually growing under anaerobic conditions. This would lead to the creation of a BioBrick that can be deposited back into the “toolbox” parts repository for future iGEM teams.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_M2014hs.igem.org/wiki/images/thumb/2/28/FHS_Logo.png/500px-FHS_Logo.pngMembers From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Members and AttributionsKyle Andrushko is an Eagle scout on the road to becoming an optometrist. Having recently graduated from Frederick High school, Kyle is planning on attending UMBC for his undergraduate degree with a major in biology and a minor in business. John's Hopkins medical school will be his next step after taking the OAT. Kyle will be participating in the iGEMs competition in Boston this summer. He has just finished up his first year of iGEMs and has gained expertise in the concepts of the NirB promoter gene.Dillon Kestner is a Frederick High School graduate who has been inspired by the iGEMs club as well as his mentors Mark Trice and Dave Rozak to pursue a career in neuroscience. He plans to attend a local community college in order to continue being involved in his high school's iGEMs club and mentor future participants before transferring to Muhlenberg College to acquire a doctorate in neuroscience. Dillon specialized in the 3A assembly process for his group. Dillon looks ahead to the trip to Boston and seeing his group's work pay off as well as all the work put in by groups from all over the world.Jonathon Soward is an 18 year-old who loves the study of life. Jonathon attributes his love of STEM to a string of inspiring STEM teachers at his school, including Mark Trice, Shelley Miller, and Joyce Tuten. His teachers, as well as his diligence and work ethic, has enabled him to decide upon pursuing a degree in both dentistry and microbiology. Jonathon has been a member of the iGEM team at Frederick High School for the past two years. He has also become resident expert in the light, oxygen, and voltage sensor protein(LOV). Jonathon's nickname is "master pipettor" because of his amazing pipetting skills!Alan Nguyen is a recent graduate of Frederick High School and incoming Freshman to Mount St. Mary's University. Alan has taken an interest in biology and its many forms. This interest has given him the drive to pursue a career in medicine, a choice that he wouldn't have made without his AP Biology teacher, Mr. Trice's inspiration. A two year veteran of the iGEM group, Alan is excited to see the fruits of his group's labor as it unfolds at the iGEMS competition held at MIT. He's specialized himself in microbial fuel cells and now has a fuller understanding of his group's experiments.Mark Trice is a teacher at Frederick High School, where he teachers classes such as Chemistry, Physics, Biology, and Advanced Placement Biology. He also serves as the vice president on the board for the nonprofit organization, Ars Biotechnica. Mark has worked with his iGEM club for two years and is so excited to see this project come to fruition.David Rozak is a research scientist at the United States Army Medical Research Institute for Infectious Diseases and a founding director of the nonprofit organization, Ars Biotechnica, Inc. Dave has been working with the Frederick High School Bioengineering Club for two years and served as an advisor to our iGEM team.Gary Lopez is an original founding member of the Frederick High Bioengineering Club. He graduated from Fredrick High School (with more accolades than are imaginable) and has transitioned to becoming a Hood College student. He has also taken the huge leap from curios member to knowledgeable mentor. His future goals include working in the biotechnology field, achieving a double major in biochemistry and computer science, and eventually owning your own biotechnology company.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/MemberClean Water From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Clean WaterIn addition to representing an affordable source of renewable energy, two-chamber microbial fuel cells produce water as a natural byproduct of the chemical reaction that takes place between oxygen, hydrogen, and electrons in the cathode chamber. The clean water produced by microbial fuel cells could be just as important as energy to people living in environments where drinking water is hard to come by. This is another reason why we chose to focus our efforts on developing a cheap and efficient microbial fuel cell.We hope that the oxygen-sensitive fluorescent operon, which we've worked on this year, will help ourselves and others reach this goal.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/Clean_WateMicrobial Fuel Cells From 2014hs.igem.org TEAM DescriptionMembers and AttributionsOfficial Profile PROJECT OverviewMicrobial Fuel CellsNirB PromoterLOV Domain RESEARCH Materials and MethodsLab NotebookSafety RESULTS TransformationsElectrophoresisSequencingOutlook HUMAN PRACTICES Renewable EnergyClean WaterBasic ResearchPublic Awareness SPONSORS Microbial Fuel Cells A microbial fuel cell is a device that converts the chemical reactions of bacteria into electricity. Within the fuel cell certain bacteria under anaerobic conditions will remove the electrons from organic matter and transfer them to an anode, which will then transfer the electrons through a circuit to a cathode. The current and voltage produced by this process is what creates the electricity required to power certain objects such as a light bulb. The bacteria that we are currently creating is meant to optimize the microbial fuel cell's potential, as bacteria that will glow under anaerobic conditions will reveal any weaknesses in the fuel cell, structural or otherwise, which can then be assessed and dealt with.Return to the iGEM 2014 HS Main PageRetrieved from "http://2014hs.igem.org/Team:FHS_Frederick_MD/Microbial_Fuel_Cell1259WalthamHS BioHawksWaltham Senior High School Waltham, MA USA http://www.walthampublicschools.org/High SchoolStain Removers: Creating an Environmentally Friendly AlternativeFor the Biohawks iGEM team, our primary goal is to develop an organic, safe stain remover for commonplace applications. By transforming bacteria to manufacture an enzyme that breaks down common proteins, we hope to present a new alternative to marketed removal products, which contain harsh chemicals. In theory, many enzymes could be used, varying in function to break-down different molecules such as cellulose in plant-based stains, lipids in oil stains, starches in condiment stains, and so on. However, not all enzymes can be used, as the integrity of the clothing must be preserved and the user must be kept safe from harm. Some enzymes or solutions can cause epidermal damage or respiratory problems. Our goal, using the enzyme subtilisin, is to remove protein-based stains and create a substitute for potent chemicals with a green solution for the 21st century.1211BBa_K1259999http://2014HS.igem.org/Team:WalthamHS_BioHawksBBa_K1259000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161720140High SchoolLog in   Team:WalthamHS BioHawks/Project/Results1 From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly Alternative Home Our City Outreach Project DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafety E-Notebook Team Our Poster Sponsors Results of Gel 1 Restriction Digest Results: Lanes left to right: 1.Ladder 2.BBa_J04500 3.Subtilisin 4.pSB1C3 5.RFP Control This picture shows a gel of our restriction digest (BBa_J04500, synthesized subtilisin gene, linearized plasmid backbone pSB1C3 and RFP control). The ladder is visible, but the digested DNA is not, indicating an error in our experiment. Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/Results1Design From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors DesignOur stain remover device is built of four main components; a promoter, ribosome binding site (RBS), the subtilisin enzyme and a terminator. The promoter and RBS are taken from the distribution kit under part BBa_J04500. We had the subtilisin enzyme and terminator synthesized by the company BioBasic at reasonable cost. In addition, they synthesized the Biobrick prefix and suffix with the part to make it 3A compatible.Basic Descriptions: The promoter and RBS are together in BioBrick Part BBa_J04500, which is the same part used in the practice 3A assembly kit. Subtilisin is a serine protease enzyme that can degrade other proteins. The terminator is BBa_B1006. Parts Explained: The promoter, R0010, is taken from the inducible lac operon. A lac operon is a series of genes that code for proteins that digest lactose. However in our experiment, those genes have been replaced by genes that code for subtilisin. LacI codes for a repressor that halts transcription by preventing RNA polymerase from binding. In the presence of lactose, the repressor is removed and RNA polymerase is able to bind. IPTG is a molecular mimic of lactose, and therefore in the presence of IPTG, the lac operon operon is turned on. In addition, the lac operon is sensitive to catabolite activator protein, or CAP. CAP is a transcriptional activator and is only functional when glucose levels are low and consequently cAMP levels are high. Therefore the lac operon, and in our case the promoter, is turned on when glucose levels are low and IPTG levels are high.B0034 is ribosome binding site on mRNA, which facilitates translation. Before the promoter there is a BB-prefix and after the termination sequence there is a BB-suffix. The two BB sequences are restriction sites where the the gene can be cut and then ligated into a plasmid. The promoter and RBS together are called Part A (BBa_J04500), totaling to 220 base pairs. The specific subtilisin enzyme (Subtilisin BPN’, UniProt ID P00782 6 ) we used has been commercially marketed by the company Novozymes under the name Alcalase for use in detergents. Based on the work of Ghasemi (2012) and Yamabhai (2008) we knew that to get E. coli to excrete our enzyme a signal peptide would have to be added. After our advisor consulted with colleagues, the OmpA signal peptide was chosen. OmpA signal peptide is 21 amino acids long. It helps secrete recombinant enzymes out of the bacteria, where time is an important factor in how much is secreted. Induction time requires several hours, 4 in the experiment by Yamabhai. Normally, it is used to translocate and direct enzymes across the cell membrane of the bacteria. The type of bacteria should not be a major factor, but should also not be ignored. For some enzymes tested by Yamabhai, OmpA was equally effective in secretion as the native signal peptides, however this is not guaranteed for all enzymes. Due to the large variety of enzymes that OmpA can direct, it is advantageous to use it for non-native enzyme secretion in transformed E. coli bacteria. Subtilisin is a protein-degrading enzyme. The peptide bonds are degraded with serine residue at the active site. The enzyme works in a charge-relay network, using Asp-32, His-64, and Ser-221. The three amino acids form a group in the 3D shape of the protein as is indicated in Figure 1. Ser-221 is able to cleave the peptide bond of proteins with its partially negative oxygen atom as is shown in Figure 2. In commercial use, a genetically engineered form of subtilisin is used, as harsh chemical detergents and high temperatures easily inactivate the wild type. It usually consists of 269 to 275 amino acids and its active from pH 6 to 11, with its major activity from pH 9 to 11. Subtilisin comes from the Bacillus genus and is easily found in the environment (in soil bacteria) and is readily biodegradable.Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/DesigSponsors From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors SponsorsThe Waltham Education and Beyond Foundation and the Sally Elizabeth Peters Enrichment Program provided a grant to help us fund the lab work and iGEM Jamboree. New England BioLabs generously donated enzymes and competent cells to support our project.Bio Basics Inc. supplied us with synthesized DNA.A special thank you to Waltham High School Staff, especially Mr. McKnight for making chemical solvents for the team and Mr. McCarthy for helping us create and print the poster!Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/SponsorHistory From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Our City Waltham, Massachusetts was first settled in 1634 as a farming community. 200 years later, Waltham advanced to the forefront of the American Industrial Revolution. In the early 19th century, Francis Cabot Lowell, known as the Father of the Industrial Revolution, founded the Boston Manufacturing Company in Waltham. At his factory, located along the Charles River, he created the Waltham System, a manufacturing model that transforms raw material (in this case cotton) into a completed product at one location. Before this system, the processes of carding, spinning, weaving and fulling cotton were carried on in separate establishments under different proprietors. It was in 1813 that Francis Cabot Lowell combined the processes of turning cotton into cloth under one roof, creating the world renowned Waltham System. Because of this system, Waltham became the epicenter of the American Industrial Revolution and one of the most productive cities in America. The Waltham Watch Company created millions of watches from the late 19th century through the first half of the 20th century. This is how Waltham got its nickname, “The Watch City”. The Waltham Watch factory was known for its engineering advancements in the standardization of parts, automation of assembly, and use of precision tools. These are all core ideas behind the concept of synthetic biology. Today, Waltham is still an appealing site for science and engineering advances, home to such companies as Raytheon, AstraZenaca and Nova Biomedical. The city has also become a center for research, innovation, and higher education, with Brandeis University and Bentley University leading the way.The Francis Cabot Lowell textile mill in Waltham on the Charles River. The Watch Factory in Waltham, MA Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Histor4 From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Results of Complete Subtilisin TestSubtilisin Experiment on Milk Proteins: Trial 2We then tested two other controls against the subtilisin enzyme, to eliminate liquid dispersion as a source of error and rule out E. coli's ability to degrade proteins. This time we used 2ml of skim milk on each agar plate, and added drops of water to plate one, drops of subtilisin to plate two, and drops of E. coli to the final plate. The results below show that when adding subtilisin to the milk, the areas of exposure are clear, while the other controls are barely affected. This demonstrates that subtilisin was the protein degrading factor, and not water or E. coli. Subtilisin Enzyme Test 2 Results : 0 mins Subtilisin Enzyme Test 2 Results : 10 mins Subtilisin Enzyme Test 2 Results : 20 mins Subtilisin Enzyme Test 2 Results : 30 mins Subtilisin Enzyme Test 2 Results : 40 mins Subtilisin Enzyme Test 2 Results : 50 mins Subtilisin Enzyme Test 2 Results : 60 mins Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/ResultsOurPoster'''Our From 2014hs.igem.org Media:pic.pdfRetrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/OurPoster%27%27%27OuE-Notebook From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors E-NotebookOur Journey-September 24: This was the first meeting for the iGEM synthetic biology club for people who were interested in the project and who wanted to get an overview of the club.October 8: We looked at iGEM projects from past years and learned about the jamboree.November 13: In this meeting, we learned about the basics of synthetic biology and discussed possible ideas for our project.January 28: We learned more about how synthetic biology works, as well as narrowed our options for a project. Students were assigned projects to research.February 11: People presented research on their respective topics. February 27: This was the official iGEM team’s first meeting. We narrowed our ideas down to four plausible projects. March 4: Everyone voted on a project, based on a variety of criteria. After much debate, we decided on creating bacteria that could easily break down protein based stains in a safe and biodegradable fashion. March 7: Our wiki group posted the summary of our project on our iGEM project.March 11: The lab people began to grow bacteria after school, called BBa_J04500 (Part A) and BBa_J04650 (Part B), both of which were maintained on pSB1AK3 (a plasmid backbone). This backbone made them resistant against ampicillin and kanamycin antibiotics. These two samples of bacteria were placed on separate agar plates and were then incubated at 37 degrees ºC for 24 hours.March 13: The lab people began to transfer the bacterial Parts A and B from the plates and placed them into separate culture tubes, both which were filled with 5ml LB broth. They were incubated for 37 degrees ºC for 24 hours.March 17: The lab people continued on to step 3: digest and cut Parts A and B from their backbones using the restriction enzymes provided by NEB. The enzymes used were EcoR1, Xba1, Pst1, and Spe1. These parts were incubated at 37 degrees ºC for 30 minutes and then 80 degrees ºC for 20 minutes. We then ran the product through a gel, but unfortunately, the gel didn't show any results. We could only conclude that the digest did not cut the DNA correctly.March 20: We decided to continue the process in order to practice lab techniques. The lab people then met to do step 4: ligation. This is where we had to assemble Part A, Part B, and the linearized plasmid backbone pSB1C3. We incubated the product, following the ligation, at 16 degrees ºC for 30 minutes and 80 degrees ºC for 20 minutes.March 25: The lab people began to perform the transformation. NEB donated 10 beta strain competent cells which took in our ligated plasmid. These cells were grown on a chloramphenicol plate and, from the cells that survived on the plate, the ones that were red had the correct plasmid. Our control was transformed correctly proving that our process during the ligation and transformation process was accurate.April 3: In this meeting, we decided what had to be done, as the practice was over, and assigned new positions so that the wiki and poster could be completed.April 10: At this meeting we discussed research and wiki progress.May 15: The lab people performed a digest of Part A, the synthesized subtilisin gene, the plasmid, and the control, and then ran a gel to check the digest. The gel showed that the digest was unsuccessful. The digest and gel were repeated and was still unsuccessful.May 21: At this meeting we divided up the remaining tasks between the group members.May 29: In this meeting we started to plan for the wiki, the presentation, the poster, and our final lab steps.June 5: Together we planned the next steps of our lab procedure. The wiki people began to put together our website. June 6: The lab people ran a gel of the digest performed on May 15th to double check results. Still no digest.June 10: The lab people digested DNA from the distribution kit with restriction enzymes. They ran the gel to find out if the restriction digest worked. The enzymes did not digest the DNA. The lab people also tested subtilisin's ability to degrade skim milk. Drops of subtilisin were added to agar plates covered in milk, and the reaction was incubated for one hour. Subtilisin appeared to degrade the milk.June 12: This was a meeting where our group decided to go through the wiki and edit everything to make sure it would be ready for the competition. We also began to piece together our presentation.June 16: The lab people performed the second round of the subtilisin on milk experiment. Two additional plates were used as control: drops of water on milk and drops of E. coli on milk. Subtilisin showed milk degradation in comparison to water and E. coli.June 18: All the team members met in the library to work on the wiki.June 19: All the team members met in the library to work on the wiki. We made final touches on our wiki and worked on the poster and presentation. June 20: All the team members met in the library to work on the poster and presentation.Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/E-NotebooHistory/outreach From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Outreach In the fall of 2013, a group of Waltham High School students applied to the Waltham Education and Beyond Foundation for a grant to support the creation of its first iGEM team. The team was awarded a grant from the Sally Elizabeth Peters memorial grant program, the first grant ever awarded directly to a student group! Sally Elizabeth Peters was a Waltham student who died at a young age. She was a "renaissance kind of student", interested in a wide range of subjects, from music to science. Her parents created this scholarship program to fund ideas that bring together diverse groups within the Waltham community that support both the arts and the sciences. Our iGEM team is now part of the larger Waltham High community. The whole school supports our team, helping and encouraging us to strive and achieve our goals. We interviewed students and teachers about our project and told them our main focus and goal of the club. Before each interview, we informed them that we were transporting a prokaryotic gene segment into E.coli. When this gene is in the bacteria, it can express it, secreting it for extraction and application. We also told the interviewees that the team is participating in the iGEM competition in June. We explained that iGEM is an international genetically engineered machine competition and we will be presenting our project to a team of judges via verbal presentation and poster session. Question: How do you feel this iGEM team, dedicated to the new frontier of biology, can impact the community within Waltham High School?-Mr. Diluzio (History teacher): "Amazing - competition drives ingenuity - it is innovative. It's important for WHS to be on the cutting edge of science."-Mr. Leach (Math teacher): "Awesome. This is a great thing for the school to take part in. It is an experience that's not repetitive in the classroom. An inquisitive student culture will help benefit the school as a whole. It is so nice to hear about students creating something original."-Student Colin Holmes: "You should always try to find new things to do, it expands new opportunities. Follow your dreams."-Student Courtney Paschal: "This program gets students to think more and reach their full potential. It can create better communication and leadership among students."'-Student Charlene Preys: "This program can help students become more involved and interested - really useful!"Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/History/outreacFrom 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Crying over spilled milk? Don't worry! We've got the perfect, environmentally safe stain remover for you!!Click on the tabs above to find out more about our stain removerRetrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawkFrom 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Project Throughout the fall and into the winter, our team created a list of many possible topics for our iGEM project. We narrowed our list down to five possible subjects: a pH sensor for tap water at our school, an environmentally friendly paper towel degrader, a stain remover for protein based stains, a pre-food taster that could detect any possible pathogens, and a more effective anti-allergy molecule. After a long discussion of the pros and cons of each topic, the team narrowed the debate to two topics: the pH sensor and the stain remover. There was much lobbying back and forth before the majority of people voted for the stain removal project. And so began our journey. Our project was to create an environmentally friendly alternative to the harsh chemicals in modern detergents. More and more people every day are developing negative side effects from the continuous use of the damaging chemicals. Laundry detergents can cause rashes, red and blistered skin, sun sensitivity, sneezing, and itchy or watery eyes. Our goal was to create a powerful stain remover that was safe to use for all people.Each team member was given a part of the problem to research. Some looked at ingredients in common detergents, others researched natural stain removing remedies. In the end, we discovered a protease enzyme called subtilisin, a broad range protein degrader from the genus of bacteria Bacillus. Subtilisin is an enzyme found in many common laundry detergents and dish washers, but also found in nature.[ref 1] It can be obtained in certain soil bacteria, which creates it in large amounts.[ref 2] Subtilisin is made up of approximately 269-275 amino acids, with a typical globular shape. The active site of subtilisin involves Asp-32, His-64, and most importantly Ser-221. These three amino acids converge in a 3D shape to create the active site (see Figure 1.) The Ser-221 cleaves peptide bonds with its partially negative oxygen.[ref 3] (see Figure 2)Figure 1: Figure 2:Our task was to transform the subtilisin enzyme into E. coli to create a bacteria that could rapidly produce many of the needed enzymes. E. coli has a reproduction rate of 15-20 minutes, which makes it able to quickly create daughter cells.[ref 4] This allows for a flexible bacterial host that is able to quickly test new enzyme designs. Subtilisin also has the capability to break down blood clots.[ref 5] Blood clots occurs when blood platelets mature and combine with a protein, fibrin, to form a liquid-like gel. The blood clots occur naturally in the circulatory system to prevent blood loss in damaged blood vessels and open cuts. However, the coagulation of blood can be irregular, which can be serious if developed near or in the lungs, spine, or brain. Irregular clotting can be caused by things heart problems, stroke, obesity, and smoking. Subtilisin enzymes can be used to break down potentially harmful clots. The protease nature of the enzyme can be used to hydrolyze the fibrin (an insoluble protein). Subtilisin blocks the binding site of the fibrin to prevent more clotting, as well as breaking down the peptide bonds that hold the protein together. References:1. Karl-Heinz Maurer, “Detergent Proteases”, Current Opinion in Biotechnology 2004, 15:330-3342. F. Niehaus et al “Enzymes for the laundry industries: tapping the vast metagenomic pool of alkaline proteases”, Microbial biotechnology 2011, 4(6), 767-7763. http://en.wikipedia.org/wiki/Subtilisin4. Graznya E. Sroga et al “A Strategy for in vivo screening of Subtilisin E reaction specificity in E. coli periplasm” Biotechnology and Bioengineering, VOL 78, NO 7, June 30 20025. Younes Ghasemi et al “Cloning of a Fibrinolytic Enzyme (subtilisin) gene from Bacillus Subtilis in Escherichia coli”, Molecular Biotechnology, 2012 52:1-76. http://www.uniprot.org/uniprot/P007827. Montarop Yamabhai “Secretion of recombinant Bacillus hydrolytic enzymes using Eschericia coli expression systems” Journal of Biotechnology 133(2008) 50-57Figure 1. Crystal structure of subtilisin showing catalytic triad, image from http://www.cryst.bbk.ac.uk/PPS95/course/7_tertiary/glob_enz.html Figure 2. Hydrolytic action of subtilisin, image from http://lib.convdocs.org/docs/index-130501.html?page=5Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Projec3 From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Results of Initial Subtilisin TestSubtilisin Experiment on Milk Proteins: Trial 1These sequential pictures document subtilisin's effect on skim milk (protein). In the first round of this experiment, we tested 1ml (top plates) and 2ml (bottom plates) of milk on agar plates. Drops of subtilisin enzyme were added to the plates on the right. After 60 minutes of incubation, it was apparent that subtilisin degraded the milk.Subtilisin Enzyme Test 1 Results : 0 mins Subtilisin Enzyme Test 1 Results : 10 mins Subtilisin Enzyme Test 1 Results : 20 mins Subtilisin Enzyme Test 1 Results : 30 mins Subtilisin Enzyme Test 1 Results : 40 mins Subtilisin Enzyme Test 1 Results : 50 mins Subtilisin Enzyme Test 1 Results : 60 minsRetrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/ResultsOurPoster'''Our From 2014hs.igem.org Media:pic.pdfRetrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/OurPoster%27%27%27OuHumanPractices From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Human PracticesThe enzyme subtilisin has many uses. We focused our project on the stain fighting ability of subtilisin. It has the ability to hydrolyze the peptide bonds that hold together all proteins. This enzyme is environmentally friendly, meaning that it can be readily degraded back into the earth. It also does not produce the harmful side effects or allergic reactions that the harsh chemicals in laundry detergent can cause. Subtilisin is therefore a green alternative in the detergent field. HealthSubtilisin can cause a respiratory allergy in some people when they come in contact with it in detergent. Reactions differ with the frequency, magnitude, and duration of the exposure. These cases however, are very rare. Because of the low concentrations in detergent, subtilisin is not a concern for skin or eye irritation. There are no major health concerns for the use of subtilisin in laundry and cleaning products.Alternate Uses: Blood ThinningBlood clotting, or coagulation, is the process in which mature blood platelets and fibrin (a protein) combine to form a liquid gel. This gel forms blood clots to prevent blood loss when repairing a damaged blood vessel. This process occurs naturally in the circulatory system. Sometimes blood clots form in places and at times where a person does not need it. These clots can be caused by heart problems, stroke, obesity, or smoking. If a clot forms in or near major organs, serious problems can occur, such as: Embolism: The result of an embolus (any object in the circulatory system that can clog arteries or capillaries during circulation) blocking a blood vessel in the circulatory system, causing vascular occlusion (a cut off of the bloodstream due to a blockage), which could affect a distant part of the body. Thrombosis:The formation of a blood clot by platelets and fibrin which can clog blood vessels, possibly causing oxygen deprivation if the clog is significant and leading to lactic acid accumulation (an acid that is toxic in large quantities), which can then lead to tissue cell death and prevent oxygen from reaching the rest of the body.Subtilisin has the ability to break down these blood clots. Subtilisin is a serine protease, an enzyme that degrades proteins by hydrolyzing peptide bonds. Subtilisin enzymes use nucleophilic attacks (they add an electron) to hydrolyze peptide bonds by removing an OH group from the serine residue of the protease and an O from the neighboring carboxyl group of the amino acid chain. This cleaves the peptide bonds between amino acid chains. Clots are formed partially by fibrin, an insoluble protein. Ser-221 is the part of the subtilisin enzyme that blocks the active site in the blood clots, preventing the substrate from binding. This blocks further clotting, while also breaking down the fibrin in the clot. Many potential deaths from blood clots could be prevented if the blood clots could be degraded by subtilisin.Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/HumanPractice2 From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Results of Gel 2Restriction Digest of DNA from Distribution Kit: Lanes left to right: 1. Ladder 2. DNA cut with EcoR1 3. DNA cut with Spe1 4. DNA cut with Xba1 5. DNA cut with Pst1This picture shows our gel of the restriction digest of DNA from the DNA distribution kit. If the restriction enzymes were working, EcoR1, Spe1, Xba1, and Pst1 should cut the DNA at their designated restriction sites and consequently DNA fragments should appear on the gel. The results of the gel show that the DNA was never digested, and therefore the restriction enzymes may not have been functional or we had an error in our procedure.Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/ResultsProcedure From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors ProcedureOur lab procedures consisted of three activities; practice with the 3A assembly protocols, building our stain remover device, and testing the stain remover functionality.Practicing with the 3A Assembly KitBefore we began our project, we practiced using the 3A Assembly Kit: restriction digests, gel electrophoresis, ligation, and transformation.Building the Stain RemoverAs with the 3A assembly practice, there were 4 fundamental steps to the process. The first was to use the restriction enzymes to digest the component parts, in this case BBa_J04500 and the synthesized subtilisin DNA. Our subtilisin gene and terminator were synthesized by BioBasics, and delivered in a plasmid with compatible BioBrick restriction sites. Second, a gel is run prior to ligation to verify the fragment sizes match the calculated predictions.The third was to ligate the two parts into a plasmid. And the fourth was to transform the E.coli with the plasmid. The engineered bacteria can then be incubated, allowing it to secrete subtilisin, which can then be extracted for potential stain removal.1: Restriction DigestWe first digested BBa_J04500, subtilisin, pSB1C3 and RFP control with their respective restriction enzymes. EcoRI and SpeI were added to BBa_J04500, XbaI and PstI were added to the synthesized subtilisin tube, and EcoRI and PstI to the plasmid pSBIC3 backbone tube. An RFP control tube received EcoRI and PstI, which would show that digestion worked properly. Each tube had approximately 50 ul of solution total and were incubated for 30 minutes at 37 degrees C and 20 minutes at 80 degrees C using a water bath. 2: Running the GelAfter performing the restriction digest for BBa_J04500, the subtilisin gene, the plasmid, and the RFP control, a gel was run to identify the fragment sizes and verify the digest occurred properly. The gel lanes were loaded using the DNA and loading dye, totaling 5 ul. The gel ran for 6 mins under 275v. If the digestion worked correctly, and the gel supplied evidence to support it, then the transformation could continue. However, after running the gel electrophoresis of all digested sequences, no visible results were found. We then repeated the digest and gel and still we were unsuccessful. View Our ResultsBecause the DNA ladder was present and its fragments were correctly distributed, the lack of data from the samples infered a digestion-based error of some kind. Without the vector, the experimental E. coli could not be grown, preventing the biological production of subtilisin. Due to the ultimately insufficient resources of DNA, we were unable to continue our transformation. We hypothesized that the restriction digest error may have been caused by nonfunctional restriction enzymes. To test this, we digested DNA from the DNA distribution kit with restriction enzymes. Four samples of DNA was incubated each with one type of restiction enzyme. We ran the results on a gel, and the DNA did not appear to be digested. We concluded that the restriction enzymes were nonfunctional. View Our Results3: Ligation into PlasmidIf digestion worked properly, the parts would be combined in solution and ligated. Although some variations of ligation would be arranged incorrectly, there should be sufficient correct vectors that could be inserted into E. coli. 4: Prokaryotic TransformationThe competent E. coli would be incubated on ice for half an hour. The vectors would then be forced in by heating the cells in a water bath at 47 degrees Celsius for 1 minute, then put back on ice for 5 minutes.The bacteria are then grown at 37 degrees Celsius in an incubator for 24 hours on agar plates. Following initial incubation, they would then undergo another 24 hour growing period at room temperature in a controlled environment. An antibiotic would be applied, and all bacteria not properly transformed would not grow, leaving only the subtilisin-producing colonies. The colonies are then placed in 5ml tubes, where the subtilisin will be secreted into solution and then extracted from the cells.Testing the Stain RemoverEven though our transformation of E. coli failed, we still wanted to simulate how our project idea would have worked if the experiment was successful. Since our goal was to transform the subtilisin gene into E. coli to produce enzymes that digest proteins, we re-hydrated subtilisin enzyme powder (Sigma Aldrich P5380-25 mg) and tested its effectiveness on milk. We chose skim milk because it had a high concentration of proteins.1: Initial Subtilisin Control TestWe added 1ml and 2ml of skim milk to agar plates, and placed 10 drops of 10ul of subtilisin enzyme onto each experimental dish. Subtilisin's enzymatic decomposition was successfully observed. The control plates with only milk were relatively unchanged. This experiment supported the functionality of subtilisin in the decomposition of the proteins. View Our Results2: Complete Subtilisin Control TestWe then wanted to confirm that subtilisin had protein-degrading capabilities, and not E. coli. We also wanted to eliminate liquid dispersion as a source of error. So, we repeated the experiment with three plates, each with 2ml of skim milk applied on top. The first control plate had 10 drops of 10ul of water, the second with 10 drops of 10ul of subtilisin, and the third with 10 drops of 10ul of E. coli, which was grown in LB overnight. Similar to the previous experiment with only milk, the control with water was unchanged and the water diffused into the milk without modifying it. When E. coli was applied to the milk, there were no significant changes. Similar to the last experiment, milk treated with subtilisin was degraded. This proved that subtilisin was solely responsible for protein degradation. View Our ResultsRetrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/ProcedurSafety From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors SafetyPre-Project:In the beginning of the school year, we all went through a safety training session in each of our science classes. Based on the school's Chemical Safety Manual approved by the Waltham Public Schools, we learned how to engage safely in investigations in the classroom. All of our science teachers are RCRA and GHS trained. At the end of this safety training session, we were required to sign a safety contract proving that we understood the lab rules and agree to adhere to all safety procedures. During our AP Biology class we practiced using proper scientific techniques, such as sterile technique, working with living cultures, micropipetting, gel electrophoresis and transformation. This preliminary practice before our experiment helped reduce human error during our actual project.As we began collecting supplies for our project, we reviewed and saved the material safety data sheets (MSDS) that came with the materials. By the time we began working in the lab, we were sufficiently prepared to handle the tools and materials to ensure a safe and accurate lab proceedings.Organisms and Proteins: As we experimented in the lab, our team took precautions for the safety of the lab. We used a nonpathogenic strand of E. coli, which has a biosafety level of one, and DNA from the bacteria Bacillus licheniforms, which also has a biosafety level of one. Subtilisin enzyme can sometimes cause respiratory allergies, but only at exceptionally high quantities, of which we did not use in the lab. Subtilisin can cause a respiratory allergy in some people when they come in contact with it in detergent. Reactions differ with the frequency, magnitude, and duration of the exposure. These cases however, are very rare. Because of the low concentrations in detergent, subtilisin is not a concern for skin or eye irritation. There are no major health concerns for the use of subtilisin in laundry and cleaning products. Otherwise, the subtilisin enzyme is a not a cause for health concerns. During the Project: Throughout the entire project, we all used proper sterile technique. Before and after each experiment we wiped down the lab benches with ethanol in order to sanitize the area and prevent any sample contamination. We also always washed our hands before and after each experiment, and no food or drinks were allowed in the lab area. Every time the group handled any of the supplies, we protected ourselves and the purity of the materials by wearing protective equipment, including coats, gloves, and goggles. At all times the lab group was supervised by our AP Biology teacher. Finally, all live bacterial cultures were sterilized before disposal.Retrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Project/SafetTeam From 2014hs.igem.org Stain Removers: Creating an Environmentally Friendly AlternativeHome Our City OutreachProject DesignProcedureResults of Gel 1Results of Gel 2Results of Initial Subtilisin TestResults of Complete Subtilisin TestHuman PracticesSafetyE-Notebook Team Our Poster Sponsors Waltham High School Waltham Public High School has a diverse student body of approximately 1,300 student and 125 teachers. Waltham is a city just west of Boston, MA with a population of 58,000 residents. Team Our team consists of students currently enrolled in AP Biology and a few aspiring biology students. Once we were introduced to the idea of iGEM, people who enjoy science and wanted a hands on experience in laboratories came together to form the BioHawks team. Our team consists of 8 students, our biology teacher, and a parent who is serving as our advisor. Rutvi Bhatt: Hi, I am Rutvi and I am a BioHawk. I love biology and learning about stuff we experience in everyday life. I find microbiology very interesting and lab work helps me understand the concepts. I love community service and I am involved in numerous awareness activities in India and Waltham. I am in charge of making the wiki for iGEM and helping plan the project. I look forward to presenting our project at MIT and making Waltham proud!! Besides this I love photography and Basketball. I also enjoy managing people and resources, I find it interesting to take charge. In conclusion, this project was a great idea, and I have learnt a lot. Role: In charge of designing, editing, making the wiki, planning and organizing work. Michael Perlow: I am a recent graduate of Waltham High School, in the class of 2014. Being naturally curious, especially in the sciences, I take a particular liking to biology. Last summer, I worked with a graduate student in the Biochemistry department at Brandeis University. Dealing with molecule-interactions (between RPS30B RNA and the u1 snRNP protein), we transformed bacteria and yeast to produce the desired nucleotides or polypeptides. Using that experience combined with the AP Biology class under Mrs. Maddox taken in the 2013-2014 school year, I have developed skills and experience highly adequate for use in synthetic biology. Role: Lab technician, wiki editor of the Procedure page, and OmpA researcherEdward Lo: Hi, my name is Edward Lo and I am currently a junior at Waltham High school. My hobbies include playing the piano and playing tennis. I decided to join the iGEM Club because it interested me in how high school kids could customize their bacteria to perform different tasks. Role: Lab technician and wiki editor for the History of Waltham and Results pagesMina Antic: Hi! I’m a junior at Waltham High School, and always been interested in the sciences (the reason why I’m working on this team). Biology is probably (read: definitely) going to be my major when I leave high school and I thought this project would be a good experience. But besides that, I enjoy playing volleyball, painting, and sleeping. Particularly sleeping, which I don’t do as much as I want to. Role: Lab technician and wiki editor of the Safety and Human Practices pagesMolly Wack: My name is Molly Wack, and I’m a junior at Waltham High School. I joined this club because I think synthetic biology is a really cool field and it’s an exciting time to join it. I think that learning about synthetic biology can help us understand the world we live in. In my free time I like to nap and eat, and I play basketball and lacrosse as well.Role: Enzyme researcher, wiki editor, project overviewLeon Mamish: I am Leon Mamish, take out the student and what do you get? Genius, Billionaire, Playboy (ok not really), Philanthropist. But seriously, I am a student at Waltham High School interested in the sciences, specifically biology and chemistry. I joined the iGEM team due to my strong interest in biology and plan to peruse a career in science. My hobbies include sleeping, watching movies, sports and eating. Also, sleeping. Role: Lab technician and wiki editor of Human Practices pageJason Gonsalves: I’ve always had a peculiar interest for science. I’ve completed Environmental Science, Biology, Chemistry and AP Biology in high school and my interest has only grown stronger. When I heard about the iGEM team, I just had to check it out! I love biology and wish to pursue a carrier in it. My other hobbies are track and field, writing and sleeping. Role: Lab Technician and blood thinning researcherJames Zanghi: Hello, I am James Zanghi! I am a sophomore at Waltham High and I really enjoyed biology this year.I wanted to expand my horizons with scientific experience and learn even more about the biology frontier. I am the Founder and President of the WHS History Club, hockey player, and academic. I am a student who appreciates learning and takes hold of opportunities by working hard with passion. Role: Lab technician and public relations by interviewing the Waltham High School community. Marisa Maddox: I have been teaching Introductory Biology and AP Biology at Waltham High School for seven years. In my free time I enjoy spending time with my family, traveling and sleeping. Role: Instructor Edward Wack: I am an advisor to the Waltham BioHawks iGEM team. When I'm not coaching youth sports or gardening and doing yard work, I lead the Bioengineering Systems and Technology Group at MIT Lincoln Laboratory.Role: Advisor, research librarianRetrieved from "http://2014hs.igem.org/Team:WalthamHS_BioHawks/Tea1268OLS Canmore AB CAOur Lady of the Snows Canmore, Alberta, Canada http://www.redeemer.ab.ca/Schools/Our-Lady-of-the-Snows/Pages/default.aspxHigh SchoolHeat-induced Olfactory BiosensorAs Our Lady of the Snow?s first iGEM team, we have decided upon creating a heat-induced olfactory biosensor using E. coli as a chassis. Constructed by placing a wintergreen odor enzyme generator downstream from a heat sensitive promoter, the bacteria should emit a noticeable wintergreen smell starting near a human body temperature of 37�C. As the temperature increases the heat sensitive promoter gradually decreases the effect of the repressor to allow for maximum wintergreen enzyme generation around 42�C. The creation of this biobrick will serve as a first step towards a multiple input bio-sensing system. Such a system may include the input of heat and different wavelengths for activation. Both pursuits will help achieve the goal of using bacteria in increasingly complex circuits to more effectively sense the environment.1602BBa_K1268999http://2014HS.igem.org/Team:OLS_Canmore_AB_CABBa_K1268000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162920140http://2014hs.igem.org/files/presentation/OLS_Canmore_AB_CA.pdfhttp://2014hs.igem.org/files/poster/OLS_Canmore_AB_CA.pdfHigh SchoolLog in   Team:OLS Canmore AB CA/DIY.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Do It Yourself 1. Dremelfuge 2. Shaker Table/Incubator 3. Setting Up A New Lab   Dremelfuge (Dremelfuge concept on-line from: http://www.thingiverse.com/thing:1483)Back to the Top   Link to view/download dremelfuge video: http://2014hs.igem.org/wiki/images/d/df/OLS_Diy_dremel_video.MOV In to order fulfill our lab’s need for a centrifuge, the team decided to purchase a Dremel 3000 Variable Speed Tool. The tool has a nose cap with an integrated wrench that allows the nose cap to be used as a wrench allowing us to tighten different accessories of our choice. The Dremel tool has more of the appearance of a drill as opposed to the typical centrifuge seen in a lab. For additional safety precautions, we cut a paint can to function as a protective barrier around the Dremelfuge. This prevents any objects from flying out of the device and causing harm within the lab. This was a necessity as the tool rotates at very high speeds. Since it has the ability to hold different attachments, the Dremel tool can be used for cutting, grinding, sanding, carving and polishing. Within our lab, its main purpose was a centrifuge. We ordered a 3D printed Dremelfuge piece (pictured left) to do this. The Dremel tool has the capability of rotating at high and controlled speeds, a function that makes it a suitable centrifuge. Costs for dremelfuge (shipping, currency conversion and taxes included) $67.06 - 3D dremelfuge bit from http://www.shapeways.com/ $176.09 – Dremel rotary tool and drill press stand from http://www.canadiantire.ca/en.html   Shaker Table/Incubator (Design adapted from http://openwetware.org/wiki/DIYbio/FAQ/Projects#Incubator.2C_Shaking_Incubator )Back to the Top   Detailed plans for our shaker table/incubator can be found at http://www.instructables.com/id/Incubator-Shaker-Table Link to view/download shaker table/incubator video:http://2014hs.igem.org/wiki/images/c/c7/OLS_Incubator_video-quicktime.movWe started off by purchasing a 4 x 8 foot particle board and cut it to pieces, creating 6 16x48 inch rectangles. We cut one of the the rectangles with the dimensions of 16x16, and another one in half to make the door and created a rectangular box by gluing them together and stapling them afterwards using a staple gun. We drilled 4 holes on one side being the top and this was where the platform was suspended with the use of fish lines. Next we drilled a hole on the 16x16” side to make a hole for the plugins. We used a record player to simulate the same effect as a shaker table. We used cyanoacrylate and glued nuts to the spinning platform of the record player. We then used the corresponding bolt and drilled the a hole through the platform with the diameter just a bit bigger than the diameter of the bolt.  With the box and the record player and platform assembled we just needed a heat source and a fan to dissipate the heat. We used a thermostat to set the temperature for the light bulb for the heat source. Initially we used computer fans to try and circulate the temperature. We ended up using a regular fan because the computer fans showed no progress..  However, because of fire hazards and the risk of mould growth, we had to choose different materials for the box. We ended up purchasing a plastic container with roughly the same dimensions. Since the box had a lid, we had to construct a frame to hang the fish wires that was later changed into chains. Since the record player was too big for everything to fit, we decided to strip all the unnecessary parts, (cd drive, lights, button mechanisms, etc.) and kept only the motor that spun the platform and the base. We then attached the base to the bottom of the plastic container through nuts and bolts. We attached the spinning platform with the motor using the same technique. However, since we did not have a power source for the motor, we used the 15V power supply to make it spin. We soldered one end of the alligator clips to the circuit board in order for everything to work.  Air Temperature Water Temperature   Setting Up a New LabBack to the Top   Setting Up Your Own Lab for $4500Item or EquipmentWhere Acquired FromCost   Fridge/Freezer (-20 and +4)Already at schoolFreeFume Hood Already at schoolFreePipettes (p10, 20, 200, 1000) Genefoo.com$225.00Autoclave Donated from other Junior High (JPII Okotoks)FreeCentrifuge - Bigger Tubes 2,300 rpm Donated from other High School (HTA Okotoks)FreeIncubator/Shaker TableDIY$100.00Dremelfuge - Smaller tubes 5,000 - 25,000 rpmDIY$245.00MicrowaveDonated (The Morgans)FreePCR Ordered eBay$789.49Gel electrophorisis School councilFreeLab stock University of CalgaryList Below$2931.77TotalTotal$4291.26 Back to the Top 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/DIY.htmlOutreach.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Outreach   This year, the team chose to focus on three key projects: - Creating a working biobrick by assembling two biobricks made by previous iGEM teams - Setting up a synthetic biology laboratory - Educating our community about our project and synthetic biology. The latter of which was separated into two different parts, one of them is community outreach. Once our project goal—creating a heat-induced olfactory biosensor—was decided, several different team members discussed different ways to talk to community members and get them interested in our iGEM project and synthetic biology. In the end, we decided on pursuing three different ideas: Presenting to teachers and other students at our school, connecting with friends, family, and peers through social media, and talking about our project to the town council is the third. Teacher and Student Presentations Due to the team using the school’s lab, starting a discussion about synthetic biology and the heat-induced olfactory biosensor we are creating was important to team members.   Our Lady of the Snows (our school) is a catholic school, hosts students from kindergarten all the way to grade 12; however, the student body consists only of, approximately, 440 students. This allowed team members to visit students from grade 4 all the way through to grade 9. Depending on the audience, different concepts were focused on during the presentations. The presentations for grades 4 to 6 included information on the basic science of DNA, synthetic biology, and bacteria: with the focus being placed on the heat-induced olfactory biosensor that is being made. The content presented to grades 7 to 9 changed little from the other presentations, though more information about synthetic biology was presented. The pieces of information added were concise and informational descriptions of all the steps the team will be following to join the two biobricks together.  Team members also felt the need to communicate to teachers what our project entails. This was accomplished by teaching them about the impact of synthetic biology on the world, techniques used and the science applied when genetically engineering bacteria, and how the heat-induced olfactory biosensor would be created.  These presentations proved successful, since they instilled interest in iGEM in the younger students (grades 4-7) and the team received feedback from people saying that they had learned a lot. Talking to the elementary kids about the benefits of bacteria and about the lab work the team is conducting was phenomenal since the students were very involved in the conversation and very curious. Though the junior high students weren’t as enthusiastic as the others, they were still very attentive and many expressed interest in participating in an iGEM team next year. The teachers were astonished high school students have the ability to participate in such a high level competition and were very accepting of synthetic biology.  Links to all different presentations can be found here: Presentation 1 (Grades 4-6): https://docs.google.com/a/isidore.redeemer.ab.ca/presentation/d/1t72MQKKJt-l05VcBinBHNDs5A5XETNIRqv1pKAvg8OY/edit#slide=id.p38  Presentation 2 (Grades 7-9 and teachers):https://docs.google.com/a/isidore.redeemer.ab.ca/presentation/d/1VlMln6j9twZ-IguEo9l8Oqs-6Ba2b3TYq1za0mKAyGM/edit#slide=id.p73 Social Networking As a small project, team members created both Twitter and Facebook accounts to share updates on the project. Many peers and friends followed these accounts allowing for team members to keep students and community members updated on our project.  Twitter: http://twitter.com/OLeSsence_iGEMFacebook: http://www.facebook.com/OLeSsence DIY - Community We have created our own synthetic biology lab, in addition to some of our own equipment, such as our dremelfuge and our incubator/shaker table  Dremelfuge concept on-line from: http://www.thingiverse.com/thing:1483  Incubator/Shaker Table design adapted from: http://openwetware.org/wiki/DIYbio/FAQ/Projects#Incubator.2C_Shaking_Incubator   We have also contributed to the DIY biology community. The plans for our homemade incubator/shaker table can be found here: http://www.instructables.com/id/Incubator-Shaker-TableNewspaper Article We have also been featured in our town newspaper, the Crag and Canyon, in an article about our team, project, and synthetic biology. This has helped educate our larger community about iGEM, and our project goals.  Link to the article: http://www.thecragandcanyon.ca/2014/05/22/students-take-part-in-igem-competition                Town Council As a final human practices project, a few team members visted Canmore's town council and mayor. A presentation had been scheduled in order to introduce them to our project and open doors for funding next year if the school team decides to continue participating in iGEM. The presentation focused on two main ideas: What is synthetic biology and what is our iGEM project. Even though this was most of what our project consisted of, some ethical concerns regarding Catholic perspectives on synthetic biology and safety concerns regarding E. coli and genetic engineering were also addresed. Since a 10-minute time limit was imposed, the presentation consisted mostly of key information and the mayor along with council memebers had the chance to ask questions during a 5 minute question period directly after.   At the time of the presentation, the council chambers were also open to all members of the community with a few members from the local press recording content from the presentation.   The feedback received from this was positive, and many were impressed that high school students had the opportunity to participate in iGEM. Questions asked during the question period centered on how to actually piece together the two biobricks we are using and how we set out on this endeavor. An example of a comment from during the question period is one council member remarking on the recyclng of material in the construction of our Do-It-Yourself equipment (example: the use of an old record player). All the more, council memebrs and the mayor invited us to apply for funding through Canmore's community grant system.   A link to the presentation can be found here: Town Council Presentation         2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/OutreacThe Team.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Our Team Teachers LucArvisaisA project where you can think outside of the box … I mean textbook – fantastical ! Twenty plus years of high school and I have never heard of Synthetic Biology before this one. We began our journey to experience the definition of Synthetic Biology last December at our small school nestled in the Canadian Rockies. This project definitely helps to enrich our lives if we consider ourselves to be the sum total of our experiences. When I am not working at school, I dedicate much of my time to my two kids (say hello to my daughter below). There must always be time to play in the mountains too, so, get out there and DO something (awesome snow this March)!Jessica PuurunenI'm a mother, biology/special education teacher, technology enthusiast, semi-pro (read: recreational) mountain biker, and a general grumpypants. iGEM is like no other teaching and learning opportunity I've come across in a decade of work as an educator. I studied genetics and microbiology a mere ten years ago... And my how it's changed! A, T, C, G is the same, but that's about it..... Glad I have the students below to walk me through everything. Go team!Janell ToewsI am one of the luckiest teachers on earth! I get to live in beautiful Canmore and work with amazing students at Our Lady of the Snows school. As a music teacher, I am not the first person you would think of when you mention synthetic biology. Luckily, all of my years of organizing band trips have made me a perfect fit for the logistics and PR portion of this team. I am learning more about science everyday and enjoying the journey. Who knows, I might switch my teaching assignment to science next year. Wouldn’t that be a scary thought!     Student Team Alina ArvisaisScience has always been interesting to me, and when I first heard of iGEM I was eager to participate. Since I am only in grade 9, when we first began working on this project I did not know much about the science involved. As our project progressed I learned more about synthetic biology and lab work. I am particularly interested in working in the lab. My main interests outside of school and iGEM are rock climbing and reading.Delaney BarthI am currently a grade 9 student attending Our Lady of the Snows Catholic Academy. I found iGEM to be a very unique opportunity, and did not want to miss the chance to join. By participating in this project, I have learned much about synthetic biology, and am still learning more. I believe that this project will provide opportunities for me in the future, and I am very interested in the lab work and human practices. Besides iGEM, I participate in extracurriculars such as debate and piano. Carter BehmI am 17 years old, and soon to be finished Grade 11 at Our Lady of the Snows. The iGEM team is a unique opportunity for our school, and it has provided many aspects for me to get involved. My main role on the team is the creator/manager of the online website, which I had to learn to do from scratch. I am also quite interested in the lab work. As extracurricular activities I enjoy reading, sports, and playing either guitar or piano.Frances DaviesI am currently a grade 11 student at Our Lady of the Snows Catholic Academy. I have always been involved in sports my community and school. Injuries in sports have led me to a curiosity about the human body, which in turn led to a fascination with genetics. iGem presented me with the opportunity to learn about, and experiment with genetics. Though I am not interested in pursuing a post-secondary education in synthetic biology, opportunities such as iGem allow me to gain in depth knowledge pertaining to synthetic biology, lab protocols, and bioethics. I am involved in other groups within my community, such as student council and social justice. Talia DixonI am currently in grade 9, going to Our Lady of the Snows in Canmore, Alberta. I took an interest in iGEM because it sounded very interesting, educational, and can potentially open a lot of doors for me. It also allows me to try something new and gain experience in a different field. My interests include hockey and debating. Jeremiah EllisI am currently a grade eleven student attending Our Lady of the Snows Catholic Academy. I joined iGEM with the hopes of broadening my education and having an academic experience like no other within my high school life. I believe that iGEM is a great opportunity for us to interact with the academic community and pursue our scientific interests. For those of us who seek careers in science, iGEM opens a door to the scientific world and is a precursor to our post secondary education. Even if we are not continuing with sciences outside of high school, iGEM still provides an education for us that goes far beyond our curriculum. Elisa HallI am currently in twelfth grade at Our Lady of the Snows Catholic Academy. I have always been involved with sports and my community. Through volunteering at the Canmore hospital I have been able to experience the ethics behind medicine and what synthetic biology can do for us in the future. I hope that in the future I will be able to apply the knowledge that I have learned to law, or psychology. I have always been passionate about biology, law, English, and social issues. I believe that the future will propose a lot of opportunities for synthetic biology and am elated that the opportunity has been given to me to be a part of it now and hopefully in the future.Steven Hughes I am currently in Grade 11 at Our Lady of the Snows Catholic Academy. I chose to involve myself in the iGEM team so that I can expand on what I already learned throughout my education. Science is always something that I have enjoyed no matter what type, and iGEM added to the list. The iGem team stood out for me because it offers a certain level of education that the majority of high schools do not offer, so I saw this as an unbelievable opportunity for me and my classmates. Rachel KimI am a grade 9 student at Our Lady of the Snows Catholic Academy. I have attended the school for one year now—working on my second--and I love how many different projects and teams our students can participate in. Go student involvement! I am a part of the recently created badminton club, and, of course, iGEM. Outside of school, though, I enjoy yoga and karate, as well as being involved with Girl Guides of Canada. I think iGEM is fantastic; not only do students get to learn about synthetic biology, they get hands-on experience working in a lab and organizing their own project. There are so many opportunities that become available with this kind of work. Hoping my team does well, let’s go OLeSsence!Matthew MarquezI am currently enrolled in grade 10 and soon to be finished. I am a very curious teenager and I am constantly trying to explore new territories and science is one of the most vast areas of them all. Igem is a great way to connect with pears in my school along with having fun with science. I personally believe that by further exploring this it will satisfy my hunger for knowledge. On the team my main jobs are wetlab, ethics, fundraising, and human practices all of which intrigue me.Nicole MelzerI am currently a grade eleven student at Our Lady of the Snows Catholic Academy. I was skeptical about the idea of synthetic biology due to the little knowledge I had beforehand of what it entails. However, the opportunity that was presented to me through iGEM was something that I could not pass up. Science has always fascinated me, and it is clear that there is great potential for synthetic biology in the future! I love a good challenge and iGEM has definitely challenged my ability for abstract thinking. I am very excited to be a part of the first ever OLS iGEM team and I hope to participate again in my grade twelve year. Freya MorganI am a grade 9 student attending OLS. I am fourteen years old. My favourite subject has always been science, because it is interesting and I enjoy learning about it. I have been to seven schools, and this is the most interesting science opportunity that I have come across. When I heard about iGEM I was really interested and I thought it sounded cool, but since then I have learned a lot about DNA and bioengineering and I am enjoying it lots.Noah NadalI am currently in grade 11 at Our Lady of the Snows Catholic Academy. With the participation in iGEM, I see great opportunities by committing my time to this project, and gaining the experience achieved from iGEM will give me experience in future opportunities in post-secondary and in choosing my career. My curiosity towards synthetic biology is what has driven me towards joining, and working with people as a team has always been something I love doing.Matthew San JuanI am currently in Grade 11 attending Our Lady of the Snows Catholic Academy. I joined iGEM in order to pursue my interests within science. Although my greatest interest is mathematics, I still wanted to learn about synthetic biology and join iGEM for the scientific experience. Growing up in the Philippines, I had never even heard of an opportunity like this. For me, iGEM is something different and it gives me a glimpse of post secondary education. Daniel TullyI am currently a grade 11 student at Our Lady of the Snows. My favourite science has always been biology. After I graduate from High school I plan on going to study Medicine or Bio-Medicine. I joined my school’s IGem team in order to further my experience in biology. I am enjoying the technical aspect of IGem and I am finding the Lab skills and practice very interesting. I believe that the lab skills learned from this project as well as furthering my knowledge of biology will directly relate to my post secondary studies. I am mainly working on the wet lab. My interests include skiing, kayaking, school sports, biology and science in the community. 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/The_TeaAttributions.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Attributions   The OLS 2014 iGEM Team would like to thank the following people/groups for their assistance in starting up a brand new Canmore team: - Calgary Mentors/Technical Advisors: Mr. David Lloyd, Dr. Magdalena Pop, Mr. Iain George (we are especially grateful for their patience in teaching us – we really knew nothing about synthetic biology and how any of the equipment was supposed to work ie. workshops, VROC, emails, …) - Kitwa Ng at iGEM headquarters – especially for rushing us our parts and helping with our “plasmid resistance debacle” - Sarah Lee and Jennifer Hill from Alberta Innovates and Geekstarter - Sacred Heart Parish Church for ethics contributions (Deacon West).   - Canmore Mayor John Borrowman and Town Councillors for hearing our presentation and supporting us, encouraging us to apply for a project grant next year - Maureen MacLellan (safety officer) for touring our lab and giving input to safety practices - Canmore Sweat Shop for rushing the order for our T-Shirts - John Paul II Collegiate for lending us the autoclave and Holy Trinity Academy for lending us a centrifuge - Janell Toews for organizing logistics, cooking our workshop meals, flying us to Cambridge! - Justin Parsons from the Crag and Canyon newspaper for featuring us in a May 2014 article, including a one hour interview and photo session. - Wendy Hutchins at the University of Calgary for helping us with our major lab order to get our lab started. - OLS Science Department for giving up most of their teacher lab prep space and supporting us in the school newsletter. All other OLS teachers who gave us classroom audiences to practice our presentations in front of other students. - The OLS Team listed at our web site for their individual contributions to the project each in their own way 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/AttributionBioethics From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Bioethics  The work done so far in synthetic biology may only be the proverbial tip of the iceberg. Drew Endy has stated that the “scope of material I can work with is not limited to the set of things we inherit from nature.” The growth in the number of teams involved in iGEM over the last decade has been staggering. Not many other fields of science have experienced the rapid growth that synthetic biology has. Ethics is described as “rules of behavior based on ideas about what is morally good and bad” (1), what ethical concerns may exist?   Synthetic Biology is the manipulation of DNA or genes to create new qualities or characteristics in readily controllable and standardized biological systems. As it has such groundbreaking potential for change in regards to human life, the ethics of synthetic biology should be considered before work in this field of science. The Catholic Church expresses the viewpoint that altering certain things in genetics is ethical, and not sinful, however, humankind must never attempt to create life (2). Most notably in recent history there have been attempts to create synthetic cells with various degrees of sophistication. These attempts have been described as producing a large synthetic genome, and not "synthetic life" itself. Despite such dismissals that we may not in fact have “created life” we should be mindful that “reducing life, even though it is non-human life, to merely another quantity that we control, exploit, and subject to market forces is to coarsen our sensibilities towards an important measure of our own being. In every living organism, whether humble bacterium, plant or animal, we encounter a faint glimmer of our own delicate life.” (3) The scope of the high school iGEM project exists within the boundaries of altering, and not creating, life for the betterment of humankind.   Possible misuse of synthetic biology should not dictate whether scientific advancements should be made, but rather how they are regulated. In the MIT Video “The Implications of Synthetic Biology” (4), Drew Endy was asked if a one percent misuse of this powerful technology could potentially set back the ninety-nine percent of researchers advancing the field for good. Few people would argue against breakthroughs in the form of biotherapies, more affordable pharmaceuticals, or eco-friendly bio-fuels. The Catholic Church promotes stewardship and involvement in all scientific fields to increase the potential of nature (5). Nonetheless, it is not implausible that this science can be misused, and it would be ignorant to deny this. It is however possible to support open source global communication to maintain awareness of the reasonably possible one percent of misuse. The human practices component of iGEM, including outreach, as well as the general expectation to build projects for the betterment of society forge in the direction of the ninety-nine percent of the possibly good uses.   Due to the increasing popularity of genetic engineering it is vital that ethical concerns remain as part of the human practices portion of every project whether Catholic or not. Discussing ethics in the open serves as a partial barrier to possible misuse of the science by means of awareness. Through the continual study and development in the field of synthetic biology it is envisioned “that everyone might see clearly that the Church and her Pastors are no opposed to true and solid science, whether human or divine, but embrace it, encourage it, and promote it with the fullest possible devotion (Pope Leo XIII) (6).” This devotion must include the sharing of ethical concerns throughout the diverse, global community of the world today.   1 - http://www.merriam-webster.com/dictionary/ethic 2014 2 - Catechism of the Catholic Church, 1994 Libreria Editrice Vaticana, Page 474-1954 3 - Rev. Tadeusz Pacholczyk, Ph.D. “Making Sense out of Bioethics” http://www.ncbcenter.org/page.aspx?pid=1072 4 – MIT video “The Implications of Synthetic Biology” with Drew Endy, http://video.mit.edu/watch/the-implications-of-synthetic-biology-9400/ 5 – Arthur Caplan, “Synthetic Biology: Is Ethics a Showstopper? http://www.synbioproject.org/events/archive/caplan/ 6 – Pope Leo XIII, http://vaticanobservatory.org/about-us 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/BioethicSafety.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Safety   Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety? Researcher Safety The organism we are using to conduct our project is a laboratory strain of Escherichia coli. Specifically, NEB10-beta competent E.coli, a high efficiency strain derived from New England Biolabs Incorporated. NEB10-beta is treated as a Biosafety Level 1 organism. This level is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment (Centre for Disease Control, 1997). Safeguarding our lab is accomplished by following standard decontamination and laboratory procedures such as disinfecting lab surfaces with ethanol, autoclaving, bleaching, wearing gloves, wearing a lab coat, and wearing safety goggles. Public Safety Respect for public safety is addressed since the organism we are using is non-pathogenic. Furthermore, non-pathogenic E.coli is already present in the human gut. Nevertheless we took steps to ensure genetically modified organisms would not come in contact with school teachers not involved with iGEM who have access to the laboratory prep space. We limited access to E.coli, DNA, stains, and enzymes by locking fridges/freezers, placing signs indicating “incubating bacteria,” and by using separately marked bins for collection of any bacterial contaminated waste to be autoclaved as well as a separate bin for used gels. We keep the area secure with key-only access and have not seen any examples of potential misuse of our promoter or wintergreen enzyme generator to date. There are similar risks of misuse with a number of the commonly used high school laboratory chemicals in the lab storage area. It is for this reason that we restrict access as a general best-practice for the safety of all school community members. Environmental Safety Our project poses minimal threat to the environment. Strains like E. coli K12 are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in non-laboratory environments. Many lab strains lose their ability to form biofilms (Vidal O, Longin R, Prigent-Combaret C, Dorel C, Hooreman M, Lejeune P (1998)) meaning, if the bacteria somehow entered the environment the bacteria would not likely be able to thrive. Even so, there is always an inherent risk of releasing genetically modified organisms into the wild and for this reason proper lab decontamination procedures are always followed.  Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes, did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience? We did not encounter any safety issues for the temperature promoter and or wintergreen enzyme generator as described in the parts registry; no other team who had previously used the same parts expressed any safety issues. Neither of these parts pose significant danger. The first part we are using is a generator of methyl salicylate—which is an organic ester naturally produced by many species of plants. Since methyl salicylate is an organic compound and it is used in many commercial fragrances, foods and beverages, this biobrick was considered safe. The second part is a heat-activated promoter. Being a promoter, its safety is dependent on what it is promoting. As we are using it to promote the generation of methyl salicylate its use in this project did not raise any safety concerns. Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country? Our school division safety committee does not have any guidelines with respect to Biotechnology and Synthetic Biology. Our team invited the head of our division’s safety committee to visit our lab, and we discussed the new materials, equipment and protocol used for the iGEM project while personally touring her through the lab. She agrees we are in compliance with the general safety protocols of our school division with respect to laboratory practices. We are following guidelines given to us on site by a group of mentors with experience working in the field. Familiarity with safety (hazards, possible contaminations, disposal) in the newly configured lab space require regular communication to any new staff in the lab space including custodial staff, teachers, and students new to iGEM. Have you consulted anybody regarding biosafety? We have consulted biotechnology mentors from University of Calgary and have put together the following guidelines for general use: Safety for support staff We have moved recycling out of the lab prep area and support staff access is restricted to emptying any regular garbage that we may have. Regular lab prep for high school science labs has its own separate counter space.   Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices, and systems be made even safer through biosafety engineering?  Our DIY dremelfuge is operated behind safety glass and inside of a paint can operated with a direct plug-in outside of the fume hood. iGEM teams considering utilizing this DIY plan in the future are encouraged to consider the safety requirements of such high RPM with potential for a tube to become dislodged at high speeds. DIY Incubator/shaker table risk of electrical short is minimized with fuses on the turntable power supply, breaker on the power bar, and GCFI wall plug in. We are concerned with the treatment of hazardous waste like that of DNA staining dyes. Although we are dealing with microliters of staining dye per gel, the disposal of such type of waste is outside the scope of our small municipality. Our team has made arrangements for proper disposal of such hazardous waste in Calgary. We would encourage other start-up high school iGEM teams to consider ahead of time their disposal needs, as it can be challenging in small communities to treat specific wastes appropriately. 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/SafetTimeline From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Timeline  June 13thThis Friday, team members worked on various tasks that need to be finished for the wiki freeze. The bioethics essay was reviewed. Safety questions were answered, and DIY instructions were uploaded to instructables.com. June 9th Today, the team came together for a short lunch to work on the presentation for town council, when we would stop lab work, and website deadlines were discussed.June 6thAfter school, team members worked on a presentation for our town council and mayor, as well as, performing a restriction digest. Some team members also worked on a bioethics essay. June 2ndOver the weekend, we learned that we would be presenting our project to the town council here in Canmore. For this, four team members will be missing some school next Tuesday to inform the town council about our project. Website write-ups for outreach, ethics, and lab work, along with poster design were discussed. May 30thThis Friday afternoon was devoted to running a miniprep and working on our presentation. For the presentation, our powerpoint was edited and we now have a couple different drafts from which we will pick our best. Then, the only thing left to do in regards to the powerpoint is to fine-tune all the small details. The miniprep was done to separate the plasmid DNA from the E.coli (DH5a) the parts had been sent in. Single colonies had been plucked from the plates we had streaked earlier on, which were then used to conduct the miniprep.May 26thDuring this team meeting, team members split into groups for ethics, editing our powerpoint for the jamboree, working on a poster, making a survey to test smell, making DIY videos for the powerpoint, and putting instructions for our "home-made" equipment on Instructables. The different teams will meet up during the week and present their progress at a team meeting on Friday.May 24thToday was our last geekstarter-funded workshop day. However, this day was different from our other workshops since all teams from around Alberta met for a mock-jamboree at the University of Calgary. The morning was spent presenting projects to everyone and during the afternoon, each group received feedback on their presentations.May 13thPlanning out the next couple weeks was the focus of this team meeting. We also arranged meetings after school to work on our 20 minute-long presentation for the Jamboree. Our rational for working on the presentation early is that we have a practice presentation day in Calgary on the 24th of May. The next thing on the agenda was to discuss website content: namely, writing about what we have done so far for DIY projects, human practices, ethics, and biosafety.May 9thWe were hoping to have our DNA for Friday but unfortunately, we will only have it around Monday. Instead of starting a restriction digest with the two parts, we inoculated two plates with bacteria we will use as competent cells later on. Everyone else worked on outreach, fundraising, and starting the poster and presentation.May 5thFirst on the agenda for the meeting today was to review results from our lab work on Saturday. (We had successfully grown E.coli that expressed RFP-Red Fluorescent Protein) Next, came talking about a workshop on May 24th. This workshop is a practice for our MIT presentation, where we will have the chance to present to some professors--largely professors from Calgary. After this was done, we moved on to counting the colonies that had grown on our plates.May 2ndAfter a week-long break it was time for us to start working on our construct. For this reason, today was a lab day. We streaked out some DH5 Alpha plates for when we have to transform our cells, and we streaked out some cells for the two parts we are using. Some time was also spent writing up a summary for all the DIY work we did, and we did some research regarding ethics. Let's not forget that some people also worked on finding somewhere to get team t-shirts printed.April 13thThis mentor day, our second of the year, was very productive. At the beginning of the day the team split into three groups. The first group worked on making new LB-agar plates with different antibiotic resistance. The different antibiotic resistances include ampicillin, kanamycin, and chloramphenicol. Once this team was done making plates it moved on to make liquid LB broth and to streak Part A (J04500) and Part B (J04650) on the new agar plates. The second team worked on a restriction digest for most of the day. The third team made some agarose gel for when we run an agarose gel electrophoresis. Then they, actually ran a gel electrophoresis. The day ended with everyone discussing the exact steps of putting together the two parts we selected to form our final construct.April 11thAfter looking at the agar we had streaked yesterday we found out that no bacteria had grown. Because of this we were not able to get ahead with our practice run, which consisted of assembling two different parts together. Talia, Alina, Freya worked in the lab today with Mrs. Puurunen to go through the protocol again. They also stayed after school to work on ethics and human practices. This work included preparing a presentation for younger grades at our school and contacting people to talk about bioethics.April 10thToday was the first day of lab work. Jeremiah, Carter, Tully, and Mrs. Puurunen streaked part A and part B from our 3A assembly kit onto the agar. They also autoclaved some of pipette tips.April 7thToday, many of the team members were busy volunteering at our school's hot dog sale. The students that remained worked on fundraising and human practices. We also discussed finishing our website biographies and finding a new design for our incubator/skaker-table.April 4thThis Friday, team members came together to create a project plan for ethics and human practices. Work was also done on our incubator/shaker-table, and Mrs.Puurunen VROC-ed, Magda Pop, one of our mentors.March 31stThis meeting begun with determining who could help in the lab Friday after school. However, most of it was spent split up into different groups. These groups include ethics, fundraising, building, and wiki.March 24thStarted by discussing progress on setting up our lab and when we should begin lab work. After this people broke up into different groups for ethics, protocols, building, and fundraising.March 21stThe meeting this Friday consisted of: making a lab notebook template, working with our PCR machine, writing a draft e-mail to send to ethics board members, learning how to use the autoclave, contacting companies to print a poster for the jamboree. contacting our geekstarter mentors, working on an incubator shaker table, and updating our website timeline.March 17thThis week team members split up into groups to work on different parts of the project. The different groups included ethics, lab notebook and timeline, autoclave, and a team to learn how to work our P.C.R. machine.March 10thDuring the meeting this week we were giving packages of lab protocols to read over. We also split up tasks between people interested in doing ethics work.March 7thThis week, we had an extra meeting Friday after school in order for us to write a project description. During this meeting team members also worked on building equipment for our lab, as well as starting work for the ethics part of our project.March 3rdThis meeting was very important since we finally decided on a project to pursue: a heat activated biosensor.February 24thComing back from a week break from school, the team’s task for the week was to evaluate each project idea . February 10thAt this team meeting, team members were reminded to continue research for the following week. We decided upon this since we did not have enough information to make a decision on a project yet.February 3rdDuring this team meeting we split up into groups for researching the different project ideas throughout the week. Research for the project ideas includes finding past iGEM teams that have done something similar, finding biobricks related to our project, and contacting people that could help with the project.February 2ndThis was the first workshop day we spent with our Geekstarter mentors. We were given a more in depth explanation of the science we would be applying. We were also able to try some pipetting and were taught sterilizing techniques. Lastly, we went over project ideas so far and narrowed them down to an ethanol detector, a nitrate detector and decomposer, an air freshener, and a CO detector.January 30thMuch like the previous team meeting, most of the time was spent deciding on a project idea that our team could pursue. However, we also began team members were tasked with decided what part of the project (i.e. ethics, website, lab work, fundraising) they would rather work on.January 20thThroughout this team meeting we, team members, wrote down and shared ideas for a project we could pursue. This was done on a shared Google Doc that can be accessed through the hyperlink in the paragraph above this table. Our ideas filled up 2 and a half pages typed. January 13thTeam meeting during which Mrs.Puurunen, one of the teachers helping us, gave us a 30-minute long lesson on the basics of DNA and genetic engineering. This was a very important and useful meeting since many of the team members were confused with the science we would need to apply throughout the course of this semester.January 9thFirst team meeting. It consisted mainly of finding out who would be available for a first workshop with our iGEM geekstarter mentors, and sharing ideas people had for a project.December 23rd - January 5thAfter receiving information that our team had qualified for funding, team members took time over the break to read over previous iGEM team wikis and summarize their favourite projects.Early OnAfter having first been introduced to the concept of iGEM the everyone interested in the project had to write a statement of interest in order to apply for funding through Geekstarter.  2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/TimelinLab Notebook.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Lab Notebook 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/Lab_NotebooProject.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Our Project  The objective for our school’s first iGEM project is to create a heat activated olfactory biosensor. Using E.coli NEB10-beta as a chassis, when exposed to temperatures of 37°C to 42°C, our biosensor will emit a wintergreen smell. The wintergreen smell will be quite faint at 37°C and strong at 42°C. Using salicylic acid as a fuel the E.coli will produce an enzyme that catalyzes a reaction—a product of this reaction is methyl salicylate, which smells like wintergreen. Parts BBa_K098995/BBa_J45119 To create this biosensor, we will place a wintergreen odour enzyme generator (BBa_J45119) downstream from a heat sensitive promoter (BBa_KO98995). Once the two biobricks have been engineered into a single plasmid, the plasmid can than be inserted into competent E.coli NEB10-beta. An example of an application for this project is the use of this biosensor as a promoter when using bacteria to produce endothermic reactions to prevent overheating. The promoter would enable all parts downstream from it to function, and the wintergreen smell would indicate that the promoter is in fact allowing for the functioning of other biobricks. A similar use would be the use of our project in a self-regulating temperature-dependant system similar to a thermostat.   Project Plan A considered long-term project related to our first project is creating a thermal-wavelength biosensor. This endeavour—detailed in the image below—would require the input of both heat and red or blue wavelengths. With the first project providing team members with the knowledge and skills to modify DNA, we could then continue on the path of using bacteria in increasingly complex circuits to more effectively sense and respond to the environment.   Long-term Project   2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/ProjecSponsor.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Sponsors      2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/SponsoAbstract.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Abstract   Heat-induced Olfactory Biosensor      As Our Lady of the Snow’s first iGEM team, we have decided upon creating a heat-induced olfactory biosensor using E. coli as a chassis. Constructed by placing a wintergreen odor enzyme generator downstream from a heat sensitive promoter, the bacteria should emit a noticeable wintergreen smell starting near a human body temperature of 37°C. As the temperature increases the heat sensitive promoter gradually decreases the effect of the repressor to allow for maximum wintergreen enzyme generation around 42°C. The creation of this biobrick will serve as a first step towards a multiple input bio-sensing system. Such a system may include the input of heat and different wavelengths for activation. Both pursuits will help achieve the goal of using bacteria in increasingly complex circuits to more effectively sense the environment.           2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/AbstracAbout Us.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Contact Us   Our Lady of the Snows Catholic Academy    3100A Stewart Creek Dr. Canmore, AB T1W 3M6   (403) 609-3699 Fax: (403) 609-4008   http://www.redeemer.ab.ca/Schools/Our-Lady-of-the-Snows/Pages/default.aspx   Student Liaison: Alina Arvisais alinaarvisais@isidore.redeemer.ab.ca   Teacher Liaison: Luc Arvisais larvisais@redeemer.ab.ca 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/About_UFrom 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us jQuery Slide Show by WOWSlider.com v5.2m 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_Chttp://2014hs.igem.org/wiki/images/2/2b/IGEMlogo_version64.pngLab Successes.html From 2014hs.igem.org Home Project   AbstractDescriptionTimelineLab SuccessesLab Notebook Human Practices          EthicsOutreachDIY Safety About Us                  The TeamSponsorsAttributionsContact Us   Lab Protocol Successes - Team OLeSsence  - Mastered use of pressure-steam autoclave for all sterilization/decontamination- Successful completion of the 3A Protocols, including: - Restriction digest of plasmids - Gel electrophoresis of plasmid digest (NB: No evidence of DNA on gel) - Ligation protocol - Routinely preparing our own LB-agar plates (with and without antibiotic) and LB broth for cell culture. - Successful plating techniques for E. coli – routinely growing single colonies free from contamination using aseptic technique. - Successfully growing cell cultures from individual colonies in DIY shaker/incubator - Successfully grew E. coli cells (provided from iGEM) containing our parts (K098995 and J45119), including cell cultures.- Mastered use of DIY Dremelfuge (confirming RPM with photogate) to pellet DNA- Successfully mini-prepped both our parts to extract plasmid DNA - Successfully used restriction digest protocols to cut our plasmids of interest, confirming presence of DNA strands of expected size via gel electrophoresis*Need to build gel illuminator next year- Successfully transformed RFP plasmid into competent cells (prepped by our mentors) to grow RFP colonies. 2014 Our Lady of the Snows iGEM Retrieved from "http://2014hs.igem.org/Team:OLS_Canmore_AB_CA/Lab_Successe1274Jefferson VA SciCOSThomas Jefferson High School for Science and TechnologyHigh SchoolSolving a 4-Node Traveling Salesman Problem Using the hin/hixC Recombinant SystemBacterial computing has become a feasible way to autonomously solve quantitative problems. We sought to utilize the computational capacity of E.coli K-12 to solve the Traveling Salesman Problem, a problem in theoretical computer science that asks for the shortest possible route that visits each node in a system at least once and returns to the original node. We utilized a series of initial configurations for the hin/hixC recombinant system that were previously developed by a 2006 iGEM team to solve the Hamiltonian Path Problem. In addition, we created a fourth node by splitting blue fluorescent protein (BFP) with a hixC site and reinserted this node into one of the composite hin/hixC paths. To simulate varied distance, we added a ribosome binding site of a different strength in between the initial recombinant system and the fourth node.901BBa_K1274999http://2014HS.igem.org/Team:Jefferson_VA_SciCOSBBa_K1274000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163620140http://2014hs.igem.org/files/poster/Jefferson_VA_SciCOS.pdfHigh SchoolLog in   Team:Jefferson VA SciCOS/Team From 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome   Captains Tina Ju 70px Tina Ju is a junior at Thomas Jefferson High School for Science and Technology and the founder of the Jefferson iGEM team. She is primary interested in public health, synthetic biology, and computer science. These interests are fueled by her desire to use technology, particularly synthetic biology, to innovate medicine. She plays an active role in the Jefferson research community, serving as an editor of Teknos, the Jefferson research journal, and the committee co-chair of tjSTAR, the school research symposium, as well as working on iGEM. She also founded and currently serves as the director of the Northern Virginia Hepatitis Free Initiative, a public health project to promote hepatitis B and C awareness and education in the Asian and Pacific Islander community. She will be working at the National Cancer Institute this summer. Manotri Chaubal 70px Manotri Chaubal is a junior at Thomas Jefferson High School for Science and Technology. She is primarily interested in biotechnology and this year, Manotri's passion for research prompted her to explore an alternative source of stem cells that earned her the 2nd place grand prize at Intel's International Science and Engineering Fair. In addition, Manotri is both founder and director of the Indian chapter of the Northern Virginia Hepatitis Free Initiative. Manotri also enjoys educating others about biotechnology and synthetic biology through the club, Girls in Engineering, Math, and Science (GEMS), which she is the treasurer of. This summer Manotri will be further pursuing her passion for research at Children's National Medical Center. Webmaster & Systems Modeler Lucas Kang 70px Lucas Kang is a junior at Thomas Jefferson High School for Science and Technology, in Alexandria, Virginia. He has a passion for understanding the behavior of complex systems, and often utilizes his expertise in physics, mathematics, and computer programming to gain new-found perspectives on any topic that interests him. This summer, Lucas will be one of three high school researchers employed at the new Colorado State Accelerator Facility, and will also publish his work on a new application of cellular automata under the mentorship of Stephen Wolfram. Besides his enthusiasm in the sciences, Lucas enjoys soccer, playing violin, cooking gourmet food, and reading. For Lucas' research and accomplishments, visit his Google site. Researchers Jenny Seo 70px Jenny Seo is a junior at TJHSST. She is ready to explore the fields of biotechnology and bioengineering, and she is additionally interested in pathology. She has taken extensive courses in biology and math, such as Pathophysiology and Matrix Algebra. This summer, she is going to work as an intern at Children's National Hospital. Jenny enjoys playing volleyball and she occasionally plays violin, piano, and takes time to draw out her thoughts. Sahitya Allam 70px Sahitya Allam is a junior at Thomas Jefferson High School for Science and Technology. Her primary interest is the intersection between neuroscience and immunology, which she seeks to investigate as part of an internship dealing with the pathogen infection strategies and host immune mechanisms that affect aging in Drosophila at the cellular and molecular level. She actively participates in several clubs and organizations at TJHSST, including Biology Olympiad, Future Problem Solvers, Chemistry Team, and iGEM. In addition, she has been awarded numerous distinctions related to the biological sciences, such as two time USABO semifinalist, top 50 ranked student in the National Biology Competition, Conrad Spirit of Innovation Semifinalist, NOVA Brain Bee runner up, and top 10% honorable mention for the NSTA/Toshiba Exploravision competition. She also actively participates in her community as a volunteer at a hospital and non-profit autism care center, and as a tutor for regular/AP Biology, regular chemistry, and Spanish 2. Eric Bo 70px Eric Bo is a junior at TJHSST. He has a general interest in science and technology, but especially synthetic biology and bioengineering, and his senior technology lab is Biotechnology. However, he is also interested in physics and computer engineering. Besides academics, Eric also spends a considerable amount of time on extracurricular activities, mainly on Environmental Impact Club and Chinese Honor Society. William Woodruff 70px William Woodruff is a junior at TJHSST. He has taken classes in a wide variety of sciences, including DNA Science, Organic Chemistry, Physics, Marine Biology, and Astronomy and will be conducting research in the Chemical Analysis lab as a senior. Over the summer, he will be interning at NIH and he intends to continue his education in biology and chemistry in college. In addition to his scientific interests, he enjoys playing the piano, learning languages, and participates in a number of extracurricular activities such as the Environmental Impact Club, Model United Nations, Biology Olympiad, and Writing Center tutoring. Richard Wang 70px Richard is a junior at Thomas Jefferson High School for Science and Technology. He is primarily interested in biotechnology, chemistry, and beauty of language. He explores these interests through advanced courses at Jefferson and through iGEM. He is also a varsity debater on the Jefferson policy debate team and will be the captain of the team next year. He will be doing something business/law-related this summer in New York City. Peyton Randolph 70px Peyton is a junior at Thomas Jefferson High School for Science and Technology. He is fascinated by science, especially synthetic biology and chemistry. He intends to pursue a career in synthetic biology and hopes to meaningfully contribute to the field. He furthers his study of science by participating in iGEM, Environmental Impact Club, and Teknos, the Jefferson research journal. He will be researching at Georgetown University this summer and will be volunteering with the Northern Virginia Hepatitis Free Initiative. He discussed his passion for synthetic biology with the TJHSST Dreams team, who posted it in all the bathroom stalls at school. That's called dedication! Daniel Carris 70px Daniel Carris is a junior at TJHSST. His area of interest is Chemistry, which he has studied for 3 years. In addition to chemistry, Daniel enjoys physics and mathematics. Outside of school, Daniel is a member of TJ's crew team, devoting 10+ hours a week to the team. He also enjoys music, playing the guitar, piano, viola, and violin. Nihita Manem 70px Nihita Manem is a rising senior at Thomas Jefferson High School for Science and Technology. Her interest in neuroscience, medicine, and synthetic biology has promoted her to join the iGEM team. As an active leader in her community, she volunteers at the local stembassadors program, as an instructor for the NeuroInspire program, as well as a member of several clubs at school. She will be working at the Krasnow Institure this summer, researching the impact of allatostatin expression in specific hippocampal neurons related to hippocampal-dependent learning and memory abilities. Arjun Iyer 70px Arjun Iyer is a rising senior at Thomas Jefferson High School for Science and Technology. He is mainly interested in Biotechnology and Chemistry, and has taken advanced classes in both subjects to pursue this passion. Additionally, he plans to use the knowledge that he has acquired in these classes to a research internship at the National Cancer Institute this summer. In his free time, he runs track and Cross Country for TJ, draws cartoons, and listens to music. Also, Chris Brown is his idol and he is very skeptical of the moon landing. Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCOS/TeamNotebook From 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome  2014 iGEM NotebookOctober 2013 – January 2014We started raising awareness of the iGEM competition in the TJHSST community by discussing the competition during weekly meetings of Synthetic Biology Society and delivering lectures on basic concepts of synthetic biology, such as transcription and translation, synthetic biology terminology, and methods of assembly to construct a part. We required that all members of Synthetic Biology Society who were interested in joining the iGEM team submit a proposal for the 2014 project by December 15th. We selected six of those proposals for advanced consideration and invited the members who submitted the proposals to join the 2014 iGEM team. We also received the One Question Grant during November, for which we had to conduct an outreach program.January 2014 – February 2014We finalized the idea for our project this year: the construction of a 4-node bacterial computer that solves the Traveling Salesman Problem. We reviewed about ten ideas submitted by members of Synthetic Biology Society, which dealt with topics as diverse as an insulin regulation system for the human body and a ciguatoxin poison attenuator, before we settled on this idea. We felt that it was the most feasible project to complete with the resources in the TJHSST lab and provided abundant opportunities to backtrack if a component was not functioning properly. Moreover, due to the combinatorial nature of the problem we were attempting to solve, we believed it had the most potential in terms of mathematical modeling. We also received a generous donation of $1000 from Dr. Brian Becker of Precision Economics, LLC during the month of February.February 2014 – March 2014As part of our outreach program, the Mini-Jamboree, we conducted prep lab sessions during meetings of Synthetic Biology Society. The prep labs were intended as a way to introduce freshmen and sophomores to the Biotechnology Lab and to basic research techniques so that they may be able to formulate their own research ideas with ease. The prep labs were based on the protocols that we received from iGEM headquarters. Although the end goal of the prep labs was to create a part that fluoresces red through the transformation of control RFP, we were not able to do the restriction digest or ligation with the participants due to the erratic weather on the east coast this year. Instead, we first taught the students antiseptic techniques, how to grow bacteria on plates, and how to make these plates using agar powder, distilled water, antibiotic solution, and an autoclave. As we neared the end of March, we taught them a transformation protocol using competent cells we had already purchased from New England Bio Labs.April 20144/3/14 – We received 5-alpha Competent E.coli, a BioBrick Assembly Kit, a Q5 High-Fidelity 2X Master Mix, and a Quick-Load Purple 2-Log DNA Ladder from New England Bio Labs. These items were distributed free of charge to all high school iGEM teams that requested them as part of a one-time deal from New England Bio Labs.4/5/14 – Techstravaganza outreach event at TJHSST. We had two booths: candy DNA and banana DNA extraction4/9/14 – We transformed wild-type GFP (BBa_E0040) with competent cells. These were cells that were made competent by Dr. Burnett the previous day, so she was not sure whether they would work or not. However, since this was just a trial transformation to familiarize ourselves with the procedure, we decided to use the old competent cells instead of spending time to make our own. We could not use the cells we purchased from the vendor because we thought they were electrocompetent (however, they were not, we just got confused due to the many boxes of cells that belonged to researchers in the Biotechnology Lab that were cluttering the -80 freezer at the time). Due to time constraints, we used the “What a Colorful World” lab procedure that is taught in an introductory DNA science class at our school. This procedure is a simplified version of the complete transformation procedure and does not yield very high efficiency. It was developed by previous iGEM teams but was highly tailored to a specific project, so it is plausible to assume that the same procedure would not work properly for all projects.4/10/14 – We checked to see if the previous day’s transformation had worked. As expected, we did not see any colonies that fluoresced green under the UV pen light, although we saw a few normal colonies under natural light. Again, we did this transformation mainly to familiarize ourselves with the basic transformation procedure and not to derive results that could potentially be useful for our project.4/11/14 – 4/20/14 – Lost a substantial amount of time in the middle of April due to spring break. We did do some advance planning for the project during this time however and ordered the DNA invertase Hin tagged with LVA (BBa_J31001) part from iGEM headquarters.4/22/14 – Received the DNA invertase Hin part that we ordered from iGEM headquarters today. Fast service!4/23/14 – We did another transformation with an RFP (BBa_J04450) part from 2012 Kit Plate 1, Well 1A. This time, we used the 5-alpha competent E.coli that we purchased from New England Bio Labs. We utilized the protocol that was provided with the purchased competent cells with 5 ul of resuspended DNA from the plate. We plated the transformation mixture on two separate plates – one without a serial dilution and one with a 1:10 serial dilution.4/24/14 – We checked the results of the previous day’s transformation. We found that the transformation without the 1:10 dilution worked. However, we only observed one transformed colony. 4/30/14 – We transformed three more RFP (all were BBa_J04450) parts from 2012 Kit Plate 1, Wells 3A, 5A, and 7A. We followed a few suggestions made by the protocol that we realized we had not abided by last time. For the previous transformation, we had neglected to set the incubator to shake. This time, we made sure to set the rotation speed per minute to at least 205 (the protocol recommended 250 but we could not use the full amount because it was making too much noise and was beyond the amount regulated by the lab). We hoped for more colonies this time considering that we had neglected to rotate the transformation mixtures last time and had still observed one colony.May 20145/1/14 – We checked the results of the last three transformations. We were not very pleased with the results – while we expected to see more colonies after rotating the transformation mixtures at 205 rpm, we actually got no yields for any of the parts. After consulting with our mentor, Dr. Burnett, we came to the conclusion that our SOC media was contaminated. We did not store it at 4 degrees Celsius after opening it as per the storage directions. As a result, the SOC media became very cloudy over time, indicating the presence of contaminants within the media. Before doing more transformations, we replaced the contaminated media with a fresh vial that we found within other competent cell kits in the lab.5/7/14 – Before proceeding further with any experimentation with regards to the actual project, we used this as a planning day to figure out when each procedure would be completed and by whom.5/8/14 – This was the official first day of project. We transformed paths HPP-A0 and HPP-A1 in order to see if the system is working using the 5-alpha competent E.coli procedure that we had used previously without a serial dilution.5/9/14 – We checked the results of our transformations. The transformations did not appear to work again, even after we seemed to have mastered the transformation protocol. After researching the paths in more detail and reading through the procedures of the Davidson/Western Missouri iGEM team, we realized that the promoter used in the paths was receptive to T7 RNA polymerase. Therefore, our solution was to use purchased T7 Express E.coli cells as they had done.5/12/14 – Submitted a purchase order for T7 Express E.coli cells, PstI restriction enzyme, and PCR Mix Superfidelity. Throughout this week, we were also in the process of ordering the PCR primers for our new part (although we ran into a bit of snafu with our school’s finance office in doing so that delayed their arrival). Since we were waiting on the T7 Express E.coli cells in order to retransform the pathways, we decided to isolate the plasmid DNA from the bacteria containing the DNA invertase Hin part that we received on a stab culture last month. We streaked ampicillin plates with the bacteria from the stab culture and grew the plates overnight in the incubator at 37 degrees Celsius.5/13/14 – We made a cell culture for DNA invertase Hin.5/14/14 – Instead of doing the miniprep, we worked on a grant application because there was not sufficient time in the lab to finish the procedure. Consequently, we had to remake the cell culture and do the miniprep on Friday.5/15/14 – We made another liquid culture for DNA invertase Hin.5/16/14 – We performed a miniprep in order to isolate the plasmid DNA containing the DNA invertase Hin part. We were not able to quantify the DNA on the same day due to lack of time, so we stored the DNA in the -20 freezer for later use. In addition, we received all the items that we submitted a purchase order for on Monday.5/20/14 – We transformed HPP-A2 (BBa_I715044), HPP-A2 + Hin (BBa_I715051), and hixC (BBa_J44000) parts using T7 RNA Polymerase cells that arrived last week. We checked the concentration of the DNA invertase Hin plasmid DNA using the gen5 quantification system 5/22/14 – We checked the transformation results and they were a bit disheartening. We expected to see a few yellow colonies interspersed with red and green colonies for A2 + Hin, but we didn’t see that. The efficiency of the new cells was obviously much higher because there were several colonies. It might have been possible that the DNA in the 2012 plates wasn’t of high quality because it was over a year old, and since that time, our school has been undergoing renovation. The DNA might have been subjected to various climate extremes due to the construction and the many days off school we got from the erratic weather. We reordered all of the parts from iGEM headquarters to make sure that the fault wasn’t the quality of the DNA itself. We also decided to remake all of the ampicillin plates that we had been using because it is possible for the antibiotic to degrade over time, meaning that the bacteria growing on the plates might not be carrying the plasmid containing the part/antibiotic resistance gene at all.5/23/14 – We made new ampicillin plates. Ratio of antibiotic to agar – 1ul/ml5/28/14 – We gave a presentation on our synthetic biology outreach efforts at tjSTAR, TJHSST’s annual symposium to advance research, as part of the One Question Grant we accepted at the beginning of this year. During the presentation, we talked about some of the activities we conducted to promote greater awareness of biotechnology and hands-on learning through the Mini-Jamboree, Prep-labs, and Techstravaganza event.5/29/14 – We received HPP-A0, HPP-A2, and HPP-A2 + Hin from iGEM headquarters. The other parts did not grow in the quality control tests. We also received Primers A and B (forward and reverse primers for first gene half of BFP).5/30/14 – Slivoskey Grant Presentation. We received Primers C and D in the morning and plated the parts we received from iGEM headquarters on both the old and new ampicillin plates. We also performed a PCR reaction for both gene halves using the primers. Primer A - 17.87 nmolPrimer B - 17.97 nmolPrimer C - 14.29 nmolPrimer D - 16.31 nmolUsed 1.4 ml to resuspend the DNA. 1ul of forward and reverse primer was used, while 3 ul of the template DNA was used. The 1 ul of each was diluted in a 1:3 ratio.June 20146/2/14 - We checked how the parts we plated on 5/30 looked. Under the UV light, it seemed that colonies on HPP-A0 glowed a distinct yellow color, which indicated that the transformation had finally worked. We also got colonies for HPP-A2 that did not glow without Hin, which was what was desired. We therefore decided to use the HPP-A2 path to construct our composite pathway, and were hoping to do a gel of HPP-A2 to confirm that it was properly constructed if we had the time. (Insert pic of HPP-A2 here)6/3/14 – Cell culture for hixC6/4/14 – We purified the PCR products using a PCR purification kit and performed a miniprep for hixC. We checked the concentration of the isolated plasmid DNA on the same day and got a very poor concentration. This was due to 2 reasons – not all of the cell culture was used for the miniprep (only 1 ml of it) and there was a mix-up on the xg and rpm units. The PCR purification protocol and the miniprep protocol used two different units but both used the same microcentrifuge. Whoever was doing the miniprep did not change the units after the person who did the PCR purification changed them initially.6/5/14 – We decided to redo the hixC miniprep and quantification. We made another cell culture for hixC.6/6/14 – We finished the miniprep for hixC.6/10/14 – We quantified the plasmid DNA for hixC and the two PCR productsWe did two restriction digests (accidentally messed up the first one by not adding a restriction enzyme to one of the reaction tubes). We cut the hixC plasmid with EcoRI + XbaI and the first PCR product (result of PCR for A and B primers) with EcoRI + SpeI.6/11/14 – We tried to do a transformation for RBS of moderate strength (B0034) and two terminators (B0010 and B0012). Due to time constraints, we had to push this back to the next day. Instead, we completed a ligation for Product 1 + hixC. Unfortunately, the thermal cycler wasn’t functioning properly on this day. The lid kept on opening, but our sponsor was able to monitor it for the most part. However, we had to keep it at room temperature overnight and then denature the enzymes at 70 degrees Celsius the next day, which may have influenced the efficacy of the reaction.6/12/14 – We redid the failed transformation of RBS and the two terminators. After discussing the project with our mentor, we realized that it is not practical to do a restriction digest on a ligated part, and thus, had to revise our plan for the rest of experimentation.6/13/14 – Without wasting another day, we redid the ligation from two days ago. We incubated the reaction tube at room temperature (couldn’t get the waterbaths to 16 degrees Celsius and the thermal cycler wasn’t functioning properly) and denatured the enzymes at 70 degrees Celsius. We transformed both the old and the new ligation mixtures that same day.6/16/14 - We checked the results of the transformations (the cells grew perfectly!) and made cell cultures for P1 + hixC, RBS, and HPP-A26/17/14 - We did a miniprep for the three parts and checked the concentrations of the isolated plasmid DNA using the Nanodrop machine. We got very high concentrations this time, which indicated progressive improvement in the execution of techniques over time. Subsequently, we did a restriction digest of the three parts as well as the second product of the PCR reaction. P1 + hixC was cut with SpeI and PstI, Product 2 was cut with PstI and XbaI, RBS was cut with XbaI and PstI, and HPP-A2 was cut with SpeI and PstI. Finally, we did two ligations, one of the P1 + hixC part and the second product, to form the composite P1 + hixC + P2 part, and another of the HPP-A2 part and the RBS to form HPP+RBS.6/18/14 - We transformed the ligation mixtures of P1 + hixC + P2 and HPP-A2 + RBS using T7 Express E.coli cells.6/19/14 - We made cell cultures of the transformed parts. However, the media was partially cloudy, which indicated the presence of contamination. We wanted to remake the LB broth to ensure as little contamination as possible, but we did not have the time to do so.6/20/14 - We did a miniprep of P1 + hixC + P2 and HPP-A2 + RBS. We did a restriction digest for both of them by cutting P1 + hixC + P2 with XbaI and PstI and HPP-A2 + RBS with SpeI and PstI. Lastly, we ligated these two parts together, to get our final pathway: HPP-A2 + RBS + P1 + hixC + P2. This was the last day of experimentation for us because our mentor was not available to supervise us after this date. We finished the composite pathway, but we could not test it through a transformation and affirm that all components were constructed properly by running a gel. Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCOS/NotebooSafety From 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome  SafetyWould any of your project ideas raise safety issues in terms of researcher safety, public safety, or environmental safety? The organism we used was the NEB-10 strain of E. coli (Biosafety level 1). If it were to be accidentally released, the risks would be minimal, because this strain of E. coli cannot survive outside of a controlled lab environment.The primary source of safety concern in this experiment would be the Bunsen burners used to create a convection current of air to prevent foreign pathogens from contaminating cultures. However, all the researchers have experience with performing experiments nearby Bunsen burners, and as such these pose minimal risk. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? The BFP split gene part that we made is based on a protocol that was developed by a 2007 iGEM team. Since the part functions as a reporter gene, we do not see any safety issues that could arise due to fluorescence. In addition, this part is connected to a pathway that was submitted by a previous iGEM team and has already been accepted by the registry. The moderate ribosome binding site that connects the rest of the pathway and the split BFP gene, B0032, is also part of the registry already.If yes, did you document these issues in the Registry?Not applicable.How did you manage to handle the safety issue?Not applicable.How could other teams learn from your experience?Not applicable.Is there a local biosafety group, committee, or review board at your institution?Being a high school, TJHSST does not have an official review board to provide oversight. However, the lab directors in charge of the biotechnology department served the same role by requiring all purchases, protocols, and experiments to be approved by them prior to any action being taken. Within our own iGEM team, we formed a subcommittee who approved any suggestions prior to sending them to the lab directors for approval, and who also familiarized themselves with the protocols necessary to guarantee a safe environment.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? We believe that organization and planning are especially important for safety. Mistakes and accidents are only more likely to occur when people are unsure of exactly what they are doing or when there are too many people attempting to perform the same task. The parts and chassis themselves are already quite safe, as only an optimum lab environment can sustain them, negating the possibility of devices being able to propagate beyond the lab by themselves. Where there is room for improvement is in the behaviors of the researchers themselves. Adequate training and preparedness is vital for researchers to maintain a safe and controlled lab environment. Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCOS/SafetProject From 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome Contents1 Solving a 4-Node Traveling Salesman Problem Using the Hin/hixC Recombinant System2 Bacterial Computing: The Future of the IT Industry All in an Agar Plate3 The Traveling Salesman Problem4 Taking a Previous Encoding Scheme a Step Further5 Engineering Our BFP Construct6 The Hin Recombinase/hixC Site System7 The Path We Chose to Solve Solving a 4-Node Traveling Salesman Problem Using the Hin/hixC Recombinant SystemOur project’s goal was to assess the accuracy, scalability, and feasibility of a novel bacterial computer paradigm for solving a 4-node Traveling Salesman Problem. Bacterial Computing: The Future of the IT Industry All in an Agar PlateWhere would humanity be without the advent and rise of the computer? The 21st century has marked humanity’s entrance into a new Digital Age; now, more than ever, does the global marketplace and humanity itself depend on a thriving information technology sector. From the pocket-sized computing devices that we use to manage our day-to-day tasks to the massive supercomputers that mirror human intelligence, from the databases that store millions of patient’s records to the Big Data servers that analyze the vast number of financial transactions that take place every second, computing has evolved into a ubiquitous science that is both humanity’s present as well as its future. For years, the physical limitations that deterred the development of faster, more efficient computing devices were easily maneuverable, allowing computing to present itself as the seemingly perpetual gale force that propelled the sails of our global economy. But now, the winds of the Digital Revolution seem to be stilling. Little do people realize that Moore’s Law, the golden standard for advances in computing, is coming to an end. Moore’s Law, a bold vision for the future originally proposed in the early 1970s, set a clear goal for the future of computing hardware: every two years, the transistor count on a typical microprocessor (our computing capacity) would double. Now, however, we’ve finally hit the physical limit where transistors are packed so close together that reliable electron flow can no longer be sustained. This dilemma has raised a hue and cry throughout the computing industry, and has led to extensive research and development into new avenues of computing. Can we really transcend this computational limit? Could it be possible that the future of the computing industry be quantum computing? Or could it be something else? Our group believes that synthetic biology, specifically the nascent field of bacterial computing, holds the key to the IT industry’s qualms. The computational capacity of biological systems is shockingly vast and amazingly robust. Unlike standard computers, bacterial computers may be able to autonomously solve quantitative problems in a massively parallel fashion; moreover, because bacteria are constantly dividing, the number of processors working on the problem grows exponentially with culture time. Also, bacterial computers use much less energy than standard computers to solve the HCP and the agar plates that they’re cultured on can even fit in the palm of your hand. The 2007 Davidson-Missouri iGEM team demonstrated an algorithm using DNA constructs and the Hin recombinase/HixC site system capable of solving a three node Hamiltonian Path Problem. However, this team’s ground-breaking research left several questions unanswered that our group found critical to the future of bacterial computing:How accurate is bacterial computing? Can the algorithm be expected to reliably converge to a solution state regardless of the initial configuration? How scalable is bacterial computing? Will our group be able to scale the technique up to larger node problems and how stable are hixC-site constructs for different proteins? Will our group be able to solve a completely different problem than the Hamiltonian Path Problem?How feasible is bacterial computing? Bacterial computing features a paradigm where the hardware is far cheaper than the software. How will this compare to other computing paradigms when we perform a cost-benefit analysis?With these guiding questions in mind, our group has proposed a project that assesses the accuracy, scalability, and feasibility of a bacterial computer paradigm for solving different initial configurations of a 4-node Traveling Salesman Problem. Our work proposes a novel way of encoding the Traveling Salesman Problem using variable Ribosome Binding Site strengths and DNA constructs that can be solved by the Hin recombinase/HixC site system. The Traveling Salesman ProblemImagine you’re a salesman tasked with delivering goods to a set of towns. How can you find the shortest possible route to travel in order to visit every town exactly once to deliver your goods? This is exactly what the Traveling Salesman Problem seeks to find. The problem itself is extremely crucial to the transportation and shipping industry, where finding the shortest route possible can mean millions of dollars saved on the thousands upon thousands of goods delivered daily. Could bacteria performing computations in an agar plate hold the answer to this longstanding question? Taking a Previous Encoding Scheme a Step FurtherThe 2007 Davidson-Missouri iGEM team proposed three encoding schemes given by Figure 3, where cities correspond to given genes and adjacent gene halves separated by hixC sites represent edges on the graph. To encode our 4-node problem we decided on using genes encoding for (1). blue fluorescent protein , (2) green fluorescent protein , (3) red fluorescent protein and a (4) terminator. To encode for the distances between graphs we decided to use varying Ribosome Binding Strengths. Using the following gene sequence to RBS calculator (http://salis.psu.edu/RBS_Calculator.shtml), we proposed a function to go from the distance between cities to RBS. The function was constructed in such a way that the shorter the distance between cities, the greater the RBS. This way the solution encoding for a path with the shortest distance would fluoresce the brightest. Engineering Our BFP ConstructIn order to carry out the encoding scheme we developed above, it was necessary to develop our own BioBrick to represent the two gene halves encoding for BFP. It was important to make sure that the protein’s fluorescent activity would not be compromised once the hixC site was inserted, i. e. our insertion could not occur within the BFP’s chromophore sequence. To discover what the chromophore sequence was for BFP, we read the documentation for Part:BBa_K592100 (mTagBFP as shown in the figure) as described by Subach et al., 2005 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2585067/). Figure from Subach et al., 2005. Letters specifying chromophore sequence are in blackAfter having found the sequence for BFP and taking due note of which region encoded the chromophore, we then performed an analysis through the online gene splitting tool provided by the 2007 Davidson-Missouri Team (http://gcat.davidson.edu/iGEM07/genesplitter.html) in order to determine the correct primer to order. The following is a selection of the output from our analysis: The Hin Recombinase/hixC Site SystemThe Hin Recombinase/hixC Site System is what breathes life into the encoding scheme we designed above. The system designed by a 2006 iGEM team is derived from Salmonella, where Hin regulates how flagellin genes are expressed. In our project, the system was used to constitutively shuffle the gene halves until a solution is arrived at. The Path We Chose to SolveThe 2007 Davidson/Western Missouri iGEM team constructed 3 initial pathways to demonstrate that the Hamiltonian Path Problem could be solved in vivo. We decided to use one of the starting constructs as a scaffold from which we could build our own composite pathway because each of the constructs contains three nodes: two split genes and a double terminator. Also, in the interest of time and resources, we were not concerned with constructing several pathways of our own as the Davidson/Western Missouri iGEM team had done. We ended up using HPP-A2 because it showed evidence of no fluorescence without Hin, which was the expected result as recorded by the Davidson/Western Missouri iGEM team. To HPP-A2, we added an RBS of moderate strength, as determined by the ribosome binding calculator and published values in literature. Compared to B0034, the RBS used in HPP-A2, B0032 is reported to exhibit 32% binding efficiency. Downstream of the RBS, we added another node: a BFP split-gene part. Thus, the order of our final part, from 5’ to 3’, was HPP-A2 + RBS + BFP1 + hixC + BFP2. We wanted to transform E.coli containing this composite pathway with Hin recombinase in order to shuffle the gene halves randomly and with varied efficiency, resulting in a solution to the TSP. Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCOS/ProjecModeling From 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome  Mathematical ModelYou can download a pdf of our model's documentation here: [1] Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCOS/ModelinFrom 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome AbstractSolving a 4-Node Traveling Salesman Problem Using the hin/hixC Recombinant SystemBacterial computing has become a feasible way to autonomously solve quantitative problems. We sought to utilize the computational capacity of E.coli K-12 to solve the Traveling Salesman Problem, a problem in theoretical computer science that asks for the shortest possible route that visits each node in a system at least once and returns to the original node. We utilized a series of initial configurations for the hin/hixC recombinant system that were previously developed by a 2006 iGEM team to solve the Hamiltonian Path Problem. In addition, we created a fourth node by splitting blue fluorescent protein (BFP) with a hixC site and reinserted this node into one of the composite hin/hixC paths. To simulate varied distance, we added a ribosome binding site of a different strength in between the initial recombinant system and the fourth node. BackgroundEstablished in 1985, Thomas Jefferson High School for Science and Technology is the result of a partnership of businesses and schools created to improve education in science, mathematics, and technology. Representatives from business and industry and staff of the Fairfax County Public Schools worked together in curriculum and facilities development for the school. In recent years, local business leaders and Jefferson parents have formed the Jefferson Partnership Fund to help raise money to maintain and equip labs and classrooms in the school. As the Governor's School for Science and Technology in Northern Virginia, the school is also supported by the Virginia Department of Education. In addition to providing a specialized education for selected students in Fairfax County, Jefferson also serves Arlington, Loudoun, and Prince William county as well as the cities of Fairfax and Falls Church.MentorsWe would also like to thank Dr. Mary Susan Burnett, a DNA teacher, researcher, and lab director who is currently employed at the Thomas Jefferson High School for Science and Technology. She guided us through the more difficult procedures in our experiment, and taught us all of the proper techniques and safety guidelines that we employed throughout our work.Website designed and created by Lucas Kang.Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCO2014hs.igem.org/wiki/images/thumb/4/43/Jefferson_VA_SciCOS_logo_colored.png/190px-Jefferson_VA_SciCOS_logo_colored.pngResults From 2014hs.igem.org Our TeamHuman PracticesSafetyModelingNotebookResultsProjectHome  Results and ConclusionsTesting We hoped to run two gels to see if our new node and composite pathway were made properly. We also planned to test our pathway and check the fluorescent properties of our node by transforming both in E.coli, but due to lack of time and resources, we were unable to determine definitively whether our efforts were successful. If we did have the time however, we would have followed this protocol to determine a false positive to true solution ratio in order to determine the efficiency of our bacterial computer in solving the Traveling Salesman Problem. Screening for all correct phenotypes If some cells do not contain antibiotic resistance or do not fluoresce a certain color under UV light (bright white in this case), then we know that they do not represent a solution to the Traveling Salesman Problem. However, with this comes the possibility of a false positive solution.Using PCR to separate false positive solutions from true solutions In the case of false positives, all the gene halves are properly aligned to produce fluorescence – the first half is followed by the second. Yet the resulting path is not logical because it requires moving between nodes without following an edge. It has already been established that the length of a pathway that solves the Traveling Salesman Problem is the length of all split genes, and that the first and last node of a pathway is never flippable. Thus, PCR primers can be designed for the first and last node, and running the PCR products on a gel should give the lengths of the amplified DNA. If the path is false-positive solution, then the resulting bands should theoretically be too small to be the most optimal solution to the Traveling Salesman Problem.Detecting Fluorescence Using Spectroscopy To count the number of colonies that fluoresced a certain color on a plate, we would have used a fluorometer. In order to use the fluorometer for our construct, we would have first run scans to find the optimal excitation and emission wavelengths for the split BFP genes that we created. Once these wavelengths have been determined, the fluorometer could be used to determine the emission wavelengths of the fluorescent colonies and we could compare them to the previously determined optimal results. Depending on the number of colonies, they could be counted either by hand or by taking pictures and analyzing them through a program such as ImageJ. By compiling the results of all these different methods, it would be possible to determine the number of fluorescent colonies, how many of them were true solutions compared to false positives, and ultimately achieve our goal of evaluating the efficiency of our system.Conclusion We successfully designed and created a 4-node pathway with varied ribosome binding ability in order to solve the Traveling Salesman Problem, but we were ultimately unsuccessful in implementing the pathway by testing its efficiency. However, this proof-of-concept experiment has the potential to demonstrate the applicability of synthetic biology to the solving of NP-complete problems, and could validate synthetic biology as a feasible approach to computing in the future. In order to demonstrate its feasibility to solve a wide range of problems in theoretical computer science however, various constructs will need to be developed that are designed to simulate other common problems, such as the clique problem and the rank coloring problem. Furthermore, it is recommended that in the future, more time be allotted to troubleshooting. Although we spent significantly more time for experimentation for this year’s experiment than we did for last year’s, we ran into several problems with the transformation and had to repeatedly fix certain variables of our procedure so that we ended up with a favorable result. To circumvent this issue, we recommend future researchers to be completely aware of the unique resources and environment within their lab so that they can adapt their protocols more easily. In addition, since we worked in a school laboratory, finding sufficient time to finish certain procedures was a bit of a hassle. For most of the year, we were only able to work during 45 - 1 hour 30 minute blocks, which is not enough time to complete a substantial portion of experimentation. To work around this issue, we recommend that future groups truly work as a team - map out everyone’s schedules and find out who is available when. Not everyone needs to be available for a procedure to get done; as long as one person can be in the lab, the job can be finished. Retrieved from "http://2014hs.igem.org/Team:Jefferson_VA_SciCOS/Result1277PEA Exeter NHPhillips Exeter Academy Exeter, NH, USA www.exeter.eduHigh SchoolDevelopment of Fracking Runoff DetectorsOur team's primary goal is to create a cell that can detect naphthalene (a toxic chemical often found in fracking runoff) and degrade it to a less harmful compound. We used a nahR gene, on plasmid NAH7 (BBa_J61051), which breaks down naphthalene in a cell environment into the more inert catechol. We then use the downstream element BBa_K118021, whose protein product, catechol-2, 3-dioxygenase, catalyzes the conversion of catechol to 2-HMS (2-hydroxymuconate semialdyhyde) a yellow-colored molecule. In addition, we created a cell that will be able to broadly identify other toxic fracking byproducts via a nonspecific SOS cell damage element. The promoter recognizes and binds to a combination of sequences of SOS repressor proteins produced after cellular stress from damaging toxins or radiation. This promoter (BBa_K518010) will be attached to a downstream element (BBa_K592009) coding for a blue-colored protein, so that bacterial cells exhibiting stress can be easily discerned.1101BBa_K1277999http://2014HS.igem.org/Team:PEA_Exeter_NHBBa_K1277000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163920140High SchoolLog in   Team:PEA Exeter NH/Abstract From 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome Abstract Three integral concepts underlie the design of our parts: upstream regulation, fluorescence and pigmentation. Upstream regulation is the idea that the expression (quantity of transcribed protein material) of a gene primarily depends on the region directly upstream of the coding region of the gene, which is known as the binding site for RNA Polymerase. In addition, expression of a gene can be regulated by other DNA sequences, called signature sequences. Signature sequences are binding sites for proteins called transcription factors. These transcription factors modify the affinity of the binding of RNA Polymerase to the promoter region, thus modifying the rate of transcription. If the binding affinity increases, then the transcription factor is an activator for that specific gene. Likewise, if RNA polymerase is less likely to bind, then the transcription factor is a repressor for that specific gene. Fluorescent proteins are those that emit visible wavelengths of light upon exposure to lower wavelengths. The chemical mechanism for fluorescence comes from the nature of quantum mechanics, which states, in short, that electrons may only occupy discrete energy shells associated with discrete amounts of potential energy. The further away an electron is from its neighboring atoms, the more potential energy it has. However, electrons far away from nuclei of the atoms will be attracted by the Coulomb force, causing a discrete decrease in potential energy. Due to the conservation of energy, this energy is then emitted as a photon. Fluorescence occurs when a step of the return of the electron to the ground state, where it has the lowest possible potential energy, causes an emission of visible light. The emitted light is necessarily of a lower energy level and thus higher wavelength than the light triggering fluorescence due to the conservation of energy; heat (high wavelength light) releasing steps along with one or many visible light releasing steps combine to convert the energy from the absorbed light back to light energy. Biologically, a fluorescent protein is typically used as smaller fluorescent particles such as metals cannot be expressed via genes. The green fluorescent protein (GFP), expressed naturally in the jellyfish Aequorea Victoria, is the first example of a fluorescent protein successfully expressed in E. coli bacteria in 1994. Since then, the gene has been chemically modified to express blue (BFP) and red (RFP) fluorescent proteins. Pigmentation is the ability for a compound to absorb specific wavelengths of light and reflect others. The chemical basis for this is, once again, quantum mechanics. As each wavelength of light is associated with a different amount of energy, certain wavelengths of light will excite electrons and others will not. Those wavelengths that do excite electrons are absorbed by electrons of the protein. The return of the electron to its ground state in pigmented molecules does not include a large quantum level change as it does in fluorescence; instead, the return of electrons to its ground state with respect to the protein contains multiple steps, each of which releases heat. Importantly, the same electron does not execute this multi-step release; indeed, it is unfeasible for this to occur due to the quantization of energy. Instead, the electrons’ energy release trigger the excitation of other electrons, in a chain reaction eventually dissipating all of the initial energy of the photon as heat. In addition to this, some wavelengths of light are instead reflected, which occurs when the light cannot be absorbed by any electrons in the compound. This occurs when the light has an inappropriate amount of energy so that no change in quantum state for the electron would conserve energy upon photon absorption. Since white light is a combination of multiple wavelengths of visible light, a pigment will reflect some of the constituents of white light and reflect others. This causes the pigment to be visualized by the color that it reflects. Biological examples of pigments include the chlorophyll of plants giving plants their green color, the melanin of animals that determines skin color, and the hemoglobin in vertebrate red blood cells. The gene-regulated production of specific fluorescent and pigmented protein products allows for visualization of gene activity, corresponding to the presence of an activator or the absence of a repressor. This visualization is a concept integral to the identification of catechol and lead. In order to detect catechol, we will use the downstream element BBa_K118021, whose protein product, catechol-2, 3-dioxygenase, catalyzes the conversion of catechol, in an oxygenated environment to the fluorescent yellow pigmentation molecule 2-hydroxy-cis,cis-muconic semialdehyde. The catechol detection mechanism can be extended to detect naphthalene via the catabolism of cytosolic naphthalene into catechol. The nahR gene, on plasmid NAH7 (BBa_J61051), controls naphthalene degradation. It is organized in two operons which encodes six enzymes in nahA-F, and eight enzymes in nahG-M. The “upper operon” or nahA-F on the NAH7 plasmid degrades naphthalene to salicylate and pyruvate. Then, the “lower operon” or nahG-M encodes enzymes that degrade salicylate into intermediates of the TCA cycle. Thus, this gene breaks down naphthalene in a cell environment into detectable catechol. In addition to detection of the compounds listed above, our cell will also be able to unspecifically identify other toxic fracking byproducts via a nonspecific SOS cell damage element. The promoter for this gene is a combination of signature sequences insertable on sulA. These sequences bind to SOS repressor proteins that E. coli typically destroy after detecting stress from damaging toxins or radiation. This promoter (BBa_K518010) has been isolated and will be attached to a downstream element (BBa_K592009) coding for a blue-colored protein, so that bacterial cells exhibiting stress – specifically from the presence of toxic or radioactive runoff from fracking – can be easily discerned. Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_NH/AbstractOutreach From 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome Educating Our Peers and Igniting Passion Synthetic biology is a growing field, with boundless potential to change thousands of lives and to improve the world we live in. At PEA, we want to help advance the public's perception of synthetic biology by not just confining it to the lab, but by introducing it the masses, facilitating discussions and igniting interest. We've done this by guiding school-wide research projects, and raising awareness among our peers. We've set up si-RNA research in nematodes for our peers, and we hope to publish our findings this spring. We've organized club trips to bird sanctuaries, “Learn about Meiosis/Mitosis” sessions, and countless other things. As members of a distinguished synthetic biology team, we can all remember the first experiences in a lab that inspired us, and we hope to recreate this experience for others. By expanding the reach of synthetic biology, we can have an educated populace consider the benefits of the field of synthetic biology. Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_NH/OutreacTeam From 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome The Team Christina Savvides Christina Savvides is an 11th grader at PEA from Shaker Heights, Ohio, and the student team leader of Exeter's iGEM team. She has been fascinated with biology and experimentation from a young age with mosquitoes’ larvae and crayfish as common subjects of study in her homemade basement lab. While taking AP Biology three years ago, her interest turned to molecular genetics. Alex Song Alex is a member of the Phillips Exeter Academy Class of 2015 from Waterloo, Canada. He became interested in biology through computer programming, being introduced to the field through a seminar on computational biology. He then took a full-year high school biology course, which gave him a broader view of the study of biology with laboratory methods. He aspires to be an applied mathematician. Charis Edwards Charis Edwards is a 14 year old from Duluth, Minnesota. She has a special interest in studying drug-resistant bacterial infection and would like to work to decrease stigma and increase education about disease, medical care and hygiene in the Middle East, after graduate school. Cat O'Donnell Cat is a lover of cats and clunky centrifuges. She hails from Alaska, where she spends her time skiing and wrestling bears. She is a firm believer that what can go wrong will go wrong, but that has given her a special skill in creating contingency plans. She one day hopes to take over the world using fracking runoff detecting e. coli cells. Reginald Lamaute Reginald Lamaute is a sophomore from Chapel Hill, NC. After he was diagnosed with a rare esophageal disorder, he intrigued by descriptions of his own disorder and soon started reading more texts on physiology and neurology as well. After that, he was accidentally put on a Science Olympiad team in seventh grade and fell in love with biology. He eventually hopes to join Doctors Without Borders. Melody Nguyen Melody is a sophomore at PEA from Vietnam. She enjoys fumbling on the piano, working in the lab (for iGEM), socializing with friends, and community service. When she's not overloaded with work, she spends her downtime either sleeping or drinking a cup of matcha green tea while browsing the internet. She's looking forwards to pursuing a career in medicine. Connie Cai Connie Cai is a current ninth grader at PEA. She loves biology because of its ability to explain our origins, and how everything came to be. Other than reading about biology, she loves to play tennis, violin, piano, and she listen to music. She hopes that she can continue to do iGEM throughout her years and continue to grow as a scientist and biologist and be able to conduct more fascinating experiments. Janice Chung Janice Chung, an upper at PEA, is excited to join the iGEM experience. She has always had a deep interest in the sciences and is delighted to work in the lab. Outside of iGEM, she enjoys playing violin, golfing, skiing, and painting. A special thanks to Mr. Janicki for advising and mentoring Exeter's iGEM team, and to Ms. Rotondo for research support! Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_NH/TeaNotebook From 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome Lab Notebook Starting in the 2013 Winter term, our team learned laboratory techniques and protocols. We began research on the effects of fracking and the chemical characteristics of toxic water contaminants: Catechol, naphthalene, and lead. Over the spring semester of 2014, six full time team members were working tirelessly on the project. Here, we present a summary of our daily progress and experiments. GROWING CELL CULTURESNames: Alex S, Reginald L, Cat O, Melody N, Janice C, Connie C, Christina SDate: April 19th, 2014Species: E.ColiPart Number: BBa_J04500, BBa_J04650Results: TBALIGATIONNames: Alex S, Reginald L, Cat O, Melody N, Janice C, Connie C, Christina SDate: April 20th, 2014Species: E.ColiPart Number and plasmid: BBa_J04500, BBa_J04650, pSB1C3Results: TBATRANSFORMATIONNames: Alex S, Reginald L, Cat O, Melody N, Janice C, Connie C, Christina SDate: April 24th, 2014Antibiotic: Amp and KanPart number and plasmid: BBa_J04500, BBa_J04650, pSB1C3Results: TBAGROWING CELL CULTURESNames: Christina S, Janice C, Melody NDate: April 29th, 2014Species: E. ColiPart number: BBa_K118021, BBa_J61051, BBa_K518010, BBa_K592009Results: TBAPICKING CELL CULTURESNames: Alex S, Reginald L, Cat O, Melody N, Janice C, Connie C, Christina SDate: April 30th, 2014Species: E. ColiPart number and plasmid: BBa_K118021, BBa_J61051, BBa_K518010, BBa_K592009, pSB1C3Results: TBARESTRICTION DIGESTNames: Alex S, Reginald L, Cat O, Melody N, Janice C, Connie C, Christina SDate: May 18th, 2014Species: E. ColiRestricted: BBa_J61051, BBa_K118021Results: TBAIncubate 20 hours at 37°CGROWING CELL CULTURENames: Christina SDate: May 20th, 2014Species: E. ColiPREPARATION OF ANTIBIOTIC SOLUTIONNames: Christina SDate: May 20th, 2014Antibiotic: ChloramphenicolMINIPREPNames: Alex S, Janice C, Melody NDate: May 21st, 2014 Mini prep: BBa_K518010_pSB1C3, BBa_J61051_pSB1C3Species: E. ColiResults: Worked Well LIGATIONNames: Charis E, Reginald L, Christina S, Cat ODate: May 21st, 2014Species: E. ColiResults: TBATube 1- BBa_J61051_BBa_K118021_pSB1C3MINIPREP:Names: Christina S.Date: May 22nd, 2014Cultures for Miniprep: BBa_K518010_pSB1C3 (SOS), BBa_J61051_pSB1C3 (NAP), BBa_K118021 _pSB1C3 (CAT), BBa_K592009_pSB1C3 (BLUE) [samples prepped in duplicate]Results: TBAMEASURING DNA CONCENTRATION FROM MINIPREPNames: Alex S, Christina SDate: May 23rd, 2014Samples from MiniprepResults: DNA ConcentrationsNAP1: 26.25 ng/ulNAP2: 33.50 ng/ulCAT1: 39.50 ng/ulCAT2: 21.00 ng/ulSOS1: 29.75 ng/ulSOS2 (from mini prep on 5.21.14): 30.50 ng/ulBLUE1: 70.25 ng/ulBLUE2: 20.50 ng/ulRESTRICITION DIGESTNames: Christina S, Reginald L., Alex S.Date: May 23rd, 2014Samples from MiniprepGEL ELECTROPHORESIS ON DIGEST PRODUCTNames: Christina SDate: May 25th, 2014Samples from restriction digestLoading Order of Gel:100 base pair ladderAmpicillin Backbone Digest 1Ampicillin Backbone Digest 2Kanamycin Backbone DigestNAP1NAP2CAT1CAT2BlueSOSLIGATIONNames: Alex S, Cat O, Connie C, Reginald L, Janice CDate: May 25th, 2014Samples from restriction digestMAKING AMPICILLIN/KANAMYCIN PLATESName: Christina SDate: May 26th, 2014TRANSFORMATIONNames: Cat O, Christina SDate: May27th, 2014Samples from LigationGROWING UP CELLSNames: Christina SDate: May 30th, 2014 Species: E. ColiResults: TBASETTING VARIABLE ENVIRONMENTS FOR CELLSNames: Christina S, Cat ODate: June 1st, 2014 Species: E. ColiResults: TBAEXPERIMENTATIONDate: June 2nd to 5th, 2014 Species: E. ColiResults: TBA Experiment Result to Be Shown At Jamboree!See our poster! (click link) Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_NH/NotebooProject From 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome See our poster! Introduction Fracking, or hydraulic fracturing, is a process by which highly pressurized liquid is used to create hairline fractures in rocks, particularly shale. Water, natural gas, brine, petroleum and other fluids then migrate into the well via the fractures, where it is harvested and refined into the natural gas and petroleum that we use everyday. Fracking has gained popularity due to its ability to extract deposits that were previously unreachable. Worldwide, there are more than 2.5 million fracking jobs. However, this method of extraction comes at a price. The fractures created during the fracking process could act as a conduit for petroleum and natural gas to migrate into groundwater. This intrusion affects both the environment due to lake and pond contamination and human health due to drinking water contamination.The hydrocarbons associated with petroleum and natural gas are often very difficult to detect in drinking water, making the consequences of groundwater contamination even greater. Additionally, new studies have linked fracking with the presence of radium in water runoff. Current laws require only infrequent testing for radioactivity in public drinking water systems. Therefore discovery of the contamination before widespread consumption of the radioactive water is unlikely. Exposure to radium can result in anemia, cataracts, cancer, and death. Therefore rapid detection of hydrocarbon as well as radiation contaminants is crucial.Our team's goal is to produce bacteria that can detect toxic runoff and break down poisonous hydrocarbons. To that end we are using a catechol breakdown gene that will convert catechol into a yellow waste product, allowing contamination to be easily visualized. In addition, we are transforming the bacteria with a naphthalene breakdown gene that will first convert naphthalene to salicylate, and then convert the salicylate to catechol. We will also transform the bacteria with a cell damage promoter (that can be activated in the presence of radiation) attached to a downstream element coding for a blue colored protein. In order to outline a means of detection for each, we shall review the chemical properties of our contaminants: catechol and naphthalene. What is Catechol? Catechol (molecular formula C6H6O2), pyrocatechol or 1,2-dihydroxybenzene, is a toxic chemical that can cause depression of the central nervous system in large doses. In addition, it is classified by the International Agency for Research on Cancer as possibly carcinogenic to humans. As a result, our team intends to detect and eliminate catechol using the xylE gene that encodes catechol-2,3-dioxygenase. This enzyme catalyzes the transformation of catechol to a bright yellow product, allowing us to visually confirm the presence and elimination of catechol." What is Naphthalene? Naphthalene (molecular formula C10H8) is a toxic hydrocarbon found in crude oil. When ingested, naphthalene is known to cause potentially fatal hemolytic anemia, in which red blood cells are destroyed, affecting the body’s ability to transport oxygen. In addition, naphthalene can induce jaundice, tachycardia, and convulsions. Naphthalene can potentially leak from sites of fracking into groundwater. In order to combat this public health threat, our team intends to transform E. coli with a gene that will induce the degradation of naphthalene. In order to effectively detect naphthalene, we rely on its biochemical relationship to catechol. Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_NH/ProjSafety From 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome Lab Safety 1. Would any of your project ideas raise safety issues in terms of researcher safety, public safety or environmental safety?The Phillips Exeter Academy iGEM team works in a science laboratory with Biosafety Containment Level 1, because of our work with non pathogenic E.Coli. However there are some potentially poisonous and irritating reagents that we work with in the lab. Some examples are the reagents used in lab protocols such as PCR and Ligation, as well as the naphthalene we use for testing our bacterium's functioning. To minimize the risks associated with these chemicals and enzymes, team members are expected to follow standard lab protocol. Laboratory coats, gloves, close-toed shoes, pants must be worn at all times while working in the laboratory. Students must wash their hands before and after working in the lab. Additionally, personal protective equipment is not allowed to be worn outside the lab. Team members are expected to maintain a sterile working environment by wiping down the benches with 70% ethanol before and after procedures. Fire extinguisher, emergency shower, and an eye-wash station are readily available in our lab. While working in the lab, a faculty supervisor is always present. All high school participants have completed lab-specific safety training prior to starting our project. 2. Do any of the new devices that you made this year raise safety issues?Our transformed bacteria, containing a naphthalene degregation gene, a SOS promoter, a lead promoter, and a gene for lead detection do not raise any significant safety concerns. If all bacteria, DNA, protein samples, and chemicals are handled appropriately, our project is quite safe. 3. Is there a local biosafety group, committee, or review board at your institution?Our iGEM research team works closely with the school’s Science Department and Phillips Exeter Academy staff who provide us with biosafety and lab training. Our training workshops provide us with the necessary tools to work saftely in the lab, however should our instruction prove to be inadequate in a scenario, our faculty suprevisor will guide us in making the best choices. Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_NH/SafetFrom 2014hs.igem.org IGEM 2014 - The Fracking Run-Off Detector Project Phillips Exeter Academy OutreachSafetyThe TeamNotebookProposalProjectHome Team Goal: To Create a Fracking Run-Off Detector Fracking, or hydraulic fracturing, is a controversial process that is commonly used to extract natural gas from underground deposits. However this process can result in toxic runoff. Our goal is to create a bacteria that could identify the toxic runoff thus notifying people of the danger and limiting its consumption. We are using naphthalene and catechol breakdown genes to convert naphthalene into catechol and catechol into a benign yellow waste product, allowing contamination to be easily visualized as well as neutralized. See our poster! Contact Us: peaigem@gmail.com Address: 20 Main St. Exeter, NH 03833 peaigem.com/igem2014 Tel: 603-772-8311 Retrieved from "http://2014hs.igem.org/Team:PEA_Exeter_N1258WHHS Cincinnati OHWalnut Hills High School Cincinnati, OH, USA http://www.walnuthillseagles.com/High SchoolArsenic BiosensorIn many developing countries, arsenic-contaminated water remains a cause of many problems. Although the WHO and EPA both recommend a maximum level of contamination of 10 ppb, many areas, like West Bengal and Bangladesh, have a much more relaxed 50 ppb. Arsenicosis, arsenic poisoning, has severe effects, including headaches, vomiting, hair loss, and skin lesions. Arsenic has also been known to lead to heart disease, stroke, cancer, and eventually death. A reliable biosensor would allow these areas to observe arsenic contamination. This inspired the WHHS iGEM to design a project similar and just as effective, yet with our own spin. Instead of pH, a pigment output would also be a viable method. Using the lycopene plasmids that were created by the 2009 Cambridge team, we are engineering an E. Coli strain that will produce a visible solid yellow pigment (PNPG) when it comes in contact with toxic arsenic concentrations.1601BBa_K1258999http://2014HS.igem.org/Team:WHHS_Cincinnati_OHBBa_K1258000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161620140High SchoolLog in   Team:WHHS Cincinnati OH From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:WHHS_Cincinnati_OH/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:WHHS Cincinnati OH logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:WHHS Cincinnati OH team.png Your team picture Team WHHS_Cincinnati_OH Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project In the United States, arsenic levels in water sources are not high enough to raise eyebrows, but in most developing countries, arsenic levels remain a prevalent cause of many problems. Although the World Health Organization (WHO) and the US Environmental Protection Agency (EPA) both recommend a maximum level of contamination (MCL) of 10 parts per billion (ppb), many areas, such as West Bengal and Bangladesh, have a much more relaxed MCL of 50 ppb. In these countries, tube wells, which tend to be deeper than 100 meters, were unknowingly drilled through large concentrations of arsenic, subsequently giving those who drank large quantities of water from the wells arsenicosis. Arsenicosis, arsenic poisoning, has severe effects, including the likes of headaches, vomiting, hair loss, and skin lesions. Arsenic has also been known to lead to heart disease, stroke, cancer, and eventually death. A simple, highly performing yet cost efficient biosensor would allow these areas to observe the levels of arsenic contamination before continuing the water use. Many previous arsenic biosensors have required knowledgeable technicians and expensive equipment, which is not easy to use in the field. Some methods, for example the one used by the 2006 Edinburgh iGEM Team, involved a sensor that gave a pH response, which is much more practical for field use. This inspired the WHHS iGEM to do something similar and just as effective, yet with our own spin. Instead of pH, a pigment output would also be a viable method. Using the lycopene plasmids that were created by the 2009 Cambridge team, we are engineering an E. Coli strain that will produce visible red pigment when it comes in contact with certain toxic arsenic concentrations. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:WHHS_Cincinnati_OH1281AMSA WeColiAdvanced Math and Science Academy Charter School Marlborough, MA, 01752 amsacs.orgHigh School1301BBa_K1281999http://2014HS.igem.org/Team:AMSA_WeColiBBa_K1281000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164420140High SchoolLog in   Team:AMSA WeColi From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:AMSA_WeColi/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:AMSA WeColi logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:AMSA WeColi team.png Your team picture Team AMSA_WeColi Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project What are you working on this semester? Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:AMSA_WeColi1249Montgomery Cougars NJUSAMontgomery High School Skillman NJ, 08558 USA www.mtsd.k12.nj.usHigh SchoolAcne Vulgaris Prevention through Dehydrogenation of Sebaceous LipidsAcne vulgaris affects around 85% of American adolescents. The four main causes of acne are comedogenesis, sebum production, proliferation of Propionibacterium acnes, and inflammation in the form of papules, pustules, and nodules. Triglycerides, wax esters, squalene, free fatty acids, and sapienic acid are the main components of sebum. Our team has created a biosystem that reduces the presence of sebaceous components (primarily wax esters). Our mechanism includes two proteins of interest-Medium Chain Alcohol Dehydrogenase and Medium Chain Aldehyde Dehydrogenase in two identical biological circuits to change the structure of wax esters and other lipids, rendering them insufficient for bacterial consumption. We included a basic promoter, RBS and terminator, making the protein-coding domain the only variable between the two circuits. We hope this mechanism facilitates sebum breakdown and reduces inflammatory responses of the P. acne bacteria.3801BBa_K1249999http://2014HS.igem.org/Team:Montgomery_Cougars_NJUSABBa_K1249000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=160220140http://2014hs.igem.org/files/poster/Montgomery_Cougars_NJUSA.pdfHigh SchoolLog in   Team:Montgomery Cougars NJUSA/Extras/Human Practices From 2014hs.igem.org (Redirected from Team:Montgomery Cougars NJUSA/Human Practices) Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Contents 1 Our Project2 Extras3 Human Practices 3.1 Our Mission3.2 Our Impact3.3 Our Outreach 3.3.1 Community Outreach 3.4 Leadership 3.4.1 What makes a good leader? Human Practices Our Mission Our Mission Statement: Montgomery iGEM’s fundamental goal is to inspire the next generation of synthetic biologists, doctors, biomedical engineers and STEM professionals through immersion in a challenging and innovative program that allows students of MHS to explore concepts of applied biology. In Montgomery iGEM, students will be exposed to real life laboratory experiences beyond the classroom. Students learn the fundamentals of synthetic biology through experiments, community networking and sharing products at the international competitions at the conclusion of every season. By working directly with cells, genes, plasmids and bacteria, students can become inspired to work in applied sciences such as medicine, biochemistry and engineering. In addition to the hands-on aspect of iGEM, students were encouraged to actively research and understand the science behind the labwork. Upperclassmen and teacher advisers gave class lessons to teach the underclassmen, which successfully peaked both their interest and understanding of the biology part of iGEM. The goal of Montgomery iGEM is not only to inspire students’ futures, but also to give them opportunities to contribute to the scientific community in the present. We hope that this program will positively impact the STEM education within the community. Through working with the other STEM clubs, teams and activities within the Montgomery co-curricular system, Montgomery iGEM hopes to become fully integrated into STEM within our school. We, however, hope to provide a different side of STEM education than the Robotics teams and other STEM clubs do. Instead of working with gears, wheels and drill presses, students will be able to work with DNA to program bacteria and cells, potentially drawing the blueprints for the next radiation detector, synthetic circuit, or even a cure for cancer. Our Impact One of the most prevalent problems plaguing adolescents around the world is acne. Acne is understood as skin blemishes caused by clogged pores as a result of dead skin cells and oil. With our project, we aim to reduce the occurrence of acne by targeting one step in the way acne is produced. Through this goal, we strive to create a product that is more effective and safer than leading commercial acne reducing creams. In addition to our project, we hope to provide research that helps others understand the causes of acne and how it develops. As the only hands-on biology-related extracurricular activity in Montgomery High School, we have the opportunity to spread awareness on not only acne, but also on synthetic biology as a whole. Through these efforts, we aim to expand students' interests in and the community's exposure to biological studies. Our Outreach Montgomery iGEM was founded on the premise of inspiring the next generation of STEM professionals. One way we seek to do this is through community service. We currently hold a biannual Shoprite fundraiser to raise awareness for the organization and earn money for our club. In the future, we plan on doing presentations at our Montgomery Board of Education meetings and freshman orientation to broaden our spectrum. We also plan on doing presentations throughout our school and participating in the district wide STEM board to increase the curriculum in our entire district. Community Outreach In our school, we are able to promote our team through the school newspaper by writing articles about our mission and our project for the year. This allows us to reach out to a large audience within our high school. In addition to this, we have a close tie with the local Shoprite supermarket; this gives us the opportunity to promote our team in the local community. For fundraising, we participated in Shoprite bagging. We handed out small slips of paper about the club and spoke to customers about our project. Through this, we were able to meet specific individuals who were knowledgeable in synthetic biology. We have developed into a sustainable team with over 20 avid members that are eager to take part in synthetic biology. Emily Ma (Class of 2015, Secretary) Ankit Shah (Class of 2016, Underclassmen Representative) Kevin Shen (Class of 2015, Operations) Bharghav Vemulapalli (Class of 2016, Operations) and Mr. Resch (Advisor) Sarah Oh (Class of 2015, Librarian) Leadership President: In charge of all activities and coordinates all team projects including website, financial administration, team structuring, meeting curriculums, lab procedures and organization. Vice President: Assists the President and helps with all responsibilities. Systems Manager: Responsible for promoting the team on the team wiki, and online documentation. Will work with the Operations to support team structure. Required web design skills. Community Liaison: Responsible for community outreach including fundraising, sponsorship, and promotion of STEM in the community. This will include presentations for organizations, documentation of monetary exchanges, organizing fundraisers and promoting the team. Secretary: Responsible for taking notes and informing absent club members of what they missed. Secretary will also be responsible for documenting attendance for members. Librarian: Responsible for organizing and distributing information packets to members. It is the librarian’s job to make sure all members have an idea of what they are doing. The librarian will also work with the advisor to create packets and lesson plans. (Recommended Biology knowledge). Underclassmen Representatives (2): Conveys underclassmen opinions to the board. Works in conjunction with the librarian to distribute basic information to underclassmen. Ideally, one freshman and one sophomore. Responsible for making sure all underclassmen understand material. Research and Development Subteam Leader: Responsible for overseeing and controlling the quality of laboratory work. Responsible for sharing laboratory procedures and making sure all procedures are safe and uncontaminated. Must be present for most labs from start to finish. Operations Subteam Leader: Responsible for overseeing the functions of the team and keeping the internal structure strong and organized. Will work closely with the Community Liason, Secretary, Librarian and Underclassmen representative to improve the functions of the team. What makes a good leader? Self-Awareness. You have an intimate knowledge of your inner emotional state. You know your strengths and your weaknesses. You know when you’re working in flow and you know when you’re over worked. You know yourself, including your capabilities and your limitations, which allows you to push yourself to your maximum potential. Self-Direction. You’re able to direct yourself effectively and powerfully. You know how to get things done, how to organize tasks and how to avoid procrastination. You know how to generate energy for projects, to calm yourself when angered. You can make decisions quickly when necessary, but can also slow to consider all the options on the table. Vision. You’re working towards a goal that’s greater than yourself. It could be something small, like the success of the team, or a larger vision like world peace. Working towards a vision is far more inspiring than working towards personal gain. Ability to Motivate. Leaders don’t lead by telling people what they have to do. Instead, leaders cause people to want to help them. A key part of this is cultivating your own desire to help others. When others sense that you want to help them, they in turn want to help you. Social Awareness. Understanding social networks and key influences in that social network is another key part of leadership. Who in the organization has the most clout, both officially and unofficially? Who moves the hearts of the group? These are some of the most important characteristics of good leaders. While we have defined leadership positions, our board acted more as a joint effort to organize all aspects of the club this year. Everyone picked up slack where needed and worked coherently to build up the team that we have today. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/Human_PracticesTeam/Robert Dembinski From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Robert DembinskiRobert Dembinski is a Junior in Montgomery High School and is the Vice President of Montgomery iGEM. Together with Susan Liu, he was a founder and a crucial contributor to the structure of the team. In the future, he aspires to enter the field of medicine and save lives through cutting edge research. He hopes that iGEM will give him an insight into and kickstart his career. Aside from iGEM, Robert is a phenomenal mid-long distance runner and a key member on the Montgomery Track Team. In his free time, he enjoys playing YuGiOh and Clash of Clans with his friends. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Robert_DembinskFrom 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Project Learn more about our project this year Labs Learn more about our labs and procedures this year Team Structure Understand how our team is organized and run Pathogenesis of Acne Learn the mechanism behind and development of acne Safety Discover how Montgomery iGEM team members keep themselves safe in the lab Human Practices Our mission, impact and outreach Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Montgomery iGEMMotivated by the lack of outlets for students to explore their passions for biological research, students from Montgomery High School created their own iGEM team in the 2013-2014 academic year to gain firsthand experience in synthetic biology. While the beginning of the season presented numerous obstacles, Montgomery iGEM has developed into a successful club with over 30 active members who are all motivated by the endless possibilities that scientific innovation presents. Our club stemmed from a desire to provide students with hands-on laboratory experience. While popular clubs in our school such as Robotics and Science Olympiad provide great opportunities for students to explore and learn about new fields of science and engineering, students previously did not have the opportunity to engage in wet laboratory practices. We created this team to provide students with a firsthand look into the strikingly innovative field of synthetic biology and an opportunity to innovate. We strive to further student interest in science and increase students' exposure to fields that they might not have explored otherwise. Our iGEM program is creating the next generation of doctors, biomedical engineers and innovators, a generation with a genuine desire to help make the world a better place. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUShttp://2014hs.igem.org/wiki/images/thumb/0/08/IGEM_Logo.png/250px-IGEM_Logo.pngTeam/Emily Ma From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Emily MaEmily Ma is a currently a junior at Montgomery High School. Her interest in synthetic biology began because she wanted to do more extensive research outside of biology class. Aside from participating in iGEM, she is also on the tennis and track team. She is actively involved in many programs in the MHS extracurricular system such as Amnesty International, NHS and Interact. Outside of school, Emily’s favorite hobbies are watching NCIS, eating macarons, and taking frequent naps. Rumor has it that Emily is the master of iPhone games. She consistently sets high scores in Fruit Ninja, 94 Seconds and has completed every level of Candy Crush imaginable. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Emily_MProject/Labs From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Labs Contents1 Our Project2 Extras3 Labs3.1 Growing Cultures3.1.1 Materials3.1.2 Procedure (Streaking Agar Plates)3.1.3 Procedure (Growing Up Cultures)3.1.4 Our Experiences3.2 Miniprep3.2.1 Materials3.2.2 Procedure3.2.3 Our Experiences3.3 Restriction Digest3.3.1 Materials3.3.2 Procedure3.3.3 Our Experience3.4 Ligation3.4.1 Materials3.4.2 Procedure3.4.3 Our Experience3.5 Transformation3.5.1 Materials3.5.2 Procedure3.5.3 Our Experiences An iGEM member holding a beaker Micropipettes Streaking a cell culture Micropipetting Using Spin Columns Disposal of PlasticWare Prepping a Cell Culture Streaking on Agar Prepping a Cell Culture Safety First! Growing Cultures Materials Preparing the Culture TubesAgar Stab for Promotor BBa_R0010Agar Stab for RBS BBa_B0034Agar Stab for Protein Coding Domains BBa_K398006 and BBa_K398005Agar Stab for GFP BBa_E0040Agar Stab for Terminator BBa_B0015Inoculating TubesLB Agar PlatesMarkerInoculating LoopsSnap Cap TubesLB Broth Procedure (Streaking Agar Plates)Clean the table and clear a lab space to have a contaminant-free cultureLabel each of the 4 plates with the appropriate part name, in this case, BBa_B0034, BBa_K398005, BBa_K398006, and BBa_E0040Use the inoculating loop to take a sample from each of the 4 agar stabs where the hole is visibleStreak the loop with the bacteria sample onto the agar plate in zigzags, and once again 90 degrees rotatedRepeat these steps for all 4 parts onto different agar platesTape plates together and turn upside down so as to avoid condensation contaminationStore in incubator at 37 degrees Celsius for 14-16 hoursAgar plates, once incubated, can be stored in the fridge Procedure (Growing Up Cultures)Clean table and clear a lab space to have a contaminant-free cultureTake out the 4 plated agar samples from the incubator, there should be significant growthUsing a pipette, put 5ml of LB nutrient broth into 4 snap tubesUse an inoculating loop to take colonies from each agar plate and swirl into a tubeClose tubes and incubate for 14-16 hours Our Experiences Preparing the Culture TubesThis part of the lab was really the first time since our run-through pGLO lab that students had a chance to have first hand practice. During the Agar Streaking procedure, we encountered a significant setback to our already tight schedule. We had looked at the protocol for the 3A assembly kit, since parts were analogous, and streaked the bacteria onto Ampicillin/Kanamycin plates that were provided for us. We did not think that the parts we had ordered came with this resistance. So, we waited a full day to find that there was absolutely no growth. Thus, we had to repeat this all again on plain LB Agar.For growing up the cultures, although we completed the procedure correctly, we did not end up using these cultures for our experiment. The one day waiting period was simply too long for our tight schedule. Our students talked with our advisor and we determined that the bacteria on the agar plate should be just as good as those that would grow within the next day. So, all of of subsequent experimental data comes from the growth on the original agar plates. Miniprep MaterialsQiagen Miniprep KitCell Culture1.6 ml Microcentrifuge tubes (2 per a miniprep)TE (1:10) ProcedureWe opted not to use our grownup cell cultures. So we inoculated LB broth with the 4 respective parts and spun that culture in a centrifuge to pellet the cells, emptying the supernatant when completeResuspend pelleted bacterial cells (all 4 samples) in 250 µl Buffer P1 and transfer to a microcentrifuge tube (we used just regular ones to accommodate our centrifuge)Add 250 μl Buffer P2 to the solution and gently mix. This allows for the lysis process to occurAdd 350 μl Buffer N3 to each sample and gently mix.Centrifuge for 10 min at 13,000 rpm (~17,900 x g). In the end, a white pellet formed in each of the 4 samples we were miniprepping.Apply supernatants from step 4 to the QIAprep spin column by pipetting.Centrifuge for 30–60 and discard all the flow-through.Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min. Our Experiences Using the CentrifugeWhile we did not encounter many problems with this step directly, we did not use the standard cell cultures that were supposed to be used. We instead substituted for a faster mechanism which we did not see as significantly different. Due to our spin tube deficiency, we were forced to scale down our volumes to 1/10th their original values but kept all the proportions the same. In the end, we just ended up making very little of the purified DNA for the restriction digestion and ligation processes. We hope that our last-minute substitution plans do not interfere with the outcomes of our lab. Other than that, we followed all of the standard protocol but skipped the optional but recommended steps. We ran out of time to do these. Restriction Digest Materials Ice and bucket/containerPromoter (BBa_R0010) (Purified DNA, > 16ng/ul)RBS (BBa_B0034) (Purified DNA, > 16ng/ul)Med-Chain Aldehyde Dehydrogenase (BBa_K398005)(Purified DNA, > 16ng/ul)Med-Chain Alcohol Dehydrogenase (BBa_K398006) (Purified DNA, > 16ng/ul)GFP (BBa_E0040)(Purified DNA, > 16ng/ul)Terminator (BBa_B0015) (Purified DNA, > 16ng/ul)Linearized plasmid backbone (pSB1C3)(25ng/ul)dH2ONEB Buffer 2Restriction Enzymes: EcoRI, SpeI, XbaI, PstIThermal cycler Procedure Keep all enzymes and buffers used on ice to prevent degredationThaw NEB Buffer 2 at room temperature (We did not have BSA and instead used distilled water to fill volume. Collect solutions at the bottoms of the tubes.Add 250ng of DNA (from all 4 parts) to tubes. Add distilled water to the tubes for a total volume of 16ul in each tubeAdd 2.5ul of NEB Buffer 2 to each tube.In the Promoter tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.In the RBS tube: Add 0.5ul of XbaI, and 0.5ul of SpeI.In the Aldehyde Dehydrogenase tube: Add 0.5ul of XbaI, and 0.5ul of SpeI.In the Alcohol Dehydrogenase tube: Add 0.5ul of XbaI, and 0.5ul of SpeI.In the Terminator tube: Add 0.5ul of XbaI, and 0.5ul of PstI.In the GFP tube: Add 0.5ul of XbaI, and 0.5ul of SpeI.In the pSB1C3 tube: The pSB1C3 tube was already precut with EcoRI and PstI.The total volume in each tube should be approximately 20ul. Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture to the bottom of the tube.Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermal cycler with a heated lid.Use ~2ul of the digest (25ng of DNA) for ligations. Our Experience R&D Leader PipettingIt took us a while to figure out which restriction enzymes should be used to cut which parts; those difficulties and eventual solutions will be detailed in the space provided for the Ligation lab. The restriction digestion lab was completed smoothly, except for some issues with calculating appropriate molarity and volumes for components in the lab procedure. For example, we were not exactly sure how much distilled water to pipette into the reaction tube so we decided to pipette 6 ul of dH20 based on the protocol on the iGem website. Unfortunately, we may have used too little dH20, as it was difficult to retrieve the restriction digests for the ligation lab. Ligation Materials Restriction digests produced from previous labT4 DNA ligase bufferRehydrated DNA from DNA Kit well platesT4 DNA ligaseThermal cycler Procedure Add 3ul of promoter digested fragment.Add 3ul of RBS digested fragment.Add 1 ul T4 DNA ligase buffer.Add 0.5 ul T4 DNA ligase.Add water to 10 ul.Ligate 16C/30 min, heat kill 80C/20 minRepeat steps 1-6 with the Aldehyde dehydrogenase, GFP, and terminator restricted digests.Repeat steps 1-6 with the Alcohol dehydrogenase, GFP, and terminator restricted digests.Repeat steps 1-6 with the resulting ligated parts and 2ul of digested plasmid backbone (Promoter/RBS and ORF/GFP/terminator and pSB1C3).Transform with 1-2ul of productThe below diagram should further elucidate our procedure in creating the plasmid in interest. Our Experience Student preparing restriction tubeWe were not surprised that when two parts are cut with the same enzyme, they could be successfully ligated together as their sticky ends were complementary. However, we were confused by the reason parts cut with XbaI and SpeI could also be ligated together, because that was how the mechanism seemed to work based on the protocol found on the iGEM website. When we researched further, we realized that both XbaI and SpeI cut to produce complementary sticky ends; only a few nucleotides upstream and downstream of the restriction site differed. By keeping the standard structure of Biobrick parts and the fact that parts cut with XbaI and SpeI could be ligated together in mind, we were able to design a procedure that would ensure proper parts were constructed in the order needed. (Please note that the ORF and the GFP genes could have been switched in the ligation process, as we ligated 3 parts at the same time, but in this case the order should not matter because they will both be transcribed and translated regardless) Transformation MaterialsResuspended DNACompetent CellsIce 2ml tube 42ºC water bathSOC mediaAgar PlatesGlass beads37ºC incubator ProcedureAdd 50 µL of thawed competent cells into pre-chilled 2ml tube. We did not make these but rather took them from the BIORAD pGLO lab kitAdd 1.5 µL of the resuspended DNA to the 2ml tube. Aspirate to mix, keeping the competent cells on ice.Close tubes and incubate the cells on ice for 30 minutes.Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.Incubate the cells on ice for 5 minutes.Add 200 μl of SOC mediaIncubate the cells at 37ºC for 2 hoursLabel two petri dishes with LB agar.Plate 20 µl and 200 µl of the transformation onto the dishes, and spread with glass beadsIncubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. Our Experiences Plated out TransformationsOur transformation procedure differed from the one listed on the registry. Due to our limited time, we opted to do only the absolutely necessary steps and combined practices seen from the pGLO lab. We did not include the RFP control or use antibiotics on our plates. The last time we used antibiotics, our parts failed to grow so we decided to run the risk of overgrowth versus nothing at all. Additionally, since we had extra ligated DNA, We inoculated 4 tubes of competent cells and plated all of them. We could not figure out why the experiment called for 20 and 200 microliters to plate so we plated all at 20. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Project/LaTeam/Susan Liu From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Susan LiuShuning Liu, Susan, is President of the Montgomery iGEM team and is a Junior at Montgomery High School. She found out about iGEM through an MHS alumni attending a university with a phenomenal iGEM program. She is extremely interested in biological and biomedical research and aspires to enter the field of neuroscience in the future. Along with the rest of the iGEM 2014 board, she has worked hard to develop the foundation to the iGEM program at MHS. Aside from iGEM, she is an avid participant on the school Robotics team, Cross Country Team and Slam Poetry Club. In her free time, she likes to rewatch Grey's Anatomy, sleep and has a weakness for blueberry bagels. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Susan_LiTestimonials From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Testimonials Neha Chhugani"For me, this year in iGEM has been a gratifying experience. IGEM has given us the opportunity to act on our passions in ways that I never thought possible. I was sure that we wouldn’t have the opportunity to conduct biological experiments with recombinant DNA or research RNA and protein systems for ourselves until college. However, through the student-run nature of iGEM, we were able to accomplish both these tasks in an atmosphere that realistically resembled the real world. Unlike carefully controlled school labs where we were basically given the answers, iGEM gave us the unique opportunity to experiment in a trial, error, and reflection process. My lab group’s initial failure in the pGLO experiment actually inspired us to research more into bacterial transformation. Then our initial plight in researching a potential solution to Acne Vulgaris after referencing several scientific studies did nothing but motivate us to continue on with our quest. The entire iGEM experience has instilled in me an excitement with learning, synthetic biology, and the research process, as well as an enhanced appreciation for the complexities of life. Even though my future is a mystery, I can now say with certainty, its path is being pulled by the forces of biology." Pankti Kothari“I joined iGEM at the beginning of this year unsure of what to expect besides what I had heard about it from my brother who actually founded the iGEM team at his college and pursued the major of bio-medical engineering as an undergraduate. As a junior, the time to begin college applications, determine what college I would like to attend, but most of all determine what field I would want to enter, ominously approaches. I was lost for a majority of this year as I could not seem to find my ‘passion’. However, I decided to take part in this club to see what my brother found so fascinating about it and also to see if it would help me find a direction for my future. To say the least, iGEM has influenced me in a way I had really hoped it would. Through this club, I have learned so much about the fundamentals of synthetic biology, such as the processes of gene expression. While participating in the pGLO lab I was given a hands-on opportunity to insert a plasmid into the DNA of E.coli bacteria. This lab showed me how delicate biological processes are because our group had unfortunately failed to accomplish this task. However, it was all a learning experience and from that lab I have been more eager to know what went wrong and to redo it successfully. I also learned a lot about the pathogenesis of Acne Vulgaris which is the main issue we are targeting. I found it so interesting that we can use biology to create something that could attack problems everyday humans face and this has allowed me to appreciate science, specifically biology as a whole. I remember one day my team members and I were researching furiously to find a way we could use alcohol dehydrogenase to solve our main problem and after a significant period of disappointment we discovered how wax esters could act as the connection we needed. My friend had jokingly told me, ‘So this is how scientists will feel when they find the cure to cancer’. Of course that is a little different, but that experience got me really excited and it allowed me to immerse myself into the project. Overall, iGEM has truly allowed me to see biology in a new light and has influenced me to possibly continue this field of study in the future." Junlan Lu“iGEM this year has been such a wonderful experience. Even though I still have much to learn, iGEM has exposed me to so many new topics. I really enjoyed the labs we performed, especially the pGLO lab, because it helped me realize that research requires much patience and meticulous planning. The labs also helped me gain an understanding of how bacterial gene regulation differs from eukaryotic gene regulation, and how the products of gene expression can in turn affect regulation. It’s interesting how many of the sciences come together in biology; chemistry is essential for understanding the molecular properties of a system, while physics is essential for understanding energy, the fundamental laws governing existential things, and building the tools used in biological research. Math and computers are also extensively used to model systems and perform calculations. Biology integrates all of these fields and applies that knowledge to understand the implications for a living system. I find this level of collaboration fascinating, and it’s something I want to explore in the future. I have iGEM to thank for broadening my horizon." Mritika Contractor"iGEM at Montgomery High School was a lot fun and I learned many things through my involvement. I didn’t understand things at first, never having taken a formal course in biology, and I failed to see biology for what it was. I at first saw it as a body of boring facts to memorize, however, as we gradually started to learn about enzymes, proteins, and the process of engineering biological circuits, I became fascinated by the junction between biology and engineering. Before I thought biology was just about understanding cellular and organismal processes, but iGEM has allowed me to see biology in a different perspective; biology can be highly innovative and used to make tangible products. The pGlo lab was one of my favorite activities of the year since it was cool to see the numerous colonies that grew and how they glowed under the UV light! Overall, Montgomery iGEM had a great start for its first year and I greatly enjoyed my time! I highly look forward to next year." Bhargav Vemulapalli“When my brother came back from college (or as I like to say, rejoined my household), he came back with many stories and one of them was about his "adventure" of finding a major. In fact, he changed majors three times; however, he did find a major that he was truly in love with - Biomedical Engineering. I decided to pick up one of his biology textbooks and have loved synthetic biology ever since. I have always been a science/math person, but at times, the school curriculum seemed like a series of closed doors. However, when I first encountered synthetic biology through iGEM, I saw how much future potential the field had. I now see biology as a new frontier of knowledge, and, following in the footsteps of my brother, I plan on studying Biomedical Engineering to satisfy my love for synthetic biology and helping others. I attribute my decision to iGEM.” Murray Chen"Joining iGEM as a senior, I was laughed at by a few of my friends, since we would be graduating high school this year. However, it was a decision that I do not regret. After taking AP Biology with Mr. Resch during our junior year, I became extremely intrigued by cellular systems and DNA/RNA pathways. The material we learned as part of the AP curriculum seemed to just barely brush upon the surface of biotechnology and innovations to come. iGEM has allowed me to apply my knowledge and inspired me to independently learn more about the true potential of biology. All in all, iGEM has made a positive impact on my life and pushes me to strive toward higher goals." Aman Kishore"iGEM has significantly impacted my life. While I was new to biology and scientific protocol prior to my involvement, I was able to learn about the cellular properties of bacteria, their interactions in a colony, and how tiny changes at the molecular level can lead to large-scale changes through iGEM. I have also learned about the fundamentals of lab procedure and safety, which I hope to apply in future lab experiments. While I have only scratched the surface, I’ve gained a full appreciation for the vast body of knowledge that science constitutes, and I’m motivated to continually expand my understanding. This has been a highly rewarding experience for me, as I greatly enjoyed having fun with friends, completing labs and learning about biology. iGEM will provide with valuable experience as I pursue my dream of becoming a biomedical engineer." Mayank Kishore"Being a first year club, Montgomery iGem has really grown in leaps and bounds. This club is organized helps all of the grade learn the concepts of bio medical engineering. Learning about how genetic engineering works is much more interesting than I thought it was going to be when I first joined the club. Already being geared towards science and math, this STEM related program has helped me delve farther into my personal interests. In my opinion Montgomery iGem is a great program to teach children a subject which is not covered in school." Avi Boppana"iGEM teaches many valuable laboratory and research skills, including common biotech procedures such as DNA ligation, the proper usage of lab implements such as the micropipette, the formation and evaluation of hypotheses and proper experimental design. I have also realized that scientific research involves a significant amount of time and hard work and often results in failure, but also that it is ultimately very rewarding. Looking at the final results of our lab, I can’t help but smile and feel proud of what we have accomplished. Synthetic biology has huge potential for improving people’s lives, and iGEM provides an experience that is highly relevant for those pursuing careers in science, one that has sparked my passion for synthetic biology." HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/TestimoniaFun! From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Fun! Species of Interest The Montgomery iGEM species. What an enigma. The Montgomery iGEM team species can often be found gathered in a mystical place called the "library". They exhibit a variety of peculiar behaviors including sitting with their textbooks propped open but not actually reading what's in them. Despite the rule banning food and drinks in the library, these young delinquents secretly munch on delectable snacks while hiding from scary librarians.In their free time, the members of the Montgomery iGEM species can be found doing various things, including procrastinating on homework, watching Sherlock, Korean dramas, or anime. They also derive much pleasure in carrying out normal human behaviors such as eating (sweet things preferably) and sleeping (more than 6 hours if lucky). Researchers still cannot understand how these creatures can function with an average of only 5 hours of sleep. Their colleagues in the Robotics and Science Olympiad teams are equally perplexed by this strange behavior.The Montgomery iGEM is a voracious species. Certain members can be found eating all throughout the day. They mostly prey on junk food, such as the Cheetos Fantastix: Chili Cheese and kettlecorn popcorn. On rare occasions, such as birthdays, when the members are feeling particularly productive, they bake and feast on homemade brownies and macarons.A large majority of members of the Montgomery iGEM species participate in track and field. Although this activity frequently results in shin splints and unfashionable sock tans, they still continue to do track. Why they would willingly suffer through countless hours of workouts and abs remains a mystery to researchers...      Our very own Cindy Fan and Robert Dembinski!This past May, the species was traumatized by a plague known as the APs. After the AP plague, a few of the members were infected with a fatal disease called senioritis. Symptoms of senioritis include prolonged daytime naps, indifference, and severe procrastination. Research shows that the average amount of time spent watching TV increased by 200% in those unfortunate enough to be infected. Consumption rate also increased immensely, and the body fat to muscle ratio became alarmingly large. All in all, the Montgomery iGEM species can be characterized as docile, amicable creatures that revel in the fact that science can better their lives. They may sometimes steal your chips and fries, but other than that, they are pretty much harmless (this is an extremely broad generalization, so please note that there are exceptions). They are an intelligent group of creatures (again, this is a generalization). Perhaps one day, they will be able to genetically alter their brains to actually do well on tests. Resch's Angels to Save the Day! Our Photobook Vivian, Cindy, & TiffanyAnkit & RobCindy, Vivan, Tiffany, & Emily Emily & VivianYesh & AmanJunlan & JoannaShannon & JoannaSusan, Vivian, Tiffany, Matt, & AviCindy, Susan, Emily, & Tiffany Sarah & YuanPankti, Neha, & SusanCasey & David HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/FunTeam/Ankit Shah From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Ankit ShahAnkit Shah is a sophomore at Montgomery High School and serves as one of two Underclassmen Representatives on the Montgomery iGEM team. He is very interested in STEM education and research, as he also participates in Science Olympiad. Though unsure of his future career path, Ankit enjoys being part of iGEM because it is very different from other STEM programs in Montgomery and he also is interested in the research aspect. Apart from STEM programs, Ankit is the president of the local Interact Club and constantly gives back to the community. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Ankit_ShaTeam/Vivian Hu From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Vivian HuVivian Hu is a Junior at Montgomery High School and is the Leader of the Research & Development Subteam on Montgomery iGEM. Vivian enjoys biology and is interested in independent research. Prior to her participation in iGEM, she has had laboratory experience at the Rutgers Toxicology lab programs. Outside of iGEM, Vivian is an avid dancer on the MHS dance team, trained in both western ballet and traditional Chinese dance, and is a member of the school's FBLA chapter. Although unsure of a career in science, Vivian still contributes her interest and expertise to the iGEM team. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Vivian_HProject/TheProcessofSelectingtheGene From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Selecting the GeneInitially, we planned to create a cocktail of enzymes that would break down components of sebum (triglyceride oils, wax, squalene, and metabolites of fat-producing cells). However, as a first year team, we decided that choosing one enzyme would be the most practical. Genes that Code for Enzymes to Break Down Components in Sebum Enzyme Gene Triglyceride Lipase: lipases that hydrolyse ester linkages of triglycerides PNPLA2 gene provides instructions for making an enzyme called adipose triglyceride lipase Triacylglycerol Lipase: the fat-splitting enzyme in pancreatic juice; it hydrolyzes triacylglycerol to produce a diacylglycerol and a fatty acid aniontriacylglycerol + H2O diacylglycerol + a carboxylate LIPC gene encodes hepatic triglyceride lipase Squalene epoxidase: an enzyme released by the dermatophyte fungi to break down Squalene. SQLE gene encodes squalene epoxidase HMG CoA Reductase: an enzyme that catalyzes the production of mevalonate from HMG CoA. It is a rate controlling enzyme in the mevalonate pathway that regulates the synthesis of cholesterol and other isoprenoids. HMGCR gene encodes HMG CoA Reductase Lipoprotein Lipase: water soluble enzyme that hydrolyzes triglycerides into two fatty acids. LPL gene encodes Lipoprotein LipaseIn the beginning, we chose to use lipase as the enzyme to eliminate the bacterial food source. Because sebum is composed of 57.5% triglycerides and fatty acids, we decided that lipase which breaks down triglycerides into free fatty acids would be the optimal protein. However, through further research, we learned that when lipase converts triglycerides to free fatty acids, it only exacerbates the inflammatory response. Free fatty acids increase clumping of bacteria, which in turn facilitates the colonization in the duct. When inflammatory mediators penetrate the skin, an inflammatory reaction is initiated. P. acnes lipase (GehA, glycerol-ester hydrolase A) is in fact a virulence factor involved in the pathogenesis of acne. In Shuichi Higaki’s research paper, Higaki asserts that a lipase inhibitor can help treat acne. We contemplated the idea of trying to find a lipase inhibitor; however, few of the inhibitors are proteins. We contemplated using vitamin A, but it proved too complicated with too little standardization of use in accordance to the iGEM registry. In another approach, we thought we could break down the fatty acids before they were absorbed into a cell. The challenge was that acne vulgaris is a multi-factorial disease. Other Potential Targets Target Notes Comedones IL-1 alpha (causes hyperkeratosis). IL-1 alpha receptor antagonist? Androgens Association of acne + high androgen concentration is less consistent.Correlation not clear and is possibly dangerous/harmful to female users Sebum(change in qualitative change in sebum lipids induce alteration of keratinocyte differentiation and induce IL-1 secretion) Target the fatty acidsThe lipolysis of lipids comes from a signal transduction pathway initiated with epinephrine, norepinephrine, growth hormone, testosterone etc that triggers a GPCR that initiates lipolysis. There’s this protein called serum albumin that acts as a carrier of fatty acids. If they’re carried away into the bloodstream, they cannot be fed on by bacteria. Get this, there’s a gene for it too :)Target the squalene.Target the sebaceous linoleic acid (more linoleic acid=less sebum production). P. acnes Use of antibiotics (perhaps we could synthesize one of them) Vitamin A→(hypothesis that hyperkeratosis is caused by local deficiency of vitamin A) It doesn’t look promising because vitamin A is an ingestible molecule found in plants and beta carotenes like carrots Fatty acids Potential enzyme: Serum Albumin, carries fatty acids and hemin through human respiratory system. Coded by ALB gene.While we had all of these possibilities, many were completely unfeasible and unrealistic to try to accomplish. In the end, we decided on Medium Chain Alcohol and Aldehyde dehydrogenase. See our final decision. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Project/TheProcessofSelectingtheGenAttributions From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Contents1 Our Project2 Extras3 Attributions3.1 Our Team3.2 Research and Development3.3 Public Relations3.4 Technology3.5 Operations3.6 Photography Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Attributions Our TeamMontgomery iGEM seeks to spread the message of scientific research, exploration, and discovery as well as inspire students to pursue STEM careers. In order to do this, we try to immerse students as much as possible in all procedures and limit adviser/instructor participation to a bare minimum. This is not to say that our adviser/instructor was not crucial in the successes our team has achieved, but only to say that the vast majority of our accomplishments are the direct result of our members' hard work. Research and DevelopmentOur Research and Development Team, also known as R&D, works directly on the scientific side of our research. From the beginning of our competition season, they have been working hard to devise, and work through a project. From the months of January to February, the R&D team was carefully selected from all of the members based on their expertise and experience in synthetic and general biology. From there, the R&D team has worked diligently on proposing many projects of interest. Between the projects suggested, such as nicotine addiction control, stress relief, photosynthetic facilitation and others, we decided to go with Pankti Kothari's idea of acne reduction.After the R&D team chose the project track and idea, we worked hard to develop mechanisms of action pertaining to acne reduction. As you can see from our project page, we made many changes before finally settling upon our final solution. Our big break came by one day in May, when members Neha Chhugani, Yuan He and Pankti Kothari confirmed that our final proposed method would indeed facilitate the reduction of acne.From there, we began laboratory procedures. Many of our members contributed to these challenges and we are proud to say that our advisor did not have any direct contact with the lab. All procedures were carried out by students, with our advisor guiding us through the process. Thus, throughout the course of this year, our R&D members have become trained lab members in the processes associated with Synthetic Biology. Public RelationsAs a first year club and organization, there were many administrative tasks to be handled in building up the structure. Our Public Relations Team worked hard to make sure that our club could run smoothly and efficiently. Managing all aspects of administration from logo design to financing, our PR Team is an integral part of Montgomery iGEM and also exposes people with different interests to STEM organizations. Our PR and Tech team has been successful in putting together fundraisers for this year's registration and material costs. They are also our liaison to the community and district. We hope that in future years, we can become more involved in presentations through our Board of Education. While we have not significantly advocated our club within our community this year, it is something we plan to work on over the summer and in future years. So far, the Public Relations Team has done a phenomenal job in keeping our team afloat through sending out emails, keeping members updated and managing the team's finances. PR Team member Tiffany Lin was the sole designer and creator of our team logo featured on our website. TechnologyYou may have noticed this extremely clean and modern web design. This is all attributed to the hard work our Technology Team has accomplished. We are proud to say that all of the web elements were hand coded and done solely by the students. In the beginning of the season, we boarded out our website design and had members of the team contribute what their vision of the website would be. After weeks and months of deliberation, coding, and debugging, we have pulled together this final product. With tremendous contributions from Casey Chow, Lucy Zhang, David Fan and Sarah Oh, this website embodies the pride we take in our team and reflects the professional attitude Montgomery iGEM seeks to advocate. We did not consult any professional designers or developers in the creation of the website, and have thus learned a lot through experimenting with the MediaWiki syntax.Below is a list of code libraries we used in our site, many of which helped considerably to allow our team to create our website more quickly and easily. We observe all licenses associated with the software used. jQuery, the go-to javascript library for the web. Pure CSS, a great little CSS framework. Unslider, a great little slider that just works. Pikachoose, the slider we use within content pages. jquery.event.swipe, which gives our gallery a little touch magic on tablets and phones. ie9.js, a compatibility layer for Internet Explorer browsers. toc, for the cute little table of contents scoller on the right. Sticky Sidebar, for making the scroller pin. DTU-Denmark's iGEM Wiki HOWTO, for getting us started on this path. Open Sans, the font we use for much of the website. This font is hosted locally. OperationsOur Operations Team was extremely instrumental in the documentation of events, including our laboratory notebook. The majority of our website content was written directly by members of the Operations Team. This team makes sure that we communicate our achievements, learned lessons and image for all in the iGEM community. In fact, they have played a monumental role in raising awareness of the growing STEM program at Montgomery High School as a result of iGEM. The Operations Team made sure that all articles written on our website were accurate and grammatically correct (a more challenging feat than expected). PhotographyAside from our cited photos taken from the internet, our team photographs are all taken by students on the team. Photographers Emily Ma, Joanna Li and Shannon Lu have worked tirelessly to capture the environment and work that our team has done through digital documentation. Throughout the year, they have captured not only the fun carefree moments of team bonding, but also the scientific lab work performed by our R&D team. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/AttributioNotebook From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Contents1 Our Project2 Extras3 Notebook3.1 November 22nd, 20133.2 December 13th, 20133.3 December 20th, 20133.4 January 17th, 20143.5 January 24th, 20143.6 January 31th, 20143.7 February 3rd, 20143.8 February 20th, 20143.9 March 7th, 20143.10 March 14th, 20143.11 March 14th, 20143.12 March 21th, 20143.13 April 4th, 20143.14 April 11th, 20143.15 May 30th, 20143.16 June 2nd, 20143.17 June 14th, 20143.18 June 16th, 20143.19 June 17-18th, 20143.20 June 19th, 20143.21 June 20th, 2014 Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! NotebookOur documentation of activities, labs, and lectures for each day. November 22nd, 2013 Mission Statement We wanted to implement a new attendance sign-in setup, announce participation consent forms (principle consent form), and organize sub-teams.Meeting Overview We started by electronically taking attendance and introducing the two sub-teams. All members were allowed to choose their sub-teams (may cut down R&D later). We talked about fundraising because of the December 31st deadline of registration and payment.Additionally we came up with a couple of possible fundraising ideas which include: Hot chocolate sales after school on a winter day Snow-hat sales ShopRite BaggingIt was also the last “introductory” meeting. We announced the deadline for leadership applications which were due Wednesday, November 27 and we also reminded members to fill out consent forms.Goals/Tasks for Next Meeting We planned to come up with an idea for fundraising and collect all the participation consent forms.During the meeting, we collected and finished the members' principle consent forms.Other Notes After the last meeting, we encouraged members to pursue financial backing from local sponsors. If they found any potential ones, they had to send iGEM write-ups to them. Thus, we found sponsors quite quickly and focused on small companies in Montgomery.In addition, we checked with the supervisor to see whether he had any ideas or grants. December 13th, 2013 Mission StatementWe aimed to effectively organize the team and keep progressing with the fundraising and sponsorship efforts, as well as assemble the 2013-2014 board members.Meeting Overview We began the meeting by announcing the 2013/2014 board members, followed by a presentation of a video about synthetic biology to help members understand the importance of the team and as well as the basics of biological sciences since they have never been exposed to the knowledge. The team will consist of the board as well as two sub-teams. The board collected member emails and planned fundraising events for the registration fee.Goals/Tasks for Next Meeting We hoped to start teaching the basics of synthetic biology, organizing the ShopRite bagging fundraiser, creating online accounts for everyone, and cleaning up attendance lists. The majority of the meeting was devoted to polishing up last minute issues and building the foundation for the team. December 20th, 2013Mission StatementWe aimed to design a biological circuit from DNA sequence.Meeting Overview We received confirmation from ShopRite about our fundraiser. The college advisor, Gavin, from Purdue University came in to discuss synthetic biology and safety regulations. The R&D team learned how to use micropipettes while the Operations Sub-team discussed fundraiser ideas. .Goals/Tasks for Next Meeting We aimed to organize and execute the ShopRite bagging fundraiser. January 17th, 2014 Mission StatementWe aimed to become more knowledgeable the basics of synthetic biology, including DNA sequencing, promoters, and plasmids. Additionally, we made it a goal to become organized for the future.Meeting OverviewThe meeting began with a presentation on synthetic biology. We discussed cutting down the R&D team, introduced plasmids and gene cloning, and explained the Operations Sub-team structure. We also researched the wiki websites.Goals/Tasks for Next Meeting There were two goals for the meeting: we went over the lab procedure for the pGLO lab and established a bank account for Montgomery iGEM.Other Notes We created a curriculum for the next few weeks, began discussing T-shirt designs and organized a board meeting at the Princeton Public Library on Sunday, 1/19/14. During the board meeting, we finalized the lab practical test and had people do a mock examination to gauge its level of difficulty. January 24th, 2014 Mission StatementWe aimed to teach members about the pGLO Lab and the specifics of the procedure.Meeting Overview We explained how the iGEM Google Drive folders worked and showed a bacterial transformation video. We ran a Q&A session. Each member had to demonstrate knowledge of the steps of the pGLO lab through the laboratory practical.Goals/Tasks for Next Meeting We will understand the process of bacterial transformation and introduce laboratory techniquesOther Notes We announced the t-shirt design competition to the members, saying that the deadline was February 14th. The t-shirt design winner would receive a free t-shirt. January 31th, 2014 Mission Statement We wanted to go over the pGLO lab technique and properly inform team members of all safety procedures.Meeting Overview We reviewed the aseptic technique and the pGLO lab and went over how to clean a Bunsen Burner (if metal use plastic loop).Goals/Tasks for Next Meeting We accomplished the pGLO lab successfully without contaminating the chemicals and learned to make our own plasmids to put into bacteria. February 3rd, 2014 Mission Statement We continued preparations for the pGLO Lab.Meeting Overview Members reviewed how the the lab procedure would be conducted and also reviewed how the plasmid would be affected during the process.Goals/Tasks for Next Meeting There were shipping complications with the materials that prevented us from carrying out the lab. Thus, we had to do the lab next meeting. February 20th, 2014 Mission Statement We changed the dynamics of iGEM by facilitating team bonding activities. Furthermore, we administered the lab practical.Meeting Overview We collected t-shirt money which provided the funding for our materials, and Robert gave a speech on dynamics which gave constructive criticism and promoted better cooperation within the team. There was a Q&A session about the lab practical and iGEM.Goals/Tasks for Next Meeting We prepared for pGLO lab once again by gathering materials and reviewing the lab.Other Notes It should be noted that ShopRite bagging is taking place in March. We also needed to take the yearbook soon and noted that the meetings were mandatory. March 7th, 2014 Mission Statement We finished the pGLO lab today. Additionally, we also organized the Operations team by designating tasks among logistics, tech, and science factions.Meeting Overview We formatted the team organization, finalized acne project description, and administered the practice lab documentations. Board member profile pictures for the team Wiki page were also taken.Goals/Tasks for Next MeetingWe started DNA ligation lessons and researched more on the acne project.Other NotesWe started website structure, headed by the Systems Manager. March 14th, 2014 Mission Statement We played an icebreaker to unify the team, which included bonding over common interests, went over lab results, and did post-lab calculations.Meeting Overview We went over t-shirt sizes, reviewed team members' names and favorite Pokemon, went over results from pGLO lab, counted number of colonies growing under UV light, and calculated the transformation efficiency.Goals/Tasks for Next Meeting We will learn about DNA ligation.Other Notes Although some students failed because of mislabeling and contamination, they worked hard on the lab. March 14th, 2014 Mission Statement We played an icebreaker to unify the team, went over lab results, and did post-lab calculations.Meeting Overview We went over t-shirt sizes, reviewed team members' names and favorite Pokemon, went over results from pGLO lab, counted number of colonies growing under UV light, and calculated transformation efficiency.Goals/Tasks for Next Meeting We will learn about DNA ligation.Other Notes Lab results turned out to be rather favorable despite the failed attempts by some of the students. March 21th, 2014 Mission Statement We aimed to learn about DNA Ligation.Meeting OverviewWe received a lesson on DNA Ligation from Mr.Resch, detailing the scientific aspects including sticky ends, blunt ends, and restriction enzymes among others. April 4th, 2014 Mission Statement We continued research on project.Meeting Overview We continued researching appropriate promoters, ribosome binding sites, and terminators on the iGem registry.Goals/Tasks for Next Meeting We will receive a lesson on mathematical modeling from Dr. Fishman. April 11th, 2014 Mission Statement We had a presentation of mathematical modeling by Dr. Fishman, our school's AP calculus teacherMeeting Overview Dr. Fishman came to the meeting for an overview of mathematical modeling by introducing differential equations, differentiation, and logarithmic functions.Also, R&D researched further for an enzyme that would break down fatty acids. Goals/Tasks for Next Meeting We hope to further understand mathematical modeling. May 30th, 2014 Mission Statement We wanted to refine the Team Wiki and order the materials for our project.Meeting Overview Board Members and other juniors gathered to finalize the Wiki page as well as take care of materials. We all continued researching promoters and ribosomal binding sites for our project.Goals/Tasks for Next Meeting We continued progressing through the steps needed to finalize the project and synthesize our desired protein. June 2nd, 2014 Mission Statement We administered t-shirts, and went over ideas and further planning.Meeting Overview We distributed team t-shirts, went over upcoming deadlines, brainstormed poster ideas, and began planning the layout for the poster.Goals/Tasks for Next Meeting We will assign tasks for the poster to different members as well as begin working on the poster. June 14th, 2014 Mission Statement We aimed to make as much progress on the Wiki as well as the poster.Meeting Overview Board Members worked on adding information and editing the wiki. We also streaked bacteria onto agar plates to grow so that we will have more of the plasmids inside them. We were also able to continue to add content to the poster.Goals/Tasks for Next Meeting Our first priority was to continue to finalize the Wiki and poster. June 16th, 2014 Mission Statement We grew culture cells and did lab prep in addition to working on the team website.Meeting Overview Team members grew cells in culture tubes and worked in parallel with starting the mini prep. We know that this is out of protocol but we decided to take a two-pronged approach in order to increase efficiency as deadlines are rapidly approaching. Additionally, other team members worked on sorting through team photos and editing/contributing to the team wiki. In terms of the presentation, voice-overs were recorded today and we began editing the final presentation.Goals/Tasks for Next Meeting We will continue with the mini prep and move on to restriction digestion if possible. June 17-18th, 2014 Mission Statement Our goals are to both finish the lab, and the poster/video presentations.Meeting Overview Team members finished the mini prep, restriction digestion, ligation,and transformation. We have finally achieved our finished product!We're very excited to test it out tomorrow. Additionally, members completed our poster while our video editing team has finished the video presentation. We have begun preparations to ship the poster out by this weekend and are excited to finally showcase all the work we've done this year!Goals/Tasks for Next Meeting Study final project by testing it under UV light. Additionally, we will ship the poster next meeting. June 19th, 2014 Mission Statement Our goal was to finish up the transformation, incubate the cell cultures and make progress on the video/poster.Meeting Overview Team members finished the transformation and even dedicated their time during final examinations. We have finally achieved our finished product and it only pends evaluation the next day. Now we turn our sights onto the website and poster presentation, in order to finish up all work for the seasonGoals/Tasks for Next Meeting Study final project by testing it under UV light. Additionally, we will ship the poster next meeting. June 20th, 2014 Mission Statement Our goals are to evaluate the cell cultures and make our final conclusions for the seasonMeeting Overview Team members evaluated the growth of cell cultures under UV light. Unfortunately, we found that our mechanism was ultimately a failure, as not a single colony displayed the GFP we had put as the signaling moleculeGoals/Tasks for Next Meeting Finish up the poster and presentation with correct information. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/NotebooProject/MathematicalModel From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Contents1 Our Project2 Extras3 Mathematical Model3.1 Purpose3.2 Defining our Model3.2.1 Assumptions3.2.2 Parameters3.3 The Math3.4 Enzyme Kinetics Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Mathematical Model PurposeIn order to obtain optimal results from each one of our experiments, the Montgomery iGEM team has undertaken the challenge of using differential calculus concepts to model the rates of growth of protein. First, we seek to model the theoretical amount of proteins expressed by the bacteria's gene. The quantity of protein yielded by the bacteria is especially important due to the exacting amount of protein necessary to effectively alter enough sebum molecules and cut off P. acnes's nutrient source. Because enzyme productivity is dependent on protein output, we will be able to model the enzyme kinetics, the rate at which conformations of sebum components are broken down. Defining our Model AssumptionsWe assume that bacteria follows logistical growth. The bacteria expresses the gene to produce the protein. In the actual experiment, we failed to effectively transform the bacteria. However, because this may be due to human error, we assume that the recombinant plasmid is effectively transformed into the bacteria. Thus the model demonstrates purely theoretical results based on optimal lab procedure. We assume that we are dealing with a single-step enzymatic reaction. ParametersDoubling time of bacteria: 30 minutesThe maximum rate at which DNA is transcribed in to mRNA in E.coli, as determined by the 2009 Beijing iGEM team is 4200nt/min. The average speed of translation in E. coli is 2400Aa/min, as calculated by the 2009 Beijing iGEM team. The MathThe rate of transcription is constant and independent of the concentration of any species involved. The rate of translation (from mRNA to protein) is dependent on the amount of mRNA. We must determine the rate constant, k, experimentally. We must also take into account the degradation of mRNA and protein. The equations become: Enzyme KineticsEnzymes are proteins that catalyze chemical reactions. The typical enzyme (E) converts a substrate (S) into a product (P), as exemplified by the chemical formula: The rate at which the concentration of a product increases in proportion to the concentration of reactants. Thus:In which:Enzyme (E): DehydrogenaseSubstrate (S): LaclProduct (P): Altered wax ester.By increasing the enzyme concentration (the dehydrogenase) while keeping the substrate concentration constant, we can increase the initial rate (v) at which the product is formed (which is the altered sebum components rendered unfit to be P. acnes's nutrient source). The optimal result from our bacterial transformation would be to create a machine that can express the creation of the enzyme that increases the rate until the addition of more substrate no longer has an effect and the enzyme is saturate with substrate. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Project/MathematicalModeProject/ErrorAnalysis From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Contents1 Our Project2 Extras3 Error Analysis3.1 Laboratory Problems3.1.1 Time Management3.1.2 Growing Cultures3.1.3 Contamination3.1.4 Failed Experiment3.2 Administrative Problems3.2.1 Finances3.2.2 Team Structuring Error AnalysisAs a first year team, we have run into many problems throughout the course of the season from financial hardship to laboratory mistakes. Here we share our experiences throughout the year in hopes that other teams can learn from our mistakes and have an easier time through the season. Laboratory Problems Time ManagementThe lab procedures took much longer than we would have expected. In inspecting the equipment that we had, we realized that we could not ligate at different times since our thermocycler only has one setting. Additionally, when we ordered parts on the registry, we did not follow up until almost two weeks later. At that point it was already June. Therefore, many of our lab procedures were as basic as possible and we skipped many of the "optional but recommended" steps. Growing Cultures Team member streaking culturesIn our first attempt to culture the bacteria, we made the mistake of trying to culture the bacteria on a Ampicillin and Kanamycin LB Agar plate. We were following the protocol from the 3A assembly but forgot that the parts we ordered were not exactly identical to the parts in the distribution. We waited two or three days for the bacteria to grow before we began analyzing what we had done wrong (since expected growth should happen within 24 hours of inoculation). Upon closer analysis, we realized that the parts we had streaked were not antibiotic resistant and repeated the inoculation on plain LB Agar plates. Two days later, we had our bacteria. Contamination Contaminated Bacterial Growth)We also need to take into account the contamination that occurs during lab procedures. Although we follow all safety cautions to maintain accuracy of our lab results and keep ourselves safe from the deleterious chemicals, the environment where we perform our lab experiments is not as pristine as a professional laboratory, so errors due to contamination were unavoidable. Since our facility is seldom open over the weekends, our competent bacteria grew extremely rapidly throughout the days we were absent. When we came back to check on the growth, there were various samples of mold inside the plates. This demonstrates the fact that the plates were initially contaminated. We bleached the contaminated plates out for disposal and were more careful in our future endeavors. Failed Experiment Our finished and grown culturesUltimately, this year our biobrick mechanism failed to deliver the results we were hoping for. We attribute this to a variety of reasons. The biggest reason lies in the fact that we were extremely pressured for time, since our parts did not come when we expected them to. To help this, we cut many procedures as short as possible, and tried to get everything done in a timely fashion. Our final transformed bacteria did grow but did not display any of the characteristics we predicted for our mechanism. First, the most obvious reason comes from the fact that the lacl promotor may not have actually been stimulated because we used plain LB Agar plates. Also, since we were ligating 5 parts and the plasmid together, we had to devise our own restriction digest mechanism that may or may not have actually worked. Finally, our lab was also not carried out in a controlled, sterile and professional setting. Therefore, much of our errors could have stemmed from the fact that we were inexperienced with the equipment and materials. Administrative Problems FinancesBecause we are in the nascent period of the STEM program, we did not receive adequate pecuniary support from our school district. In order to earn money to purchase the registry parts and lab equipment, we reached out to our local supermarkets and stores. As we informed our community of our iGEM project, we asked for their donations to contribute to our cause. One way we earned money was through our Shoprite fundraiser. Members of Montgomery iGEM volunteered their time in two hour shifts and helped bag for our local Shoprite. However, our mistake lay in our failure to find a corporate sponsor or similar sponsor. The support would have given us much more time to worry about the scientific side of iGEM and allowed us to purchase more advanced equipment and accommodate for multiple reactions. Team StructuringMany of our team leaders have had a background in the FIRST robotics program. The FIRST program tries to involve as many students as possible, making for large teams. When the same concept was applied to iGEM, we encountered numerous problems. The iGEM club organization works best with anywhere from 8-15 people. Our 35+ person team, while supported the spread of STEM, was slightly less than optimally productive. For future years, while we want to try to keep everyone involved, we seek to find different roles for everyone on our team. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Project/ErrorAnalysProject From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Contents1 Our Project2 Extras3 Project Overview3.1 The Role of P. Acnes in Acne Vulgaris3.2 Description3.3 Enzyme/Gene Selection: The Process3.4 Final Design3.5 Results and Conclusion Project Overview The Role of P. Acnes in Acne Vulgaris P. Acnes (PHIL 3083, from the CDC)The bacteria Propionibacterium acnes is responsible for Acne Vulgaris, more commonly known as acne. Although P. Acnes is not an issue for most people, there are certain virulent strains that exacerbate acne inflammation. The bacteria thrives in the presence of lipids, specifically sebum, which serves as the primary source of the bacteria's energy and sustenance. An increase of sebum production attracts bacteria into hair follicles, where the bacteria can multiply and colonize, resulting in an inflammatory reaction of cysts and pustules, which can lead to acne scars if not treated properly. DescriptionFor the 2014 Competition season, Montgomery_Cougars_NJUSA aims to create a better solution for acne reduction. Acne is a problem that affects nearly 80% of American adolescents. Unfortunately, this widespread problem does not have a concrete solution. Leading acne medication companies, such as Proactiv, use benzyl peroxide and/or salicylic acid alike in their products, in which the harsh chemicals often shed the skin. Although this method may unclog pores from the patient’s face, it is moderately painful and is not always 100% effective. Therefore, to combat this widespread ailment, Montgomery iGEM has decided to take a different approach.In an effort to create a less invasive treatment, we decided to design an enzyme to break down the sebum on human skin, thereby reducing the bacteria's source of energy and ability to colonize. Because sebum is made of triglyceride oils, wax esters, squalene, and metabolites of fat-producing cells, we sought a group of enzymes that would break down these components. Through our research, we have observed a definite positive correlation between patients with acne and presence of triglycerides and wax esters. Our mechanism ultimately focused on targeting these substances. In reducing these substances, we effectively deplete the food that the P. acne bacteria needs to thrive and elicit the inflammatory response we perceive to be acne. Enzyme/Gene Selection: The Process ResearchWhen we first started our product research, we had many complications to address. At first, we planned on creating an antibiotic that would directly combat the bacteria. Once we realized that designing a bacteria, namely E. Coli, to synthesize a protein to break down other bacteria was much beyond our scope, we decided to focus on what exactly makes the bacteria thrive. Thus, we moved onto examining the sebum within the skin that allows bacteria to grow and indirectly create the inflammatory response known as acne. As mentioned before, sebum is composed of wax esters, triglycerides, squalene among other lipids and fats native to the skin of humans. We hypothesized that if we limited the food supply for the bacteria, the persistence of acne would decrease.Finding a way to break down the components of squalene was a challenge in itself. Our research revealed that sebum is primarily broken down through beta oxidation, which involves the removal of hydrogen by dehydrogenase enzymes. However, it was extremely difficult to find a suitable dehydrogenase for our mechanism. The most prevalent component of sebum is sapienic acid, which plays a key role in the formation of acne. Sapienic acid is difficult to break down because of its carboxylic functional group, which is rarely targeted by the body's natural array of enzymes. We unsuccessfully tried to find an enzyme to target carboxyls. Our breakthrough came in late May, when our research brought us to the conclusion that another component of sebum, wax esters, could be broken down by the method we had previously proposed: alcohol dehydrogenase. Wax esters are a major component of the lipids produced by skin cells, and are composed of a fatty acid and a fatty alcohol, connected by an ether linkage. The fatty alcohols each contain a single alcohol functional group, which is broken down by the alcohol dehydrogenase. Theoretically, this chemically alters the composition of the wax esters in sebum, which creates an alternative method to reduce acne production in humans. Final DesignOur team decided to create two biological systems, each designed to produce a specific protein. The two engineered plasmids contain the same promoter, RBS, and terminator; the only part that differs is the actual Protein Coding Region. One plasmid will produce the Medium Chain Alcohol Dehydrogenase; the other will produce the Medium Chain Aldehyde Dehydrogenase. The function of both of these proteins is to change the shape of the wax esters, a central component of sebum, and thus limit the P. acnes' nutrient source. Wax esters contain both alcohol and aldehyde groups, which are the targets of the enzymes that we plan to express. Our main purpose for both of these plasmids is to harvest a protein, so we needed to choose parts that would ensure a maximum efficiency rate. The promoter chosen is the naturally found LacZYA operon in E.coli (BBa_R0010). Because it is an inducible, multiregulated operon (positive CAP control and Lacl negative control), using this promoter can give us a higher level of regulation over the plasmid. We may be able to control transcription efficiency rates by changing the environment that the E.coli colonies grow in. The Elowitz RBS (BBa_B0034) is the definition of efficiency 1.0, as tested by team Warsaw in 2010. We chose this RBS to maximize the translation efficiency rate that will produce the protein of interest. We chose a double terminator (BBa_B0015) that would ensure high efficiency in terminating transcription and less error when we harvest the protein.Promotor: BBa_R0010RBS: BBa_B0034Protein Coding Domain: BBa_K398005 or BBa_K398006Signal Protein: BBa_E0040Terminator: BBa_B0015 Results and Conclusion Our completely grown transformed bacteria)While we were extremely hopeful that our lab would work, our bacteria unfortunately did not glow. This indicates that our mechanism was ultimately unsuccessful. When we checked on our bacteria cultures, we found that the bacteria did indeed grow but did not display the correct reaction when viewed under a UV light. We can only conclude that our bacteria either did not accept the plasmid, the plasmid was ligated incorrectly or the promoter was not stimulated. Our suspicions lie in the fact that our promoter was not stimulated. We realized that the lacl regulated promoter would not be initiated in the LB Agar plate. However, due to our limited time, we could not obtain what was needed for our project. Also, during our transformation process, we had to forego the RFP control in order to finish the experiment in time. Our ligation procedure was much more complex than the procedure given, so we had to experiment and create our own ligation process. All in all, our first year laboratory was very shaky and our lack of experience forced us to take many risks. The lack of professional laboratory setting could also have contributed to our failure to create our desired protein.We hold in our belief that our project, when done correctly could have remedied the acne that plagues thousands of adolescents in America. We hope that next year, our endeavors will be more successful. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/ProjecReferences From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! ReferencesAcne Vulgaris [Image]. Retrieved June 5, 2014, from http://smartgirltips.com/acne-vulgaris-definition-causes-and-treatment.htmlBhatia A, Maisonneuve JF, Persing DH. PROPIONIBACTERIUM ACNES AND CHRONIC DISEASES. In: Institute of Medicine (US) Forum on Microbial Threats; Knobler SL, O'Connor S, Lemon SM, et al., editors. The Infectious Etiology of Chronic Diseases: Defining the Relationship, Enhancing the Research, and Mitigating the Effects: Workshop Summary. Washington (DC): National Academies Press (US); 2004.Death Note Ryuk [Image]. Retrieved June 14, 2014, from: http://deathnote.wikia.com/wiki/File:Death_Note_Ryuk.jpgEating is Important [Gif]. Retrieved June 15, 2014, from: http://blog.icpl.org/channel/teens/page/6/?print=1Higaki, Shuichi. "Lipase Inhibitors for the Treatment of Acne." Journal of Molecular Catalysis B: Enzymatic 22.5-6 (2003): 377-84. Print.Lee WL, Shalita AR, Sunthralingam K, et al. Neutrophil chemotaxis to Proprionibacterium acnes lipase and its inhibition. Infect Immun. 1982;35:71–78.Lynch, Kevin (Blogger). (2013). Pad Thai [Photograph], Retrieved June 14, 2014, from: http://www.closetcooking.com/2008/11/pad-thai.htmlNewton, Elaine (Photographer). (2012). [Untitled photograph of homemade vegan kettle corn with coconut oil], Retrieved June 14, 2014, from: http://www.therisingspoon.com/2012/11/homemade-vegan-kettle-corn-with-coconut.htmlOberemok, Steve S., and Alan R. Shalita. "Acne vulgaris, I: pathogenesis and diagnosis." CUTIS-NEW YORK- 70.2 (2002): 101-105.One Does Not Simply, Avoid Senioritis [Image]. Retrieved June 14, 2014, from: http://www.troll.me/2012/04/10/uncategorized/one-does-not-simply-avoid-senioritis/Ozer Arican, Ergul Belge Kurutas, Sezai Sasmaz. Mediators Inflamm. 2005 December 14; 2005(6): 380–384. doi: 10.1155/MI.2005.380Picardo, Mauro, Monica Ottaviani, Emanuela Camera, and Arianna Mastrofrancesco. "Sebaceous Gland Lipids." (n.d.): n. pag. NCBI. Web. 31 May 2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835893/#__ffn_sectitle>.[Untitled photograph of a box of colorful macaroons]. Retrieved June 14, 2014, from http://macaroons-plate.blogspot.com/You Who Came From the Stars 6 [Image]. Retrived June 14, 2014, from http://www.koreandrama.org/?p=31118Additional team photos taken by members Emily Ma (Class of 2015), Joanna Li (Class of 2016) and Shannon Lu (Class of 2016). HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/RefereAcknowledgements From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Acknowledgements First and foremost, we would like to thank our college advisor Gavin Kuziel for introducing us to iGEM and helping to get our team started. Without his initial support, iGEM would not have existed. We also extend our gratitude toward our advisor Mr. Resch, who was kind enough to supervise the team every week. He was constantly filling out our paperwork, and we are grateful for his time commitment. He also provided the room and lab equipment with which we worked during the labs, and for that we are truly grateful to be able to have our own meeting space. Additionally, we appreciate the help and expertise of Mr. Pendleton and Mr. Sullivan, whose advice facilitated our introduction into laboratory research. Because of the guidance of the aforementioned advisors, we greatly enjoyed our first ventures into synthetic biology amd gained a lot of insight into scientific research. Many of us hope to pursue careers in science and our experiences in iGEM will be extremely valuable as we strive to make a difference in people’s lives - science has great potential for solving the world’s problems and iGEM has allowed us to begin to realize its capacity. Gavin Kuziel Mr. Resch Mr. Sullivan, Science Supervisor HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/AcknowledgemenTeam/Sarah Oh From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Sarah OhSarah Oh is a junior at Montgomery High School and has been an active participant in STEM programs since her freshman year. She enjoys biology and research and joined iGEM to further pursue her passion for science outside the classroom. During the summer, she volunteers at Schepens Eye Research Institute in Boston and has contributed to laboratory research. On the Montgomery Team, she serves as the Librarian and contributes to the website with her previous HTML knowledge from Tumblr. In school, she participates in Science Olympiad, NHS, and Track. Apart from academics, she enjoys listening to Korean Pop music, eating food, and playing sports. Currently, she is watching a Korean variety show called "Running Man" and highly recommends it. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Sarah_OTeam/Avinash Poola From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Avinash PoolaAvinash “Avi” Poola is currently a junior at Montgomery High School. Aside from being a part of iGEM, he also participates in FIRST Team 1403 Cougar Robotics and the Technology Student Association chapter at Montgomery. He is eager to study biomedical engineering in college and this has sparked his interest in joining Montgomery iGEM. In his free time, he enjoys playing basketball and watching the TV show Modern Family. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Avinash_PoolTeam/Joanna Li From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Joanna LiJoanna Li is a sophomore at Montgomery High School and serves as one of two Underclassmen Representatives on the Montgomery iGEM team. She is very interested in science and is involved in Science Olympiad. She joined iGEM because she wanted to contribute to the research that fellow students were pursuing and is eager to apply the concepts she has learned in school to real world problems. Outside of iGEM, she enjoys playing on the girls Volleyball team, reading through HONY posts, and eating. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Joanna_LTeam/Gavin Kuziel From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Gavin KuzielGavin Kuziel is an alumnus of Montgomery High School and currently attends Purdue University in Indiana. He was the first on the team to hear of iGEM in his own school, and contacted Susan Liu in the beginning of the 2013-2014 season. He works as Montgomery iGEM's college advisor, giving guidance from his experiences in college laboratories and biology. Outside of iGEM, Gavin participates in Habitat for Humanity, research in a professional laboratory, and is also an alumnus the Montgomery Robotics Team. Gavin is pursuing a career in biomedical engineering and enjoys the innovation and curiosity science presents. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Gavin_KuzieTeam/Cindy Fan From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Cindy FanCindy Fan is a student in her third year at Montgomery High School. When presented with the opportunity to apply what she learned in her high school biology class to her extracurricular activities, she became interested in iGEM. Although biology is not necessarily her forte, she always welcomes new challenges. Aside from being a member of the team, she also enjoys food indulgence, painting, running, and escaping her problems through sleep. In her free time (which really should be known as procrastination sessions), she can be found enthusiastically browsing shows on BBC. Currently, she is watching BBC's Orphan Black and highly recommends it. When Cindy is actually participating in educational pursuits at school, she can be found dillydallying around at MHS's Amnesty International club, NHS, and NAHS. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Cindy_FaTeam From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Our TeamThe Montgomery iGEM team is a relatively young team, comprised of mostly juniors, sophomores, and freshmen. What we lack in seniority, we make up for in passion and dedication! When starting up, our club divided members into different sectors, which would function together like a well oiled machine. The four sectors in which members specialized were Research and Development, Operations, Tech, and Public Relations. As our very first year as a team progressed, we discovered that having all members contribute to each sector equally was far more successful than restricting each individual to only one. Leadership Cindy FanClass of 2015Leader of Operations Susan LiuClass of 2015President Robert DembinskiClass of 2015Vice President Vivian HuClass of 2015Leader of R&D Emily MaClass of 2015Secretary Chris ReschAdvisor Sarah OhClass of 2015Librarian Casey ChowClass of 2015Systems Manager Lucy ZhangClass of 2015Reporter Avinash PoolaClass of 2015Community Liaison Ankit ShahClass of 2016UC Representative Joanna LiClass of 2016UC Representative Gavin KuzielClass of 2013College Advisor Membership R&D Operations Public Relations Tech Yuan He Kevin Shen Matt Durik Jonathan Baptiste Neha Chhugani Sushil Bhandaru Tiffany Lin Abhinav Gurrala Dylan Lynch Yesh Datar Shannon Lu David Fan Pankti Kothari Ankur Metha Bhargav Vemulapalli Shawn Bhattacharya Mayank Kishore Nikhil Maganti Ian Lee Srikar Tallavajala Pranav Reddy Murray Chen Tejas Jha Mritika Contractor Junlan Lu Aditya Mehta Avinash Boppana Aman Kishore Ritwik Sanyal ContactingOur team is readily available at [montgomeryigem@gmail.com]. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/TeaSafety From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Safety iGEM Safety Questions 1. Would any of your project ideas raise safety issues? The use of goggles in a lab environment is mandatory in our school.Montgomery High School's iGEM team works out of a high school laboratory with Containment Level 1. We use non-pathogenic strains of E. coli and do not work with any hazardous chemicals. Nevertheless, we ensure that all students tie up any long hair and wear gloves, safety goggles, pants, and closed-toed shoes during lab activities. All students underwent a lab safety demonstration in early January and practiced streaking bacteria, pipetting, etc. to ensure the use of proper technique. In regard to our lab environment, the classroom we use has multiple showers, a fire extinguisher, an eye-wash station, multiple sinks, and a first aid kit.Our project is contained from public use, and is unattainable for any purpose other than pure research. In an effort to better the current acne medications we only want researchers to have access to this mechanism, and therefore strive to keep the bacteria secure. 2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? All science rooms in our school are equipped with an emergency shower and eye washing station.Unfortunately, we have not created any new BioBrick parts this year. However, our biobrick parts are all from the iGEM registry and are quality controlled to behave as listed. 3. Is there a local biosafety group, committee, or review board at your institution? There is no biosafety group at our high school. Our iGEM team is responsible for safety. As such, we have completed a mandatory lab safety quiz as well as practiced with certain equipment to ensure mastery.We follow the World Health Organization Laboratory safety regulations: http://www.who.int/csr/resources/publications/biosafety/en/Biosafety7.pdf We use gloves to protect both the team members and sterility of bacteria 4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?In the future, we hope to have access to input from an experienced synthetic biologist or past iGEM advisor. With this aid, we can avoid experimental and lab error as well as ensure safety. Perhaps each team could be assigned an "iGEM headquarters adviser" who would guarantee the validity and security of their team's project. This would both increase the organizations involvement with the teams, as well as provide another expert opinion on top of each team's faculty adviser and college adviser. Disposal A team member spraying cultures with bleach. A team member spraying cultures with bleach.We spray contaminated cultures with bleach to sterilize before disposal so that it does not contaminate the rest of the garbage. This insures that in the off chance that the illegitimate biobrick is harmful, the bacteria carrying it will not grow and proliferate into a biohazard. Even when there is no visible evidence of contamination, we take precautions to decrease the risk of unpredicted difficulties. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/SafetProject/PathogenesisofAcne From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Pathogenesis of AcneAcne Vulgaris is caused by four main factors:1. Blockage of sebaceous follicles. Sebum BuildupSebaceous follicles are exocrine glands found in the skin of humans, concentrated within the facial areaSebum is produced to keep the skin moisturized and acts as a the first immune system barrierThe oils and lipids in skin build up around sebaceous follicles and irritate the emerging hair shaftThe extreme growth and build up creates cysts and nodules that are visually unappealing and painful when irritated2. The epithelial cells in sebaceous follicles undergo abnormal desquamation (shedding of the skin). Comedogenesis is the obstruction of sebaceous follicles due to greater cohesiveness of the follicular epithelial cells and sebum. As keratinous material gathers in the sebum filled follicle, the wall thins and swells. While comedogenesis is a natural process in humans, the overstimulation through androgens and other hormones contributes greatly to the proliferation of P. AcnesKeratinocytes actively exacerbate acne formation and sebum production3. The anaerobic, commensual bacterium Propionibacterium acnes proliferates in a lipid rich environment, such as when there is an overproduction of sebum. Excessive sebum production is caused by androgenic stimulation of sebaceous glands. 4. P. acnes produces proinflammatory mediators that cause microcomedones to inflame and become papules, pustules, and nodulocystic lesions. A microcomedone is the precursor to acne, an accumulation inside a pore, causing the pore to be plugged by dead skin cells and sebum. Lipases, proteases, and hyaluronidases, produced by the bacteria, act as proinflammatory products. Acne can manifest itself on the skin in a variety of manners, from mild comedonal form to severe inflammatory cystic on the face, back, and chest. When androgen is released throughout the body, it precipitates activity in the sebaceous glands, promoting comedone and seborrhoea formation, propagating the inflammatory P. acnes. P. acnes is actually present in the pilosebaceous ducts as an anaerobe. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Project/PathogenesisofAcnTeam/Lucy Zhang From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Lucy ZhangLucy Zhang is a Montgomery high school junior who enjoys watching anime. She is an editor of her school newspaper and Literary magazine. She does not particularly like school, but is obligated to go due to insurmountable reasons. Lucy is somewhat interested in biology, which is a statement not to be taken lightly when taking into account her lack of interest in many other things. One thing is certain: she is interested in money. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Lucy_ZhanTeam/Chris Resch From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Chris ReschMr. Christopher Resch is the Advisor for Montgomery iGEM and is also the school's AP Biology teacher and has been teaching for the past 8 years. After attending Susquehanna University for Undergrad, he went to TCNJ for Graduate school. In MHS, Mr. Resch also runs the Red Cross Club, Environmental Club, and Gamer's Guild. Aside from being an integral part of the MHS extracurricular program, Mr. Resch's hobbies include woodworking and watching Sci-Fi TV shows. To relax after a year of AP preparation, Mr. Resch and his AP classes take "field trips" to the school courtyard to garden organic vegetables. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Chris_RescTeam/Casey Chow From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! << About Our Team Casey ChowCasey Chow is a Junior at Montgomery High School and serves as Montgomery iGEM's Chief Technical Operator. Casey's skills in web design, programming, and conveying information effectively motivated him to join the iGEM team. Outside of iGEM, Casey enjoys participating in student-government type clubs such as MUN and YAG and also uses his technological savvy for the Montgomery Robotics Team. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Team/Casey_ChoProject/RegistryParts From 2014hs.igem.org Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Registry Parts Name Type Description Designer(s) Length BBa_R0010 Regulatory Promoter (lacl regulated). An inverting regulator sensitive to Lacl and CAP. Antiquity 200 BBa_B0034 RBS Based on Elowitz repressilator Mahajan,Marinescu, Brian Chow, Wissner-Gross and Carr 12 BBa_K398006* Coding Medium-Chain Aldehyde Dehydrogenase; functions as an octamer. Mathias Voges 1494 BBa_K398005* Coding A medium-chain alcohol dehydrogenase from the thermophile Bacillus thermoleovorans Mathias Voges, Hugo F. Cueto Rojas 750 BBa_B0015 Terminator Double terminator (B0010-B0012) Reshma Shetty 129 BBa_E0040 Coding Green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP jcbraff 720*Note: The aldehyde and alcohol dehydrogenases will both be tested separately as to determine which is more effective. The GFP is the marker that will distinguish this. HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Project/RegistryParFun! From 2014hs.igem.org (Redirected from Team:Montgomery Cougars NJUSA/Fun!) Montgomery iGEM HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! iGEM Our Project Project Overview Pathogenesis of Acne Selecting the Gene Registry Parts Mathematical Model Error Analysis Extras Human Practices Acknowledgements Attributions Testimonials References Fun! Fun! Species of Interest The Montgomery iGEM species. What an enigma. The Montgomery iGEM team species can often be found gathered in a mystical place called the "library". They exhibit a variety of peculiar behaviors including sitting with their textbooks propped open but not actually reading what's in them. Despite the rule banning food and drinks in the library, these young delinquents secretly munch on delectable snacks while hiding from scary librarians.In their free time, the members of the Montgomery iGEM species can be found doing various things, including procrastinating on homework, watching Sherlock, Korean dramas, or anime. They also derive much pleasure in carrying out normal human behaviors such as eating (sweet things preferably) and sleeping (more than 6 hours if lucky). Researchers still cannot understand how these creatures can function with an average of only 5 hours of sleep. Their colleagues in the Robotics and Science Olympiad teams are equally perplexed by this strange behavior.The Montgomery iGEM is a voracious species. Certain members can be found eating all throughout the day. They mostly prey on junk food, such as the Cheetos Fantastix: Chili Cheese and kettlecorn popcorn. On rare occasions, such as birthdays, when the members are feeling particularly productive, they bake and feast on homemade brownies and macarons.A large majority of members of the Montgomery iGEM species participate in track and field. Although this activity frequently results in shin splints and unfashionable sock tans, they still continue to do track. Why they would willingly suffer through countless hours of workouts and abs remains a mystery to researchers...      Our very own Cindy Fan and Robert Dembinski!This past May, the species was traumatized by a plague known as the APs. After the AP plague, a few of the members were infected with a fatal disease called senioritis. Symptoms of senioritis include prolonged daytime naps, indifference, and severe procrastination. Research shows that the average amount of time spent watching TV increased by 200% in those unfortunate enough to be infected. Consumption rate also increased immensely, and the body fat to muscle ratio became alarmingly large. All in all, the Montgomery iGEM species can be characterized as docile, amicable creatures that revel in the fact that science can better their lives. They may sometimes steal your chips and fries, but other than that, they are pretty much harmless (this is an extremely broad generalization, so please note that there are exceptions). They are an intelligent group of creatures (again, this is a generalization). Perhaps one day, they will be able to genetically alter their brains to actually do well on tests. Resch's Angels to Save the Day! Our Photobook Vivian, Cindy, & TiffanyAnkit & RobCindy, Vivan, Tiffany, & Emily Emily & VivianYesh & AmanJunlan & JoannaShannon & JoannaSusan, Vivian, Tiffany, Matt, & AviCindy, Susan, Emily, & Tiffany Sarah & YuanPankti, Neha, & SusanCasey & David HomeTeamProject Project OverviewPathogenesis of AcneSelecting the GeneRegistry PartsMathematical ModelError Analysis Labs Growing CulturesMiniprepRestriction DigestLigationTransformation Lab NotebookSafetyExtras Human PracticesAcknowledgementsAttributionsTestimonialsReferencesFun! Thanks to our team, our advisors, our school, and our community for their continued support.This site is available under a Creative Commons Attribution 3.0 Unported License. Log InUpload File Retrieved from "http://2014hs.igem.org/Team:Montgomery_Cougars_NJUSA/Extras/Fun1275Taipei FhjhTaipei Fu-Hsing Private School Taipei Taiwan Website: www.fhjh.tp.edu.twHigh School001http://2014HS.igem.org/Team:Taipei_Fhjhhttp://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163720140High School1271St Pauls LondonSt Paul's School Barnes London SW13 9JT http://www.stpaulsschool.org.uk/High School401BBa_K1271999http://2014HS.igem.org/Team:St_Pauls_LondonBBa_K1271000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163320140High SchoolLog in   Team:St Pauls London From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:St_Pauls_London1267Consort AlbertaConsort School Consort, AB, Canada https://sites.google.com/a/plrd.ab.ca/consort-school/High SchoolECOS (Environmental COntaminant Sensor)Our goal is to design and develop a construct that will detect levels of Xylene which is well correlated with the presence of carcinogenic benzene/benzene derivatives. During the extraction of petroleum, oil spills are a potential risk, and contaminated soil is harmful to the agricultural industry. Xylene is not overly harmful in the concentrations naturally found, however benzene derivatives related to xylene are, which is why we test for xylene in our lab. Our prototype will involve using alginate beads, which are porous. The matrix is big enough to contain e.coli, but small enough to prevent bacteria from escaping. The XylR transcriptional activator is a protein which in the presence of m-xylene will bind to the Pu promoter which activates the reporter genes. We are attempting to use two reporters: GFP and amilCP. We are evaluating which of these reporter genes are the most convenient and efficient to use.801BBa_K1267999http://2014HS.igem.org/Team:Consort_AlbertaBBa_K1267000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162820140http://2014hs.igem.org/files/presentation/Consort_Alberta.pdfhttp://2014hs.igem.org/files/poster/Consort_Alberta.pdfHigh SchoolLog in   Team:Consort Alberta/attributions From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact Us Attributions Our team would like to thank all of the people and businesses that contributed not necessarily through monetary donations, but that contributed to our team through their donations of supplies, equipment, advice, and expertise that played a huge role in our development. Your support is much appreciated! We would like to thank... the iGEM Headquarters and Competition themselves (for everything that is happening!), Dr. Ron Watson from Wainwright Dental Care (autoclave and alginate), Carla Stringari (for designing our logo and taking photos), Mark Riopel (advice, centrifuge, micro-pipettes, and plastic ware), Consort Enterprise newspaper (media coverage), David Lloyd and Himika Dastidar (for support, advice, and a great starter workshop), Emily Hicks and Robert Mayall (for their support, expertise, a secondary workshop, help late at night with modelling and the presentation to get ready for the Calgary Workshop, and for not minding when we phoned or Skyped them looking for help on multiple occasions), Amrinder Grewal from Lethbridge (gave advice and troubleshooting tips for the website), Miss Kara Strobel (bus driver and great teacher support), geekStarter (for the valuable workshop and cool notebooks), the Peking 2013 Team (for inspiring our alginate beads idea), Daniel McClernon and BioMONTR (supplied us with our PCR machine, spectrophotometer, gel electrophoresis equipment, several micropipettes, and the vortex mixer at a very low cost to us, basically for the price of shipping from North Carolina), and finally on behalf of the students of the team, we would especially like to thank our mentor, supervisor, and cool science teacher Mr. Gerry Bourassa for bringing his love of science to our classrooms along with the start of the iGEM in Consort, which in turn sparked other small towns to start their scientific endeavours as well. Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/attributionsproject From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors|Contact UsOur Project   Introduction   Science  Comparison    Mathematical Modelling    Prototyping   Introduction...     ECOS (Environmental COntaminant Sensor) is a biological sensor theorized to detect the presence of aromatic hydrocarbons by producing an indicator protein when exposed to xylene. We chose xylene as our trigger because it is very well correlated with the presence of other more dangerous and carcinogenic compounds such as benzene and its derivatives. The danger of this class of compounds is in the fact that they can intercalate into DNA and cause a variety of mutations. Our biobrick will, in effect, create different amounts of fluorescent protein in the presence of xylene bound to XylR- which the first portion of our plasmid will constantly produce. As the economy in our rural community is based largely on agriculture and oil and gas industries, oil spills have detrimental effects on the environment, economy, and general health. To be able to detect the presence of these aromatic hydrocarbons on site would greatly benefit all members of our community. This year we will be testing out three different indicators to allow options concerning scale and intensity of colour change. In future years, our project will allow early identification of contamination will facilitate rapid clean-up and minimize health risks to members of our community and to the consumers who rely on the food we produce.The Science...    Our resulting biobrick consists of two main portions. The first portion of our plasmid will be responsible for producing the protein XylR. This is essential because the bonding of m-xylene undergoes a conformational change when bonded to XylR which then allows it to bind to, and positively regulate, the Pu promoter. At a 4:2 XylR to Xylene ratio the bonding starts the production of our reporter protein. The second portion of our plasmid expresses the indicator protein when in the presence of bonded XylR and m-xylene and essentially allow us to know if m-xylene is present in our soil sample or not. The structure of this biobrick is shown below.     Parts: J23100 - which is a constituent promoter. B0034 - the RBS for our XylR gene. I723017 - the XylR coding region which encodes for the transcriptional regulator XylR protein. B0015 - the double stop codon for this sequence. I723020 - This is the Pu promoter. B0030 - RBS.1 strong. Reporter: E0040 - GFP wild-type, will glow when exposed to UV light. K592009 - amilCP, This chromoprotein from the coral Acropora millepora, naturally exhibits strong color when expressed. I732005 - lacZ, Beta-galactosidase cleaves X-gal and ONPG into colorful products. B0015 - lastly another double stop codon. pSB1C3 - Our plasmid backbone which includes the resistance towards the antibiotic, chloramphenicol.Comparison...     Where the comparison portion comes into play is the second part, which will be chosen from any one of three indicators: amilCP which when produced expresses a strong blue color, GFP when under a UV light source fluoresces green or, LacZ which when a substrate is added it cleaves it into strongly colored blue products. We will be looking at which indicator functions with the most efficiency and produces results of the highest clarity.Mathematical Modelling...     Making a sensor involves more than just the creation of the basic biological circuit, as there could be many factors that might improve the performance of the system. An example of these could be the strength of the RBS, the spacing between the promoter and the reporter gene, or the copy number of the plasmid used to house the circuit. While we couldn't nearly address all of these points in our project, we did want to look into the optimization of our system. To do this, we turned to mathematical modelling to create a visual representation of our system.     We worked off of a basic framework published by Koutinas, M., et al.,[1,2] which was modelling the expression of the Ps promoter with XylR induction. The Ps promoter is the natural promoter found with the xylR gene, and the XylR protein can interact with both it and the Pu promoter we used in our project. Due to similarities between the Ps and Pu promoters, we assumed the deactivation rate of the two components were alike. For remaining values, we simply replaced the values of the Ps promoter with known values of the Pu promoter, keeping constants reported for XylR the same. In the end, we created five equations to represent the action of our system. Our model shows our biobrick producing the protein XylR, binding with xylene and the relationship to the output of our respective proteins. Each of our formulas are constructed as a rate in which a concentration or output is given of a specific substance in respect to time- otherwise known as a derivative. We will now expand upon the equations we modified and used.      The first equation above represents the transcription of DNA into the XylR RNA transcript. RNA is the representation of the transcript, with P representing the number of plasmids inside a cell. J23100TC represents the activity of our promoter controlling XylR. ta is the transcription rate of the xylR into RNA, based on the transcription rate of E. Coli and the size of XylR, and KRNAdeg represents the degradation rate in the E. Coli.      The second and third equations represent the inactive and active forms of XylR- or the concentration of XylR that has not bonded to xylene (XylRi) and the concentration of XylR that has bonded to xylene (XylRa). RNA is the substitution of our first equation. tr is the translation rate of our RNA into XylR. rXylR is the oligomerization constant of XylR, rR,XylR is the dissociation constant of active XylR, and xyl in the total concentration of xylene. αXylRi accounts for degradation of XylR.      The fourth equation represents the concentration of xylene present. XylRi and XylRa refer to the concentrations of the inactive and active forms of XylR. rXylR is the oligomerization constant of XylR, rR,XylR is the dissociation constant of inactive XylR and xyl is the total concentration of xylene. αXylRi accounts for the degradation of XylRa, which would release the xylene it held. We multiplied by seconds per hour to get a domain more realistic to our needs.      The fifth equation represents the output of the pu promoter. PuTC represents the activity of the Pu promoter or the output of protein. αpu is the deactivation rate of the Pu promoter. KXylRa is the activation coefficients of the Pu due to binding and nps,a is the hill coefficient for the interaction. β0 and βPS are the basal and maximal expression, respectively, of the Pu promoter.      In order to create an output, we used the program Scilab. By inputting these equations into code we were able to have the program calculate our estimates and graph the results. In the end our output was around 5.5 mPoPS. This is a logical output and tells us our biobrick will successfully over express the protein. We set the output of our model in PoPS due to our system's flexibility when it comes to our reporter protein. This allows us to quickly add in an extra equation to turn our current output into fluorescence, LacZ output, or pigment production without having to worry about editing other parts of our model. Below is a copy of our code that we had used in our program. The resulting graph is the output of our Pu promoter.     While we have yet to apply our modelling framework to the optimization of our sensor, we have created the foundation of a model that can be easily tweaked to test the effect of different variables on sensor performance. Through this we aim to do a sensitivity analysis of the various factors involved in our model in the future so that we can predict what to change for version 2 of our sensor. From there we can return to our model, feeding new data into it to create many future versions of our system, improving performance each time.Prototyping...      ECOS was meant to be more than just a theory; more than just a project to play out at school- we wanted ECOS to have real world applications by the time we finished making our construct. We had one major problem: How were we going to test our product in an immediate and effective manner? To answer this we designed two prototypes to design and test. Unfortunately we were unable to finish the wet-lab portion of this project, but the designing is complete thanks to the Maya program via Autodesk. These prototypes are beneficial because they 1) are cheap to build and 2) relatively quick to test. Here are how they turned out:       PROTOTYPE #1: We designed our first prototype so that it has a positive pressure source which creates a current for our xylene to travel from our sample, which is in a heated container so that the xylene is vapourized, to our ECOS container in which we bubble it through the E. Coli culture to get results. This was a plausible idea, as it would require fairly non-expensive materials and could be maintained by the average business person. However, we thought it could be more efficient in it's design, and so we kept looking for ideas. Our biggest problem with this was the fact that it is a relatively complex prototype. So we challenged ourselves to try and create a simpler prototype.       PROTOTYPE #2: Another solution we theorized was to have our ECOS impregnated into alginate and then made into small spheres so that we can simply add them into a heated sample to get our results. In a container, we combine our soil sample and our alginate beads, mix them around, and then sift out the beads. The beads produce our indicator protein from the contamination levels of the soil. This design has been inspired by the Peking 2013 Team who used alginate encapsulation beads. Their reasoning and justification for using alginate beads stood out to us, as they stated that it was stable and inexpensive and easy to shape and manipulate. Upon further research into the use of these beads, we discovered that this prototype could be more efficient to use than our first prototype that required a few different pieces to the system. This revised prototype would also be easier for the average oil worker to use compared to the larger prototype.       As time progresses with our project we plan to test and find what concentrations of m-Xylene trigger what kind of report. To do this we have purchased a couple containers of Xylene, each with a specified concentration, which we will apply to our prototypes. In-lab trials, in combination with our mathemantical model, will allow us to predict what [Xylene] is around and therefore what level of Benzene and Benzene derivatives are around as well. Once we manage to find this correlation between reporter and [Xylene] then we will be moving the project out to attempt some real-world application. Depending on our results here, ECOS may move away from rural Consort Alberta and out accross Canada and potentially the world. Of course there will always be things to fix- expecially when it comes to synthetic biology, so just like a computer programer we would try to fine tune and bring out newer version of this construct. The changes, of course, would come from our observations in-lab and customer suggestions as time progressed.    References: The regulatory logic of m-xylene biodegradation by Pseudomonas putida mt-2 exposed by dynamic modelling of the principal node Ps/Pr of the TOL plasmidCalgary 2012 ECHEM Values, Wiley Value ReferencesRetrieved from "http://2014hs.igem.org/Team:Consort_Alberta/projecteam From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact Us Consort iGEM Team of 2014 L to R: Spencer Degenstein, Mr. Gerry Bourassa, Austin Wiltse, Sage Strobel, Alex Coulton, Cole Sansregret, Kris Glasier, Samantha Davies, Nikayla Goddard      Our team originates from the general area of Consort - a rural Albertan village with a population of about 750 people. Despite being from a small town, we have taken on the challenge of creating an iGEM Team, mostly through determination, and we took on the challenges of developing our very own biochemistry lab. Inside our high school science classroom we have designed bio bricks, taught ourselves how to build a website, and most importantly we have taught our community about synthetic biology. The primary industries in Consort are agriculture and oil production, and there are often many conflicts between the two occupations. Apart, agriculture and oil are strong economy suits, but together they spell disaster, and pose health and safety risks when the two mix and contaminate the agriculture. One of the amazing aspects of coming from a small town is the support we receive from local businesses and individuals, and within no time we received financial and community support. After building our own shaker table, borrowing the Kindergarten's chick incubator, and borrowing and receiving supplies such as a food dehydrator, spectrophotometer, centrifuge, and more equipment over a two year span. As a team, we also believe that coming from a small rural community gives us a unique, distinct advantage over the other teams - the community involvement is amazingly close-knit, and we've brought new ideas and perspectives to iGEM that other city teams may not have brought before.     Alex CoultonI'm a grade ten student at Consort School who only has to take a dogsled to school every once and a while. My interest in science and the future of technology has always been an entertaining hobby because of its diversity, experimentation, and possibility to change society, along with creating life. My hobbies include carpentry, metalwork, gaming, experimenting and creating anything I can make a blueprint for. Although I don't know for sure what I want to do after high school I want to stay involved in the world of technology and science, hopefully continuing on with my crazy creating. After secondary school I hope to travel to New Zealand, Australia, and Europe for work so I can get to know the culture of another country and become a part of its community.   Austin WiltseMy name is Austin Wiltse, and I joined my High School's iGEM team because I enjoy Science in school. IGEM seemed like an incredible opportunity to learn even more about science, and to meet new people in the process. As for my activities after school, I have part time employment at the local grocery store, I curl on Fridays with the school's curling team, and on Wednesday with my Dad and his friends. During the summer, I bike and swim, and take first aid courses through my swimming lessons, Bronze Cross and Bronze Medallion. For hobbies, I enjoy playing video games, and programming mobile games for iDevices. What I consider my best achievements are passing my Bronze Medallion and learning a programming language. After school, my plan is to apply to a technical institute and enrol in a video game design course. After that, I hope to apply to work at Treyarch Studios in Santa Monica, United States.   Cole SansregretI am fifteen years old, born and raised here in Consort. I am a caring, kind, and unique individual who has a humorous and likable character. I love to question how the world works, and I want to know every small detail about everything. I spend my spare time reading, playing basketball, and reffing hockey. I have always loved science, and when I first learned about cells and DNA, I wanted to know more about it and become more involved. So, when I heard about iGEM, I immediately wanted to join, and I now have the pleasure of being involved in this amazing competition. I want to learn more about synthetic biology and DNA, but I also want to create memories and have fun. I do not have a solid plan of what I shall do when I finish high school, but, I hope that if I truly love this experience with synthetic biology, I can turn to it as a career.Canadian Myth: Contrary to popular belief, we do have electricity in our igloos. How else would we warm our bacon?   Kris GlasierI am currently attending my twelfth year in Consort School and my second year in the high school competition. I started for many reasons in which I can no longer remember, but they've now been transformed by my interests of today. Striving to take an engineering course at University of Alberta, my academic interests are mainly the maths and sciences. My minimal skill in the arts, such as English, make writing rather difficult at times. The diversity of iGEM has made it possible for every kind of character to come into play, making it a prime place for an aspiring engineer to visit and build connections. Some of my interests involve archery and other medieval-style combat to reading and the occasional video game. I also enjoy seeing how nature works as a whole- the patterns clouds make churning in the sky, to the currently unstoppable force of hurricanes, tornadoes, or even simple thunder storm or the minute gusts of wind. My taste in music has broadened over the years- from strictly rock to rock, metal, country, pop, and so forth including all but most rap songs. Overall Canadians don't particularly like winter, but we endure it for as long as we can. Only the rare few actually prefer the cold over the heat.   Nikayla GoddardI’m a student attending Consort School, in Grade 10. I joined the Consort iGEM team because I believe it’s a great opportunity for any student, and has many aspects so that anyone could find something they love about the idea. My future formerly included wanting to become an astrophysicist / astronomer, particularly studying relativity and mechanics, but I have changed to pursue either teaching or journalism instead, hoping to incorporate science into the writing and creativity aspects into whichever career I wind up choosing. I work at the local newspaper, the Consort Enterprise, for over two years, and I’ve been able to develop some experience with basic Photoshop and photography techniques both business / newspaper-like and creative photos as a hobby. I enjoy art of all kinds, including drawing pictures of my favorite book and movie characters, and my friends, and painting a variety of pictures. My favorite Canadian stereotype is that people think we say eh a lot… I don’t think so, eh? I love reading fantasy books, watching thunderstorms, listening to music, watching movies such as the Avengers, Thor, and X-Men, writing stories and articles for the Enterprise, playing Scrabble, and having a good time with my friends.   Sage StrobelHello! My name is Kali Sage Strobel, and I have a passion for all things biology. My role on my IGEM team is essentially to be involved with everything. Though I particularly enjoy lab work, I'm heavily involved with the human practices portion of our project as well.Knowing that our project is important to our community and its industries makes iGEM an even better experience - I love how we can give back to the community that raised us while having a great time and learning lots. At school, I'm proud to play an integral role on both the High School Students' Union and the Grad Executive Committee. When I'm not studying or working on iGEM related projects, I'm typically found escaping reality through reading or writing.Other activities I enjoy outside of my school work include horseback riding, traveling, jogging and hiking. I absolutely love spending time outdoors and having the opportunity to try new things. A few of my favorite journeys have taken me to Egypt, Greece, Italy, and (of course) Disneyland. In the future, I see myself working either as a researcher, a doctor, or a lawyer. The only thing I know for sure is that I'll be in school for a really long time. Favorite Canadian Myth/Fact: Canadians put more miles on their snowblower than their car. Fact: I do not canoe to work, and I only drink maple syrup in my coffee on Mondays and Wednesdays. :)   Samantha Davies I joined our high school iGEM team because both Science and Math fascinate me and it seemed like a great opportunity to learn about lab work, potential careers and synthetic biology. Some of my favourite books include Handel with Care by Jodi Picoult, The Woman in White by Wilkie Collins, and Pride and Prejudice by Jane Austin. My favourite movies include Gladiator, The Count of Monte Cristo, Star Trek and Lord of The Rings. Other than iGEM I also enjoy dance, band and hanging out with friends. I love living in Canada for many reasons although my favourite part is when, upon learning what country I live in, people immediately ask me if I know their third cousin once removed who lives in Toronto. Do you think I know cousin Johnny??? I'm over three thousand kilometres away and there are over 33 million people in the country!!    Spencer DegensteinI'm currently in Grade 12, going to Consort School. After high school, I'm planning on going to University, into an engineering course. I joined iGEM after my teacher told me about it, and how it was an excellent opportunity to explore a new field, and to see if it might interest me, and possibly persuade me to pursue it in my future. I find iGEM to be very interesting and if indeed I do go into a career relating to this, I can use my role in iGEM as experience. I am mainly working on the wet lab as well as designing our biobrick, and researching xylene, along with xylR. Some of my interests include: shooting, hunting, hanging out with friends and family, and iGEM .   Gerry BourassaScience has always been a passion of mine and I love learning about it and sharing that information with others. I learned about the iGEM competition and synthetic biology at our science teachers' conference and couldn’t wait to tell my students about it. The potential of this field of study is amazing! It is also a lot more difficult in practice than it is in theory. When I’m not in school I enjoy travelling, skiing and restoring my motorcycle.Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/teasponsors From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact UsSponsorsComing from a rural area, the community and business support we get is phenomenal. Sponsors play a key part in allowing the development of our project and of our students, and many of the things we do wouldn't be possible without our amazing sponsors. Our team thanks you very much for your generous support!                                                                                                     Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/sponsosafety From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact UsSafety1. Would any of your project ideas raise safety issues in terms of:    a) Researcher safety - We follow all protocols and use every precaution while in the lab. Gloves, goggles, and lab coats are utilized at all times. We are in a unique situation in that our lab doubles as a classroom, so many students are in the room throughout the day. Also, our classroom is home to a variety of fish in a sizeable aquarium. Because our "lab" plays so many roles in our school, we must ensure that it is kept especially clean and safe for all the individuals who find themselves in it. We have been diligent in terms of cleanliness and, although keeping potentially harmful chemicals in a school classroom creates more of a risk, we have ensured that the event of an accident is next to impossible. Students, visitors and researchers alike are safe in our lab. Parts derived from organisms include: amilCP derived from a coral, Acropora millepora, and GFP derived from derived from jellyfish Aequeora victoria. Neither organisms are harmful or toxic. Any organism from which our DNA is derived or micro organism with which we work in our lab belongs to Risk Group 1 - no or low individual and community risk.     b) Public safety - As mentioned previously, a wide variety of people spend time in our lab. This ranges from parents and younger siblings on parent-teacher interview nights, to iGEM members, to junior high and senior high students. We have ensured the cleanliness of our lab, disposed of any lab waste properly and locked away any potentially harmful chemicals. Since we are working with a non-toxic strain of E. coli, the threat is relatively low to begin with, but we still strive to demonstrate excellence when cleaning the laboratory workplace.     c) Environmental safety - There is no aspect of our project that could pose a threat to the surrounding environment. Not only are none of our bacteria toxic, but they are also contained in our prototype system so that they are never directly exposed to the environment. Even if they were to escape into the open, they are only designed to glow in the presence of xylene, and would hence be impossible to be harmful to the environment. 2. Do any of the new biobrick parts or devices that you made this year raise safety issues?     Our parts containing the gene XylR do not raise any safety issues. As long as we follow the protocols properly, and other people do as well, no issues regarding safety are present. People could think that since we are trying to detect xylene and benzene that this project could be dangerous, but that is not true at all. The benzene will already be in the soil, regardless of our parts. The bacteria used to contain our biobricks is a non-toxic strain of e. coli. The genes derived from organisms in our biobrick are the following: amilCP derived from a coral, Acropora millepora, and GFP derived from the jellyfish Aequeora victoria. Neither organism is harmful or toxic. Any organism from which our DNA is derived or microorganism with which we work in our lab belongs to Risk Group 1 - no or low individual and community risk. 3. Is there a local biosafety group, committee, or review board at your institution?     No, we don't have any sort of review board at our institution. In order to maintain a safe lab environment, we have relied mainly on the guidance of our instructors and the information found in our protocol books. We have reviewed the Laboratory Biosafety Manual (Third Edition), to which we found the link on the iGEM website. This means that every student has to be extremely careful and vigilant in the lab. We all take on the responsibility to be safe and smart during our lab work, and that meticulous standard has been upheld throughout the year. We have all familiarized ourselves with various safety protocols, WHIMIS, and other safety procedures regarding our work with iGEM. The entire team has been very responsible and safe in disposing of, handling, and dealing with the E.coli and other potentially harmful substances. Our supervisor has been present for the duration of all of our lab work, and we have ensured complete understanding of protocols before undertaking them. 4. Do you have any other ideas on how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices, and systems be made even safer through biosafety engineering?     We believe that, more than anything, understanding the procedures and processes is the most important key to safety and success in a biochemistry lab. As long as the researchers involved understand all safety procedures and precautions, risks are minimized and there is a very low chance of something going awry. Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/safetnotebook From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact UsOur Notebook   Introduction      Meetings      Lab Work     Workshops   Introduction...     The following notes are of our daily log book that was kept up throughout the year - the day to day activities, challenges, and solutions we come across while having our meetings or doing our lab work. Our lab work notes include the set protocols that we did and when, how, and why we did them. All of these notes are of all team member's work from several different points in time, combined together to give a full scope of what we did this year. Within the meeting notes are references to our lab procedures, which are listed below the meetings in detail of what we did, how we did it, and some photos too. The three workshops we went to are also described.  Meetings...    September 16th: 3:30-4:15… Met the team from last year and the new member. We discussed the project idea that is continuing from last year's group (See last year's wiki for more information), and what synthetic biology and genetic engineering mean. We looked at a video about synthetic biology and a few other sites. Last year's Consort iGEM Team webpage was reviewed, as well as some of the other competition teams, and we looked through some of their wikis.    September 30th: 3:30-4:30… We are getting a new Thermo-Cycler (PCR). The one we're getting is the BioRad PTC 200 (with 96 wells, like the old one, and has a heated lid). Along with new micro-pipettes (designed to measure things out in microliters). We further discussed other lab equipment that we may have to purchase. Looked over the Central Dogma of Biology, along with how different kinds of lab equipment work.    October 7th: 3:30-4:15… Checked out other wikis for ideas for ours, and saved some links that we could refer back to and look at for inspiration.    October 21st: 3:30-4:00… We are planning on doing an iGEM presentation to the Consort Lions Club on November 21st to get financial and community support. We discussed who else we could talk to about support and sponsorship too, and came up with a short list of people or businesses we could contact.    November 18th: 3:30-5:00… We looked at the iGEM poster and determined who we need to contact for the biobricks and supplies we need for our project. The new students also looked at the process of DNA.    November 25th: 3:30-4:30… Darren and Sharon from the Consort Lion's Club presented our grant to us on behalf of the Consort Lions, as well as Sharon presented a second cheque on behalf of Ronwood Enterprises. On another topic, if things go well, we'll be submitting 2 biobricks to the registry at the end of the year.    December 2nd: 3:30-4:00… A snow day at school, so a few iGEM students hung out in the science lab and began planning our wiki based on what we want, what we saw that was cool on other wikis, and what we needed for requirements. New member to the team: Alex Coulton joined late, and so we updated him on all the science and details. We are now a team of 8 students.    January 6th: 3:30-4:00… We received an amazing grant from Alberta Innovates who was impressed with our application. We each will kick in a potential $200 for the iGEM trip, and we are each responsible for meals and souvenirs. Today we discussed what are website will look like - Nikayla is going to take candid photos for alongside the bios within the next meeting or two, and with the help of Kris and Austin will work on the technical side of the site. We're going to write the bios so they are ready for the next meeting. Next day at lunch: determined about 200 words for the bio, including favourite Canadian stereotype. We're also going to do a "very Canadian" photo at lunch depicting a barbeque, shorts and t-shirts, and maple syrup… in the snow.    January 13th: 3:30-5:30… Did our photo today… it was… cold. We are going to have a workshop on February the 8th at the lab here at the school. Today we compared resources and website uploading options for the Wiki. We might go with OpenBexi. Going to come up with a shopping list for any supplies and such we need for the web lab.    January 27th: 3:30-4:00… Workshop in Consort planning in progress - date is being shuffled around… not sure when or how it's going to work yet?    February 10th: 3:30-4:30… Confirmed workshop date and time: February 23rd. Two people from the U of C are coming, and we are going to go over most of the procedures that we will be doing specifically to our needs. We tried downloading OpenBEXI to use on our website, but it's wonky and didn't work… so Kris contacted a student at Lethbridge to see what they used, and they used Microsoft Dreamspark - we'll try that out.     February 23rd: We had our first Consort iGEM Workshop of the year with David Lloyd and Himika Dastidar from 8:30-5:00. (See this tab for more information)    March 3rd: 3:30-4:00… Mr. Bourassa was gone, but we got all of our pages for our wiki drawn out as well as our main idea of our team descriptions and project info done. We hope to start experimenting with the website program soon!    March 24th: 3:30-5:00… Discussed more fund raising, and got a few replies back from the letters we sent out. Any new modelling and software ideas are due for this Wednesday. Protocols to follow once we get our DNA… A meeting to follow on Wednesday and Friday at lunch.    April 14th: 3:30-5:00… Fund raising follow-up - we received gracious grants from the Veteran Lions Club and Under Pressure. Sam conducted an interview with T&E Pumps for the Human Practices portion of our website. We also put Carla's Stringari as a credits for the logo and website. Alberta Innovates credits too, and we forwarded our articles to their contact as well. We also need to pick up supplies in Calgary! Once we get them, we should be able to start our plasmid switches and we need to schedule times and dates for our lab work. For our flights for the Jamboree - need names for everyone as they are on the passport plus our birthday.    April 27th: We had our second workshop of the year, with Emily Hicks and Robert Mayall from 8:30-5:00. (See this tab for more information)     April 28th: 3:30-5:00… More fundraiser follow-up and general part ordering and organization discussions. Website is well in progress, and a new modelling software is downloaded and begun to be experimented with.    May 5th: 3:30-4:30… We will get some supplies in later this week so we organized the cabinets. We returned our incubator to the Kindergartens too. Arranging to go to a workshop in Calgary at the end of the month, and we'll see how that will go.    May 12th: 3:30-4:30… On May 8th Kris happened to fix the autoclave… then we ran into some… uh… issues with the apparently heat-resistant bottles that we put into the sterilizing machine… they kinda melted, but they're sterilized! Website is in further progress, other teams are starting to do theirs a little too, but we are overall ahead of the game. Workshop on Saturday May 24th, and we will leave right after school to head to Calgary. We booked our geekStarter tickets and got them ready too.    May 19th: 3:30-4:30… General meeting today; getting ready to make some LB Broth and going over procedures. For making the LB Broth, SOC and SOB media, see the lab work for more information.    May 20th: 3:30-4:30… Made LB Broth with no complications. Also worked on modelling and website afterwards until 6:00. The beginning phases of mathematical modelling are appearing after the extra help from Robert Mayall.     May 21st: 3:30-5:00… Took LB Broth out of the autoclave today. Before dealing with the more serious synthesized DNA, we are following our 3A Assembly Kit and Transformation Efficiency Kit from the iGEM Headquarters, which outline basic procedures that we'll use with our synthesized DNA later on. This book will take us through step by step and show us how to, in the end result, make E. coli cells produce a Red Fluorescent Protein that can directly tell us whether or not we did the procedure properly, as it has a strong reporter. Did some agar plates (SOB) and we are growing cultures overnight on the shaker table. Made 3 plates - two from the 2014 distribution and one from the 2013. For making the Agar Plates, see the lab work for more information.    May 26th: 3:30-6:00… Today we made more plates and did agar stabs from the Kit and grew our E. coli. We did 6 agar stabs on plates, and got together the materials and procedures that we need for tomorrow. We've planned a mini-prep for Wednesday. Also worked on modelling afterwards. To look at our procedure, see the lab work for more information.    May 29th: 3:30-4:30… We used a spectrophotometer to get an optimal density reading on our culture tubes. To look at our results, see the lab work for more information.     June 2nd: 3:30-4:30… Getting prepared for our next procedures, and planning a second human practices interview with Dave Bruha, and looking into interviewing other people as well. Did a mini-prep today with our cultures. To look at our procedure, see the lab work for more information.    June 4th: 3:30-5:00… Worked on some modelling and did our restriction digest. Also completed the interview with Dave Bruha of the Consort Enterprise as a part of our Human Practices portion of our project. To look at our procedure, see the lab work for more information.     June 5th: 3:30-5:00... We used a spectrophotometer to get an optimal density reading of our newly growth cultures. To look at our results, see the lab work for more information.    June 6th: 3:30-6:00... At lunch today we did a ligation, followed by the making of competent cells and a transformation of the plasmids into the cells after school. We're letting them sit over the weekend, and then see if they glow red on Monday. To look at our procedure for our ligation followed by the competent cell transformation right after, see the lab work for more information.    June 9th: 3:30-5:30… Our RFP cells didn't glow red, and we determined that something went wrong during the transformation stage - we are going to try it again and see what exactly went wrong.                               June 10th: 3:30-5:30… We finished making the competent cells, then did the ligation, competent cell making, and transformation using the same protocol as on June 6th.     June 11th: 3:30-6:30… Our cultures still aren't glowing red after trial number two. We contacted Emily Hicks for discussing our problems, and she gave us advice and trouble-shooting tips. We think that it was the competency protocol that went wrong, as well as the level of growth that our cells were at. When we did the optical density test, our results varied from 0.7-1.2 range, and they should be at about 0.4 for the best growing rate. And so after re-growing our E. coli, we did a single optical density test and got much more optimal results, which may be to blame for the lack of RFP transformation.                June 12th: 3:30-11:00… Got several more protocols in place and reviewed our handbooks for tomorrow, including the making of competent cells and transformation using a new method, and figured out the variations of the concentration of the CaCl and the different types of growth media added to the cells. We then plated them out on different types of media including SOC, SOB, and LB.              June 15th: 10:00-5:00… After our third trial, our cells still don't have the RFP in them. Today we researched more troubleshooting solutions for both transformations and for making chemically competent cells, and then we prepared to make more competent cells using a revised method and using new values from Emily Hicks, and this was followed by some poster brainstorming and fixing up the wiki as we waited 2 hours for the LB Broth mixture to sit on the shaker table. In the photo, the plate on the left from this round shows that we do have growth, in comparison to a blank plate with no colonies or growth on it.    June 16th: 3:30-6:30… Our fourth trial didn't work, and we are for sure that there is something wrong with the transformation or ligation stage, as our Chlor resistance we put into the cells didn't stick, because there were colonies growing on the regular LB plates, but not on the plates with the Chlor on them. We phoned Emily Hicks and asked for some more help... her one suggestion was that maybe something was wrong with the DNA. She's going to talk with her colleagues tonight and let us know tomorrow. Another thing that may be wrong is that our centrifuge holds less than what the protocols ask for, so we had to split it up into smaller volumes, which could be a problem. The photo shows all 16 plates we did under a black light, along with the 2 plates on the far right hand side from the last round - one was with LB media and one was with SOC media.                       June 18th: 3:30-5:00… We have decided to leave our lab work alone for now and focus on the poster, presentation, and wiki aspect of the project. Today we conducted an interview with Lacey Ryan of CARA (Chinook Applied Research Association) and from the perspective of an Environmental Conservation and Forage specialist, she offered valuable advice. The photos below show all of our failures in completing the competent cell making / transformation stage on our plates. We did have lots growth though. We followed our procedures step by step, and everything went well in our wet lab work up until that point.                                                June 19th: 3:30-6:00… We've been working throughout the day and into the evening on the modelling and on the wiki, as wikifreeze is this Friday. We've also been designing our poster, and have ordered t-shirts for the Jamboree. Progress is going well so far, only a few more things to put up.   June 20th: 3:30-10:00… Wiki is ready to go... counting down until wikifreeze! Today we worked on the wiki and poster, and did a few last minute photos along with proof-reading on the wiki. Can't wait to see everyone at the Jamboree! Best of luck to all teams! Lab Work... The following are the protocols in which we followed for our lab work, along with the book or web handbooks references, the dates when we did the protocol, and how the protocol went overall.     ~LB Broth (May 19th): Materials: LB broth powder, Glass bottle. Protocol: Weigh out 20g of LB broth powder and mix with 1L of water. Scale up or down as desired. Autoclave (sterilize) the broth at 121°C for 15 minutes. We made 1.5ml of Broth, and we had no problem following the procedure. ("International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors" 1 , page 96-97). We also followed OpenWetWare procedures for both the SOC media and for SOB media     ~Agar Plates (May 21st): Materials: LB-agar powder mix, Plates. Protocol: 1. Weigh out 35g of LB-agar powder mix per litre of media desired. (Note: instead of using powder, we made our own LB Broth powder from a formula given to us by our mentors... 5g yeast extract, 10 g tritone, 10g sodium chloride mixed together.) One litre makes 40-50 plates. Ensure that the mixture volume does not exceed half of the volume of the flask/container used, otherwise it will boil over in the autoclave. 2. Dissolve the LB-agar, using water from one of the wall mounted Nanopure filters. Add a stir bar and use a magnetic stirrer to facilitate mixing. 3. Cover the flask with aluminium foil, and secure the foil with autoclave tape. The foil should be somewhat loose (to avoid building pressure in the flask while sterilizing), but not so loose that lots of liquid can escape. 4. Put the flask in a plastic autoclave tray, load into the autoclave, and sterilize using the 20 minute liquid program. 5. Allow the media to cool until it can be handled. 6. Once media is cool, add other desired ingredients. Use the magnetic stirrer to mix, do NOT add a stir bar now, or the media will be contaminated. (If one wasn't added before, you must do without.) 7. Add the antibiotic that will select for your plasmid. The following antibiotic concentrations are used commonly in iGEM: Ampicillin (stock 100mg/ml, final 100μg/ml); Kanamycin (stock 50mg/ml, final 50μg/ml); Chloramphenicol (stock 50 mg/ml, final 10μg/ml) Note: To achieve final concentrations, add 1mL of stock per 1L of culture media, except for chloramphenicol, where 0.6mL stock per 1L of media is added instead. 8. Pour the sterile, antibiotic-containing broth directly from the flask into the sterile Petri plates carefully to avoid trapping air bubbles. 9. Allow the plates to stand right side up overnight, and then store them at 4°C (in fridge). We had no trouble making these either. ("International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors" [1] , page 97-98)     ~Streaking From Agar Stabs and Growing Up Cell Cultures (May 26th): We made 6 plates, 3 Part A and 3 Part B, following this procedure. After our incubation, our cells were cloudy as the procedure said they would be. ("3A Assembly kit and Transformation Efficiency" [2] , page 9-11)           ~Spectrophotometer Test #1 (May 29th): Using our spectrophotometer (GeneQuant), we tested our cultures to see what their optical density was in order to judge whether they were at their optimal growing stage or after their growing was finished. Our results were as follows...Test blank: LB Broth with Amp and Kan read 0.365. Actual testing: Test Tube 1 (Part B): 0.792, Test Tube 1 (Part A): 1.762, Test 2 (Part B): 0.778, Test Tube 2 (Part A): 0.752, Test Tube 3 (Part B): 0.882, Test Tube 3 (Part A): 1.102. Observation: Part A seemed to grow faster. We were surprised at the huge optical density though, and we considered testing it further but decided not to.     ~Mini Prep #1 (June 2nd): Completed the Mini Prep with minimal complications - all of our team members were here for this step. ("3A Assembly kit and Transformation Efficiency" [2] , page 12-14)     ~Restriction Digest #1 (June 4th): Overall it went well, and we didn't have to make any modifications to the procedure. ("3A Assembly kit and Transformation Efficiency" [2] , page 15-16)     ~Spectrophotometer Test #2 (June 5th): Our results were as follows for our tubes of E. coli cells that are all supposedly the same... Sample 1: 0.948, Sample 2: 0.736, Sample 3: 0.924, Sample 4: 1.208.     ~Ligation #1/#2 and Transformation #1/#2 (June 6th and then June 10th): This procedure didn't work. ("3A Assembly kit and Transformation Efficiency" [2] , Ligation: page 17-18, Transformation: page 19-20. Note the competent cell making on page 21-26)     ~Spectrophotometer Test #3 (June 11th): Our results were as follows for our tube of E. coli cells. After talking with Emily we discovered our optical density should be between 0.4 and 0.6 in order for the cells to be at their best 'acceptance rate' for the plasmids they are supposed to take it... Sample 1: 0.404.     ~ Making Competent Cells, Ligation #3/#4, and Transformation #3/#4 (June 15th): These procedures didn't work. For #3, we followed the protocol step by step to make just 2 plates this time. But when that failed, on #4 we followed the procedure and but also used new values from Emily Hicks, and made 16 plates. Our variables for #4 were 0.1 molar and 0.05 molar of CaCl and we used both LB and SOC media for variety, and we made four with each substance to total 16 plates. We also used heat shock at 42 degrees for 90 seconds, and increased the media added to the competent cells from 250 micro-litres to 500 micro-litres. ("International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors" [1] , Making Chemically Competent E. coli Cells and Bacterial Transformation page 95-96)                  Workshops...    This year we have had three workshops with mentors from Calgary. The three of these workshops combined have been invaluable. Between meeting in person and the dozens of emails that have been sent back and forth, our mentors have been priceless to our project and our own personal learning. Despite the fact that our learning curve is nowhere near a plateau, we have come so far and are extremely grateful for the expertise and guidance of these amazing people.   The first was on February 23rd when David Lloyd and Himika Dastidar drove down to Consort in order to get us started.          We began the morning bright and early by learning about the basics of synthetic biology and iGEM, as many of our team members are new this year. After going through some basic vocabulary, procedures, and concepts, we moved on to topics more specific to our project. We spent the afternoon looking up parts in the Registry and creating a construct for our Biobrick. After we had the basics, one of our instructors suggested we synthesize a majority of our Biobrick as it would be easier and faster. After determining precisely what would be synthesized, David then proposed that we center our project on a comparison of different reporters and outputs. Enthusiastically, we researched the best choices for reporters and came up with three choices - GFP, AmilCP and LacZ. For the remaining time we had with the expertise of our mentors, we created a skeleton of the code for synthesizing our DNA.              The second workshop, held on April 5th, was when Emily Hicks and Robert Mayall came down to answer questions and get us started on modelling, more in depth wet lab procedures, and how to improve other aspects of our project.     We learned about differences and similarities between simulations and mathematical modelling, and the potential programs we could use for them. In order to create a model, we have to gather your 'species' (the possible chemicals or states in your system), create a flow chart for your species where you define how your species interact with each other, and then add general equations to your framework. As for equations, the simplest equation is “A * k = B”, where species A becomes species B at a rate of k. And after gathering the correct information and basing any assumptions that you need to, the next step is to convert your equations to code. From there, you just test and improve, and even compare your model of how you predict the species will react to the real life interactions to see how close you were.             The third workshop was in Calgary on May 31st and was focused mainly on the presentation factor of our project. Four other Alberta teams - Lethbridge, Canmore, Calgary, and Cochrane - were present as well. The morning began by simply presenting to the other teams, a panel of mentors, and several volunteers. After each presentation the judges and audience would have a chance to ask questions and give feedback on both the content of the presentation and the manner it was presented. It was incredibly neat to see how the other teams were doing and to improve upon our own presentation by listening to the judges comments and the other teams. By the time we had finished all the presentations we broke for lunch and chatted adamantly about what we liked and disliked about our presentation while preparing for the afternoon. After lunch, we were able to meet with the judges individually. Each of them were able to give us tips, suggestions, and comments in much more detail than had been permitted earlier. Finally, we had a session on our presentation skills, with general tips about how to speak to an audience, eliminate nerves, minimize long paragraphs of text, and how to answer questions. For the rest of the workshop we revised our presentations and played a couple rounds of `PowerPoint Karaoke`- an exercise to practice thinking on your feet and talking to an audience.        References: International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors, iGEM High School Handbook   Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/noteboohumanpractices From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact UsHuman Practices Introduction      Community Outreach  School Involvement     Interviews   Introduction...     Over the iGEM season, we have been focusing on community outreach. We've been striving to educate, enlighten, and engage individuals and groups in our community concerning the matter of genetic engineering and synthetic biology. In order to achieve this goal, we have done several formal presentation to the members of the PLRD school board and our school administrators, and the Consort Lion Club, from which we are still receiving many positive comments. Several of our senior members created this presentation specifically to introduce the basic premises of synthetic biology, our project and techniques/procedures used. We have shown elementary students a similar presentation and we advocate iGEM in our school. We specifically target students entering senior high who may be interested in joining the team next year. In addition, we've promoted iGEM through our local newspaper, the Consort Enterprise, by having several articles, photos, and updates put in for subscriptions across Alberta and even across Canada to see. These articles explain our project, how we are doing thus far, and the Jamboree while encouraging support, involvement, and interest in our team. Furthermore, we have had several discussions with key members of our economy concerning potential implementations, uses, and improvements to our project. While giving presentations, talking with students, conducting interviews, or casually visiting in the grocery store, our initiative has been praised and our work received with fascination. Community Outreach...    Our presentations to community groups this year have included the Consort Lions Club, the PLRD School Board, and the Veteran Lions Club. To keep our community updated and let people know what we have going on in the science lab, every workshop or event that we do goes into our local newspaper, the Consort Enterprise. Many articles and photos over the past year have gone through the paper, and so there have been many people that have read up on the articles and become more aware of iGEM and how our team is progressing. One of the issues here is the December 2013 edition regarding the starting up of this year's project and highlighting what we are in progress of doing.  School Involvement...    This year, for our school involvement, we have made presentations to our younger students at our monthly assemblies, explaining the science of synthetic biology and our project in the hopes that it will inspire them to pursue the sciences and join our project when they are older. We have also done a more elaborate and descriptive presentation to the Grade 9 class, explaining more about the concepts of synthetic biology and our project. Our older members and adviser held a presentation early in the year to the school board and the administrators, explaining to them our project and their amazing experience in Boston. They were very impressed with our project, and our school and students are standing behind us in full support. Interviews...    We wanted to have a better idea of how we should develop our project into something that the average person, an oil worker, could use. So we decided to conduct interviews with a few members of our community and get their perspectives on our project, and see if other people thought it could be useful. We would like to thank Elaine Devine, David Bruha, and Lacey Ryan for allowing us to interview them. They gave us very valuable tips, and each had a unique approach to the project - Elaine being a key role in the local oil industry, David being a rural community member who sees many projects through the newspaper, and Lacey being an agronomist and a farmer herself.Elaine Devine - Partner owner of the major oil company T&E Pumps. Elaine gracefully agreed to meet with us and discuss the practicality portion of our project. Elaine was widely impressed with the work we are doing and the dedication of both students and supervisors. After briefly mentioning potential uses for our biobrick, Elaine brought up the idea for not only using the E. Coli as a way to test soil for xylene on a industrially level but also on an individual scale. General concern for the environment and future generations coupled with the fact that many farmers and ranchers have oil wells on their land led Elaine to suggested the use of the genetically modified E. Coli as a simple way for individuals to monitor the level of pollution in their soil. When asked if detecting trace amounts of xylene would be useful she responded with a resounding 'maybe!'. After bringing up the point that certain types and amounts of oil can actually be beneficial and act as fertilizer, she mentioned that the detection of trace amounts of xylene could allow a good option for the monitoring of the concentration of xylene over long periods of time.  David Bruha - Editor of the local newspaper, the Consort Enterprise. Dave gave the team his input about the aspects of potential individual and industrial use of our device, and finds that our project is very applicable to the community. "Anything that will help with the controversy that we sometimes see between agriculture and the oil industry will be very useful," Dave enthused. "Having the ability to detect contamination quickly and inexpensively would really help go a long ways towards a better relationship. If a product could be produced using this technology, it could be used not only in this area, but around the world wherever there's petrochemical development." Dave also elaborated on the importance of having an instantaneous acknowledgement of whether or not there is potential contamination. "Many of the small communities around rural Alberta are faced with underground storage tanks and potential contamination of the soil. This type of a product could quickly identify what people are dealing with when they dig up their streets and do regular maintenance. They could know right away what they were dealing with, rather than having to wait and wait for soil samples to be transported to the city and wait and wait for the lab results to come all the way back. Sometimes it can take weeks, but with this we could almost instantly know," Dave concluded.  Lacey Ryan - Agronomist & Animal Nutritionist for the Chinook Applied Research Association (CARA). Lacey offered her advice from a producer point of view, and explained what producers are looking for when using a means of detecting contamination. “The biggest thing with being producer friendly is cost. I’m a farmer as well; you have to make sure that is product, if developed, is either similar or lower in cost to the average lab test. A soil sample costs anywhere from $25-$150 depending on the lab. Most of the sample costs are $30. It’s not necessarily that it’s really expensive; it’s just that all the way out here, we can’t always just do a soil test.” Lacey then described that when a producer goes to buy previously used land or native prairie, the first questions they ask is if it is safe. “We want to know what’s on our land, especially if there are cattle. If a well site was reclaimed on the land, I know that almost all farmers would want to get it tested. This is a really neat idea, and it sounds like it could be really quick too. It usual takes a week to get results back by email but it depends on the lab. If you send it away Monday and your order is rushed, you could possibly get it back Friday, otherwise it’s even later, so it’s a really good to have a test right away on the site, and could really benefit producers.”     Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/humanpracticFrom 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact UsiGEM 2014 ~ Consort, Alberta                OUR PROJECT                                OUR TEAM          HUMAN PRACTICES                       OUR NOTEBOOK                    SAFETY                                   ATTRIBUTIONS                SPONSORS                                  CONTACT US Retrieved from "http://2014hs.igem.org/Team:Consort_Albertcontact From 2014hs.igem.org  Home | Our Project | Our Team | Human Practices | Our Notebook | Safety | Attributions | Sponsors | Contact Us Want to learn more? Contact the Consort iGEM Team if you have any questions or comments.Phone: #(403) 577-3654Fax: #(403) 577-2112Website: Visit our School Website!E-Mail: Gerry.Bourassa@plrd.ab.ca OR consortschool@plrd.ab.caAddress: 5215 50 Street, Bag Service 1002, Consort, Alberta, T0C 1B0Retrieved from "http://2014hs.igem.org/Team:Consort_Alberta/contac1282Charlottesville RSRenaissance High School Charlottesville, VA USA www.renaissanceschool.orgHigh SchoolPolyhydroxybutyrate as a Substitute Source of Energy for Denitrifying BacteriaIn Albemarle county, Virginia, a large amount of the waste water goes to and is processed in the Moores Creek Wastewater Treatment Plant. Each year, the plant purchases 250,000 dollars worth of glycerin, which is used by bacteria to denitrify the water, in order to prevent eutrophication in the Chesapeake Bay. The UVa iGEM team from 2008 created a part which, when added, enables E.Coli to produce polyhydroxybutyrate, a biodegradable, bio-derived plastic. Our project is to make E.Coli that produces this plastic, which the plant could then use this bacteria to create polyhydroxybutyrate, filter out the E Coli, and then use the plastic as an alternative food source for their bacteria, saving them 250,000 dollars per year, as well as giving them a renewable energy source for their plant.811BBa_K1282999http://2014HS.igem.org/Team:Charlottesville_RSBBa_K1282000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164520140High SchoolLog in   Team:Charlottesville RS/Sponsors From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Acknowledgements We thank the following people and institutions for support of our iGEM project: The University of Virginia's VGem team, for letting us check out their lab, past projects, and giving us some insight on what to expect and how to prepare. The Moores Creek Wastewater Treatment Plant, for giving us inspiration for our project, and supplying us water samples and other support. Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/SponsorsProject/Applications From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Moores Creek Wastewater Treatement Plant The Moores Creek Wastewater Treatment Plant (WWTP) is managed by the Rivanna Water & Sewer Authority. The purpose of the water treatment plant is to use bacteria to purify waste water so it can be released into Moores Creek. Bacteria are used to process the waste and purify the water. However, these bacteria require nourishment, so every year the plant purchases $250,000 worth of glycerine in order to feed these bacteria. Our idea is to use genetically modified E. coli to produce a bioplastic called PHB, which the bacteria that purify the waste water could consume for energy, and thus save the city up to $250,000 on the Moores Creek WWTP and potentially apply to other WWTPs as well. How Wastewater Treatment Plants Work1) Anoxic: Polyphosphate-accumulating organism (PAO’s) are able consume simple carbon compounds without the presence of an external electron acceptor, such as NO3 and O2. The bacteria in this step are deprived of NO3 and O2 to give the PAO’s a competitive advantage. This is done to enhance the removal of phosphorus.2) Anaerobic: Denitrifying bacteria are supplied with NO3 from step 3 but are denied O2. The bacteria can separate the NO3 into N2 and O2 and then use the O2. (2NO3→N2+3O2) The nitrogen is now a harmless gas that evaporates out of the wastewater.3) Aerobic: Aerators supply O2 and the bacteria convert NH3 from the wastewater into NO3 and H2. The NO3 is recycled to step 2.4) Anaerobic: Any leftover NO3 is processed by denitrifying bacteria, but the food supply has been largely depleted and is supplemented with glycerin.5) Aerobic: Any remaining organic material is consumed by the bacteria. Sequencing Batch ReactorSince we do not have as much space as the Moores Creek Wastewater Treatment Plant, so we used a sequencing batch reactor, where instead of the water flowing from step to step, the water remains in the same location and aerators are turned on and off to alternate between aerobic and anaerobic zones. Instead of just two aerobic and anaerobic zones, we used sixteen.Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/Project/ApplicatioTeam From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Team Renaissance Advisors:Anna Minutella: Teacher and AdvisorBecky Wilbur: AdvisorStudents:Alli Ambrosini: JuniorAnder Beaurline: JuniorLauren Ewell: JuniorBailey Fernandez: JuniorJohn Grammer: SeniorNick Keen: SophomoreJames Manchester: JuniorJessica Prax: Sophomore Renaissance School Renaissance School is a college preparatory high school for high ability students in the arts, sciences, and/or humanities.We emphasize both broad and deep interdisciplinary learning through a balanced program equally strong in the arts and academics. ​Our strength lies with students who want to maximize their potential and make the most of their high school experience. Renaissance students appreciate academic and artistic rigor, and are also able to collaborate with classmates and faculty. Links:School HomeRenaissance in the NewsCooperation Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/TeaTeam Members From 2014hs.igem.org  image pageiGEM 2015 image page Home People Project Details Notebook Community Outreach Lab Safety Sponsors Contents1 Team Members2 Alli Ambrosini3 Anders Beaurline4 Lauren Ewell5 Nick Keen6 James Manchester7 Jessica Prax8 Advisors9 Anna Minutella Team Members Alli Ambrosini Alli AmbrosiniI’m a junior at the Renaissance School. I’m interested in going into forensic science after high school, and I’m currently learning how to sew! I really like moths, fog, and candy. I spend my spare time watching and caring for three cockroaches. Anders Beaurline Anders BeaurlineI am a part of an IGEM team formed from our DNA science class here at Renaissance School. I am a junior and a member of the student government. I am currently doing an independent study project on hidden agendas in the media and visiting colleges all up and down the east coast. Lauren Ewell Lauren EwellI am a junior at the Renaissance School in Charlottesville VA. I have always loved science, but I have never done anything as interesting and complex as this IGEM competition. Outside of school, my hobbies are rock climbing, charity work, scuba diving, hiking, white water rafting, and eating. I am very excited to be apart of this amazing opportunity and learn more about genetic engineering everyday. Nick Keen Nick KeenI’m a sophomore at the Renaissance School. You may find me swimming for the CYAC team in Charlottesville, or reading at the local library. I enjoy playing video games and writing programs in Java script. I find engineering and chemistry are my forte, and iGEM gives me a unique opportunity to work with and strengthen my knowledge of chemistry, and engineering. James Manchester James ManchesterI am a junior at the Renaissance School. Although my primary interest is mathematics, I have immensely enjoyed preparing for this year’s iGEM competition. I want to go to the University of Chicago. I also play the piano and sing in the Shenandoah Valley Children’s Choir. You can often find me playing The Wiki Game and watching ViHart’s videos. Jessica Prax Jessica PraxI am a sophomore at the Renaissance School, am interested in biology, and enjoy DNA Science class very much. I am currently volunteering at the local Hope Community Center for kids, and taking American Sign Language class at our local community college. Advisors Anna Minutella Anna MinutellaNew to Renaissance School in 2013, Anna Minutella was born and raised in Wytheville, Virginia on her family’s farm. She attended the University of Virginia where she earned a B.A. in both biology and psychology and spent much of her time doing research in language development. After graduation, she moved to Baltimore, Maryland with her husband. There, she taught science to seventh and eighth grade students for five years in a private school. She also served as chair of the science department and developed the curriculum for the middle school science department. She and her husband moved back to the Charlottesville area in the summer of 2012 and Anna spent that following school year teaching science and math at Stuart Hall in Staunton. Anna’s teaching philosophy is based on using a variety of perspectives and methods for each subject in order to truly involve the students in the material and insure they gain a deeper understanding. She wants her students to acquire the same appreciation for the material that fuels her passion. In her spare time, Anna enjoys being with her husband, son, and two small dogs.Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/Team_MembNotebook/Overview From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Notebook March March 10th, 2014March 14th, 2014March 25th, 2014March 28th, 2014 April April 8th, 2014April 9th, 2014April 10th, 2014April 11th, 2014April 14th, 2014April 15th, 2014April 18th, 2014April 22nd, 2014April 24th, 2014April 25th, 2014April 28th, 2014[Back]Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/Notebook/OvervieFrom 2014hs.igem.org  image pageiGEM 2015 image page Home People Project Details Notebook Community Outreach Lab Safety Sponsors OverviewIn Albemarle County, Virginia, a large amount of wastewater flows to and is processed by the Moores Creek Wastewater Treatment Plant. Each year, the plant spends 250,000 dollars on glycerin, which is used as an energy source by specific bacteria in the sewage to denitrify the water. Denitrification of the effluent from the wastewater must occur to remove nitrate from the wastewater and thus, prevent eutrophication in the Chesapeake Bay. The UVa iGEM team from 2008 created a part which, when added, enables E.Coli to produce polyhydroxybutyrate, a biodegradable, bio-derived plastic. Our project is to make E.Coli that produces this plastic The water treatment plant could then add the engineered E.coli to the wastewater where the E. Coli would produce and secrete polyhydroxybutyrate into the water., The denitrifying bacteria in the wastewater would then be able to use this plastic as an alternative energy source for denitrification saving the water treatment plant 250,000 dollars per year, as well as giving them a renewable energy source for their plant.Retrieved from "http://2014hs.igem.org/Team:Charlottesville_R2014hs.igem.org/wiki/images/8/8c/Caltech_logo.gifNotebook/material From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Methods Preparing competent cellsThe bacteria were grown to log (or exponential) phase and 10mL of bacteria were aliquoted into 15 mL sterile plastic tubes. cells were pelleted using a tabletop centrifuge at 7000 x g for 10 minutes, or ~4000 rpm. The supernatant was poured off into liquid waste container. the tube was tapped to loosen the cell pellet, and d 5 mL of cold CaCL2 solution were added to the pellet. The cell solution was incubated on ice for 20 minutes. After incubation, the cells were spun for 5 minutes at 5000 x g (2500 rpm). The supernatant was poured off into the liquid waste container. One mL of cold CaCl2 solution was added to the cells and the cells were resuspended. Rehydration of RFP and Alcohol Acetyltransferase ITen uL of dH2O were pipetted into the well of the corresponding Biobrick-standard part. This solution was pipetted up and down, then it was left to sit for 5 minutes. Five uL of the re-suspended DNA was transformed into competent cells. The transformed cells were plated, and were grown overnight. Transformation of RFP and Alcohol Acetyltransferase IThe tubes were labeled as Transformation Control, Ligation: New Part, and Ligation Control. Five uL of RFP Control DNA was added to Transformation Control tube. Two uL of New Part ligation product was added to Ligation: New Part tube. Two uL of the RFP Control ligation product was added to the Ligation Control tube. The tubes were placed on ice to pre-chill. One tube of previously prepared competent cells was thawed and left on ice. Fifty uL of competent cells were pipetted into each of the 2.0mL micro-centrifuge tubes. The DNA and cell mixtures were incubated on ice for 30 minutes. During incubation, the water bath was heated to 42˚C. The tubes were placed in the waterbath for 60 seconds, then were incubated on ice again for 5 minutes. Two hundred uL of SOC media was added to each tube. The tubes were incubated at 37˚C for 2 hours. Two hundred uL of each tube was pipetted onto respective plates. The liquid was spread on the surface of the plate surface using glass beads. The plates were incubated lates face down at 37˚C for 12-14 hours. Extraction of RFP and Alcohol Acetyltransferase ITwo culture tubes were spun down for 3 minutes at 8000 rpm. The supernantant was poured into a biological waste container. 250uL of Buffer P1 was pipetted into each 14mL culture tube, and the pellet was resuspended. The 1.7mL micro-centrifuged tubes were labeled with their respective part names, and transferred the parts were resuspended in it. 250uL of Buffer P2 was pippeted into each micro-centrifuge tube. The tubes were flipped to mix the solution. 350uL of Buffer N3 was pippeted into each tube.The tubes were flipped to mix the solution. The tubes in the micro-centrifuge were spun down for 10 minutes at 13,000 rpm. The spin columns were labeled for each tube, and the supernatant was pipetted to their respective spin columns. The spin columns were spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. 500uL was pipetted of Buffer PB into each of the spin columns. The spin columns were each spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. 750uL was pipetted of Buffer PE to each spin column. The spin columns were spun down at 13,000 rpm for 1 minute. The flow through was poured into the chemical waste beaker. The spin columns were spun down at 13,000 rpm for 1 minute to remove the remaining buffer. The spin columns were labeled “sterile 1.7mL micro-centrifuge” tubes with their respective part names. The appropriate filter tube was transferred to their respective tubes. 50uL of dH2O was pipetted to each filter tube. The samples sat for 1 minute, then the tubes were spun down at 13,000 rpm for 1 minute. The micro-centrifuged tubes were stored at 20˚C. Digestion of RFP and Alcohol Acetyltransferase INEB Buffer 2 and BSA were thawed in room temperature water. Four 0.6mL tubes: Part A, Part B, pSB1C3, and RFP Control. 500ng of DNA were added to the appropriate tube. Distilled water was added to the tubes for a total volume of 42.5uL in each tube. 5uL of Buffer 2 was pippetted into each tube. 0.5uL of BSA was pippetted into each tube. In Part A tube, 1 uL of EcoRI enzyme was added and 1uL of SpeI enzyme was also added. In Part B tube, 1uL of XbaI enzyme was added, and 1uL of PstI enzyme was also added. In the pSB1C3 tube, 1uL of EcoRI enzyme was added, and 1uL of PstI enzyme was also added. In the RFP Control tube, 1uL of EcoRI enzyme was added, 1uL of PstI enzyme was also added. The solution was mixed by pipetting up and down, and spinning it for 5 seconds in the micro-centrifuge to collect the mixture at the bottom of the tube. The restriction digests were incubated at 37˚C for 30 minutes, then at 80˚C for 20 minutes. They were then stored at -20˚C. Ligation of RFP, Alcohol Acetyltransferase I, and PSB1C3The T4 DNA Ligase Reaction Buffer was thawed. 0.6mL was labeled as New Part. 2uL of the pSB1C3 linearized plasmid backbone digest was added. 3.3uL was added from Part A digest. 3.9uL was added from Part B digest. 1uL of T4 DNA Ligase Reaction Buffer was added. 0.5uL of T4 DNA Ligase was also added. The solution was mixed by pipetting, and by being centrifuged for 5 seconds. Another 0.6mL tube was labeled as Ligation Control. 2uL from RFP Control digest was added. 6.5uL of distilled water was added. 1uL of T4 DNA Ligase Reaction Buffer was added. 0.5uL of T4 DNA Ligase was added. The solution was mixed by pipetting, and centrifuging for 5 seconds. Both tubes were incubated at 16˚C for 30 minutes, then at 80˚C for 20 minutes and then stored at -20˚C.Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/Notebook/materiaProject From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Project Idea Junk Food In Albemarle County, most of the waste water goes and is processed through the Moore's Creek Wastewater Treatment Plant (WWTP), which is managed by the Rivanna Water & Sewer Authority. Each year, the plant purchases 250,000 dollars worth of glycerin, which is then used as “food” for bacteria which degrade the other organic matter in the water. The UVA iGEM team from 2008 created a part that, when added, enables E Coli to produce polyhydroxybutyrate, a biodegradable, bio-derived plastic. Our idea is to make a type of E Coli that produces this plastic, with a reporter which fluoresces when the bacteria is successfully producing. The plant could then use this bacteria to create polyhydroxybutyrate, filter out the E Coli, and then use the plastic as an alternative food source for their bacteria, saving them 250,000 dollars per year, as well as giving them a renewable energy source for their plant. Importance of NitrogenNitrate (NO3) is a compound found in most water. It is colorless and odorless, so it can only be detected with testing. Nitrate itself isn’t bad in water, but it causes eutrophic effects if there is too much, which causes a chain reaction of harmful effects (explained further in the next section). Every living organism (as well as non-living organisms such as viruses) needs nitrogen to live; it helps to form the structure of proteins and DNA and many other molecules in cells that are essential for plants and animals to live. The majority of nitrogen in the world is held in the atmosphere, but not in its usable form. For nitrogen to be usable by organisms, it must be in the form of either ammonium or nitrate, which it is converted to in either abiotic or biotic fixation. Animals (like ourselves) get nitrogen from eating plants and other animals. Plants get nitrogen not by consuming other organisms but by absorbing it through the soil in its fixated forms. Why we Don't Want Nitrates in our Water Because everything needs nitrogen to live, we obviously want to have some in our water. The problem arises when there is just too much - because that causes eutrophication. This is a huge problem right now in the Chesapeake Bay, and since we are part of the watershed that flows into the Bay, we have a responsibility to keep it clean. The Moores Creek WWTP and other treatment plants in the area are now obligated under the law to filter enough nitrate out of the sewage water because of the condition of the Chesapeake Bay, but soon most treatment plants in the country will probably have to follow in their footsteps. The filtered supernatant of the sewage water that goes through Moores Creek will drain into the Bay, so it needs to be clean. If there is too much nitrate in this supernatant, it will further contribute to the decline of the Bay - when there is too much of a macronutrient like nitrate, everything grows too much. For our purposes, this is mostly bad in reference to algae. The huge algae blooms that eutrophication causes not only block out sunlight to the organisms living under the water, but they eventually lead to anoxic zones in the water (meaning that there is no oxygen there). This happens when the algae dies and floats to the bottom of the water, and is then eaten by bacteria. Because there is so much algae to eat, the bacteria community flourishes, and they end up using all the oxygen in the water. Animals like fish and any others that preform cellular respiration cannot live under these circumstances because they need oxygen - this often leads to large groups of organisms dying. Right now, eutrophication is probably the biggest problem for the Chesapeake Bay, and plenty of other bodies of water in the world, It's very important that waste water treatment plants do their job efficiently and without too much money spent; and using polyhydroxybutyrate instead of glycerin will help to do that. Polyhydroxybutyrate Polyhydroxybutyrate (PHB) is a polymer of glucose and a biodegradable plastic. It is a special type of polyester called a polyhydroxyalkanoate. PHB is produced by microorganisms such as Ralstonia eutrophus or Bacillus megaterium in response to conditions of physiological stress, particularly conditions in which nutrients are limited. PHB can also serve as food for other bacteria. In waste water treatment, bacteria are required to dispose of waste in the water, but feeding them is very expensive for treatment plans, and for the Rivanna Water Treatment Plant, the closest such facility to our school, it costs the city up to $250,000 a month to simply feed these bacteria. Our idea for the project involves using bacteria in order to produce PHB as food for the bacteria that are involved in waste water treatment. Since PHB is only produced when nutrients are limited, it would actually involve feeding the PHB-producing bacteria less than the waste water treatment bacteria would be fed. We believe our idea has the potential to drastically reduce the costs of waste water treatment plants around the globe.Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/Projecafety From 2014hs.igem.org Home Team OverviewAdvisors Project Overall ProjectMaterial & MethodsApplications Safety Notebook Material & Methods Sponsors Safety Questions Question: Would any of your project ideas raise safety issues in terms of:- Researcher Safety- Public Safety- Environmental SafetyAnswer: In sewage water, there are many pathogenic organisms such as Salmonella (biosafety level 2), E.Coli (biosafety level 1), and S. Typhimurium (biosafety level 2). The only part of our project that actually includes wastewater is our controlled sequencing batch reactor buckets that only we handle. We take necessary precautions whenever handling these buckets (gloves, goggles, proper sterile technique), and they would never cause harm to anyone else unless the buckets somehow fell over and the fluid fell off of our rooftop (which shouldn’t happen). In that case, the worst that would happen would be a few people getting sick, probably mildly. If the project goes according to plan, our wastewater should be clean and free of these pathogens, so there wouldn’t be any risk to anyone.Question: Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes:- Did you document these issues in the Registry?- How did you manage to handle the safety issues?- How could other teams learn from your experience?Answer:Question: Is there a local biosafety group, committee, or review board at your institution?- If yes, what does your local biosafety group think about your project?- If no, which specific biosafety rules or guidelines do you have to consider in your country?Answer: The EPA regulations concerning the water quality of outgoing water at the Moores Creek Wastewater Treatment Plant is:- Suspended solids: 22 mg/l- Ammonia : 1 mg/l (warm months) / 6.6 mg/l (cold months)- Nitrogen: 4 part per million (ppm)- Phosphorous: .3 ppmQuestion: Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?Answer:Retrieved from "http://2014hs.igem.org/Team:Charlottesville_RS/Safet1270Shasta Summit CASummit Shasta High School Daly City, Ca, USA http://shasta.summitps.orgHigh School1111BBa_K1270999http://2014HS.igem.org/Team:Shasta_Summit_CABBa_K1270000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163220140http://2014hs.igem.org/files/poster/Shasta_Summit_CA.pdfHigh SchoolLog in   Team:Shasta Summit CA From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   Introduction Located in the Bay Area, Summit Shasta is a revolutionary school that prefers to encourage students to grow from their mistakes, rather than have students memorize meaningless facts. They push their students to seek out challenges and overcome them, thus the iGem team was born, with help they hope to achieve success in bringing their ideas to life. The team is part of Summit Shasta's first year being around, comprised entirely of eager freshman. Perhaps one day they will revolutionize the world. Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CAhttp://2014hs.igem.org/wiki/images/e/e5/Shasta_Summit_CA_logo.png/Project From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   Our Project We plan to ultimately create a bacteria that would be stimulated by the presence of CO to produce a red hue. The uses of this kind of organism/bacteria would include disaster relief in areas that do not have the access to the equipment needed to monitor the concentration of CO in the area. The goal would be to have the bacteria glow red in the presence of CO, and different colors based on the concentration. For example: It would glow a darker red and/or emit a smell if the level of CO in the air was at fatal levels. It is based upon the percentage of CO in the air. Carbon Monoxide is a fatal gas that is undetectable to humans. In iGEM we plan to use biobricks to make sensors. We plan to construct two sensors, one with the sensor and one with the reporter. The plasmid with the reporter with glow red if the levels of CO are at fatal levels. iGEM also requires outreach which we have and are currently doing; the outreach includes teaching and using a website to convey information.The PlanThe NotebookEvery Tuesday we meet to make progress on our iGEM project. On Thursdays we go to the lab at skyline collage and do laboratory work, at the end of our meetings we discuss the progress we have made, and research new information for our next meeting! Making the Bacteria We will be using a two plasmid system. One plasmid will be a Sensor Plasmid and the other will be a Sensor PlasmidPromoter (constitutive) RBS (Ribosomal Binding Site) CO Sensor Gene Double Terminator Reporter PlasmidOperator for CO Sensor Promoter (inducible) RBS (Ribosomal Binding Site) RFP Gene Double Terminator When we research we were able to find an organism that was able to sense Carbon Monoxide and create a visible reaction. Using that gene we added that to the BBa_K880005 Bio Brick, which contains the sequence for the CO Sensor Gene, Constitutive Promoter, RBS, Double Terminator This is going to be the Sensor Plasmid. There is also the Reporter Plasmid which is using the BBa_K516030 Bio Brick that contains a RBS, a Double Terminator, a Inducible Promoter, Operator for the Sensor, and a RFP gene. Using the 3A assembly we will combine these two plasmids onto one plasmid. Testing The Bacteria We plan to expose the bacteria to Carbon Monoxide. We will start with a preliminary test of simply exposing the bacteria to carbon monoxide. The second set of tests they plan to do would be to create a gas mixture of 5% carbon monoxide, 1% carbon monoxide, and .1% carbon monoxide, respectively. Making sure that the mixture is correct is difficult, as measuring how much gas is release is difficult. So they filled a 2 liter graduated cylinder with water then pumped Carbon Monoxide into the container and stopped when there was approximately 100ml of water out of the graduated cylinder. With 100ml out that means there is 100ml of gas in the graduated cylinder. Then we will remove the gas and add that to a cylinder filled with 1900ml of air. Then with that mixture we will pump that out into a container containing the bacteria. 100ml in a 2000ml total would make that 5% mixture. Results/Conclusions Over the course of the year we have practiced with bacteria by making transformations and competent cells. Here is a diagram of the project we intend to complete: Learning and Practicing 3A Assembly We had a blast using the 3A assembly kit, learning all sorts of things about cutting plasmids and instituting their own. Although we made some mistakes, we were able to move on to successfully create a modified bacteria.   Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Projec/Team From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafety   The Team Professors:Chris Vulpe of UC Berkeley Nick Kapp of Skyline College Students: Albert Liu Alex Liu Aslan Nguyen Brendan Thompson Dexter Hamilton Ethan Bull-Vulpe Jen Co Jimmy Lujan Kyra Newcomb Mitchell Wong Osama Hanhan Sydney Huddleston William McEachen   Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Tea/Goal From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   Our Goals Our goal is to create a CO (Carbon Monoxide) sensor that will detect CO and create a noticeable color. This CO sensor would be more useful than its electronic cousin in many ways: 1. It would not require constant power, this means that you could use it in locations with easy access to power, such as third world countries, underground places/mines, portable/handheld, etc. 2. It would be small and compact, allowing for easy transportation, meaning it could be carried by people everywhere, providing constant protection. 3. This means that it would have many uses beyond the ordinary electric, roof-bound, carbon monoxide sensor.   Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Goa/Attributions From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   Attributions We researched the possible options for the project, including a biofuel project and a stud-finder using magnetic bacteria. We were the ones who decided which project to go forward with, making our choice based on simplicity, cost, and likelihood of success. We split into groups of two or three to find the necessary parts after discussing what would be required. Our mentors guided us towards reputable sources of information; however, we did the actual research. Once we had found the necessary parts, we obtained them from the DNA distribution kit and we synthesized the additional parts not available as a biobrick. In order to design the new parts, we attained sequence information from NCBI and renowned literature. We used tools available online for optimiziing the codons for ''E. coli''. Our mentors ordered the gBlocks at IDT that we had designed. Our mentors provided us with the lab equipment and taught us how to correctly use it, which they had acquired from Skyline College. We did all of the lab work, from pipetting DNA to running said DNA through a gel electrophoresis to plating a transformation of our DNA into bacteria. We designed multiple posters for our different outreaches, and then one of our teammate's father printed it at his print shop. During the outreach, the presentations and activities were designed and executed by us. The Wiki The wiki was primarily created and maintained by Aslan Nguyen; however, Jimmy Lujan and William McEachen proofread and revised the content. Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Attribution/Outreach From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   OutreachExpanding Your Horizon On March 15th we went to Skyline College to be part of the Expanding Your Horizons program. Expanding Your Horizons is a series of workshops designed to encourage more young women to enter the realms of science and math, as currently the gender ratios in those subjects are not 50/50. We introduced future high schoolers to the idea of synthetic biology. Over the course of our three workshops, we used an interactive model to simulate how a plasmid is synthesized and the parts in it, introducing the young women to the basics of synthetic biology. Poster Presentation at Skyline College On April 24th, we gave a poster presentation about our team's goal and synthetic biology overall at Skyline College Annual Research Symposium. There were students and professors of Skyline and of other colleges, and other invited guests from the biotech industry. It was a full day of scientific demonstrations and presentations. Poster Presentation at San Mateo Library On June 16th, we presented to a group of officials from San Mateo County and friends of the library, about our project at San Mateo Library. Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Outreac/Sponsors From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   SponsorsSkyline College for Lab Space IDT Bio-Rad Nor Cal SOT Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Sponsor/Safety From 2014hs.igem.org HomeGoalProjectThe TeamOutreachSponsorsSafetyAttributions   Safety While we were brain storming, testing, and experimenting we were at all times supervised by Dr. Nick Kapp and/or Dr. Chris Vulpe.We were required to wear a form of safety glasses and at one point lab coats when it was found necessary. We were taught and expected to hold and use each individual piece of equipment correctly, even if we were not going to use the equipment that same day. For the most part all our procedures were safe to complete without any injury or problems. Our planned outcome will have no public or environmental safety issues. Our end result will only be dangerous if you were to use it as a projectile. If the E. Coli was released into the environment there would be no dangerous effects to come of it. When we plan to test our final product of the double plasmid we will contain it in a controlled environment. We will place in the plasmid and after sealing off a containment unit we will insert the CO through a sealed tube.   Contact: shastahsigem@gmail.com Retrieved from "http://2014hs.igem.org/Team:Shasta_Summit_CA/Safet1252Nanjing NFLSNanjing Foreign Languages SchoolHigh SchoolCRISPR-Cas9 gene editing kit in eukaryotic cellsUtilizing the unique features of CRISPR-Cas9 system, our team developed a gene editing tool kit that can be effectively used in eukaryotic cells. Comparing to the traditional approach that relies upon restriction enzymes, a CRISPR-Cas9 based system offers several advantages including greater flexibility in silencing, enhancing or repairing gene expression, and the ability to introduce larger gene fragments. Our gene editing kit has three components: Guide RNA (gRNA), outer membrane protein of TMV homologous DNA sequences and CRISPR-Cas9 enzyme. The system was designed to have great compatibility with a wide range of organisms and easy to work with, and has been preliminarily validated by plasmid transfection in CHO cell line. With iGEM database in mind, the kit follows a modular design and aims to future iGEM projects with greater flexibility and compatibility in gene editing.1602BBa_K1252999http://2014HS.igem.org/Team:Nanjing_NFLSBBa_K1252000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=160720140http://2014hs.igem.org/files/presentation/Nanjing_NFLS.pdfhttp://2014hs.igem.org/files/poster/Nanjing_NFLS.pdfHigh SchoolLog in   Team:Nanjing NFLS/project.html From 2014hs.igem.org MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/project.htmlteam.html From 2014hs.igem.org Instructor: Hong Tian: Professor at China Pharmaceutical University. Graduated 2010 with PhD in microbiology and biochemical pharmacy from CPU and continued to teach there. Main research field: chemical modification of proteins, genetic code expansion technology and its applications. Instructed the CPU iGEM team in 2008 and received a bronze medal.   MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/teabiobricks.html From 2014hs.igem.org AgRNA Specific Recognition Sequence Transcription Vectors There are three sets of gRNA. The Guide-RNA sequence comes from Addgene plasmid 41824(gRNA_Cloning Vector).The Guide-RNA sequence is inserted in the DNA from into basic plasmid backbone pSB1C3（BBa_J04450）with BioBrick cloning site prefix（EcoR I，Xba I）and suffix（Spe I，Pst I）. When the plasmid is transfected into the mammalian cell nuclei, the plasmid will transcript into the form of Guide-RNA or gRNA.Three Sets Of gRNAa gRNA-A (BBa_K1252001)TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAGAATTGATCAGAGGAACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTA bgRNA-B (BBa_K1252002)TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGGAACCGGATCTTATAATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTAcgRNA-C (BBa_K1252003)TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTACGCACCACGTGTGATTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTA**The unhighlighted parts of each gRNA is identical. *The blue highlighted part is mutated from TCTAGA（EcoR I）to TCTAGT.*The yellow highlighted part is commentary to the corresponding target gene with homologous arms.*Xba I is added to the upstream sequence, and Spe I is added to the downstream sequence to be compatible with the basic backbone pSB1C3（BBa_J04450）.BthDNA Target Recombinant Sequences of VectorsUpstream and Downstream Homologous Arm with Target GeneThere are three sets of thDNA corresponding to the three sets of gRNA, with an upstream homologous and a downstream homologous in each set.thAF/thARthBF/thBRthCF/thCRThe thDNA sequence is inserted in the DNA from into basic plasmid backbone pSB1C3（BBa_J04450）with BioBrick cloning site prefix（EcoR I，Xba I）and suffix（Spe I，Pst I）. When the plasmid is transfected into the mammalian cell nuclei, the plasmid will retain its form until there’s a CRISPR-Cas9 breakage available.athAF (BBa_K1252004)TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAATCGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGAAATCAGTGTAGAATTGATCAGAGGAACCGG*Xba I is added to the upstream sequence, and Spe I is added to the downstream sequence to be compatible with the basic backbone pSB1C3（BBa_J04450）bthBF (BBa_K1252005)TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAATCGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGAAATCAGTGAGGAACCGGATCTTATAATCGG*Xba I is added to the upstream sequence, and Spe I is added to the downstream sequence to be compatible with the basic backbone pSB1C3（BBa_J04450）cthCF (BBa_K1252006)TGAGTTTTAAAAAGAATAATTTAATCGATGATGATTCGGAGGCTACTGTCGCCGAATCGGATTCGTTTTAAATATGTCTTACAGTATCACTACTCCATCTCAGTTCGTGTTCTTGTCATCAGCGTGGGCCGACCCAATAGAGTTAATTAATTTATGTACTAATGCCTTAGGAAATCAGTGTACGCACCACGTGTGATTACGG*Xba I is added to the upstream sequence, and Spe I is added to the downstream sequence to be compatible with the basic backbone pSB1C3（BBa_J04450）**The yellow highlighted part is commentary to the guide-RNA sequence with homologous arms.d thAR (BBa_K1252007) GTAGAATTGATCAGAGGAACCGGTTCAAACACAACAAGCTCGAACTGTCGTTCAAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCCCTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGTCACAGCACTGTTAGGTGCATTCGACACTAGAAATAGAATe thBR (BBa_K1252008)GAGGAACCGGATCTTATAATCGGTTCAAACACAACAAGCTCGAACTGTCGTTCAAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCCCTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGTCACAGCACTGTTAGGTGCATTCGACACTAGAAATAGAATfthCR (BBa_K1252008) GTACGCACCACGTGTGATTACGGTTCAAACACAACAAGCTCGAACTGTCGTTCAAAGACAATTCAGTGAGGTGTGGAAACCTTCACCACAAGTAACTGTTAGGTTCCCTGACAGTGACTTTAAGGTGTACAGGTACAATGCGGTATTAGACCCGCTAGTCACAGCACTGTTAGGTGCATTCGACACTAGAAATAGAAT**The yellow highlighted part is commentary to the guide-RNA sequence with homologous arms.CCRISPR-Cas9 EnzymeMicrobes rely on diverse defense mechanisms that allow them to withstand viral predation and exposure to invading nucleic acid. In many Bacteria and most Archaea, clustered regularly interspaced short palindromic repeats (CRISPR) form peculiar genetic loci, which provide acquired immunity against viruses and plasmids by targeting nucleic acid in a sequence-specific manner. These hypervariable loci take up genetic material from invasive elements and build up inheritable DNA-encoded immunity over time. Conversely, viruses have devised mutational escape strategies that allow them to circumvent the CRISPR/Cas system, albeit at a cost. CRISPR features may be exploited for typing purposes, epidemiological studies, host-virus ecological surveys, building specific immunity against undesirable genetic elements, and enhancing viral resistance in domesticated microbes. [1][1] Horvath, P.; Barrangou, R. (2010). "CRISPR/Cas, the Immune System of Bacteria and Archaea". Science 327 (5962): 167–170. MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/biobrickourpartners.html From 2014hs.igem.org MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/ourpartnersafety.html From 2014hs.igem.org MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE National Accreditation Criteria for Laboratory Bio-safety.Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/safetFrom 2014hs.igem.org MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE ●●●●●● What's this about? What are the biobricks? What's about experiments? How we ensure safety? How did we popularize it? Who are we? Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLhttp://2014hs.igem.org/wiki/images/7/73/NFLSLOGONEW.jpgnotebook.html From 2014hs.igem.org 1Design of gRNA recognition sequence: Principles of gRNA recognition sequence design: The specificity of the Cas9 nuclease is determined by the 23-nt guide sequence within the sgRNA. the target sequence must immediately precede a 3′GGN dinucleotide (protospacer adjacent motif PAM), and FUT8 gene is selected as gRNA recognize sequences based sequence motif II structure GGATAAAAAAAGAGTGTATCTGG(Figure 1-3).2Preparation of specific recognized gRNA carrierDepending on the commercial Kit instructions ,the methods of translating DH5 Alpha cells are as follows: Monoclonal antibodies are picked and cultured in 4mL LB medium at 37℃ for 16-hours. Positive cell lines LA Taq (TAKARA CAT.RR002A) are identified by bacterial PCR described above. of PCR products are separated by 1% agarose gel electrophoresis. Select 180bp electrophoresis carrier suspension samples to expand cultivation. 5Ml turbid bacterial culture fluid are collected, endotoxin-free bacterial plasmid extraction kits are used for plasmid extraction operation (CWBIO CAT.CW2106), according with the commercial Kit instructions. DNA samples are prepared for UV (260 å/280 å) quantitative and saved in refrigerator below -20℃.3Preparation of CAS9 DNA Double-strand breaks enzyme HCas9 plasmid (Addgene plasmid 41815) whose backbone are pcDNA3.3-TOPO（Invitrogen）are saved in plasmid puncture tube and cultured in 4mL LB medium with ampicillin (Ampicillin) resistance and 210rpm shocks at 37℃ for 16-hours, then, 5Ml turbid bacterial culture fluid are collected. Endotoxin-free bacterial plasmid extraction kits are used for plasmid extraction operation (CWBIO CAT.CW2106), according with the commercial Kit instructions. DNA samples are prepared for UV (260 å/280 å) quantitative and saved in refrigerator below -20℃.4TransfectionChinese hamster ovary (CHO-K1) cell lines which are saved in our laboratory are cultured in Media DMEM/F12 (calf serum containing HEPES, 10%) at 37∘C in a humidified atmosphere (5% CO2, 95% air). Cell grows adherently, with 0.25% trypsin (EDTA) digesting every 48 hours, which 10% passage. For transfection experiments, the cells were seeded in 24-well tissue culture plate at 1×106 cells every well at 37∘C in a humidified atmosphere (5% CO2, 95% air). Replace the medium at an hour before transfection. Dose of Plasmid for transfection: DNA mixture liquor: Lipofectamine2000/DNA Transfection reagents diluent preparation: gently mix, let stand for 20 minutes. Preparation of Lipofectamine2000/DNA mixture: 50 μl of diluted Lipofectamine2000 drops add 50 μl DNA mixture, gently mix, let stand for 5 minutes. Mixed droplets containing added Lipofectamine2000/DNA cell culture the hole, gently shake mix. Culture in 37∘C, 5% carbon dioxide for 6 h. Replace the medium DMEM/F12 (calf serum containing HEPES, 10%), incubating for 18h5the preliminary identification of monoclonal cellsAbout 5x106 are harvested and are centrifugalized at 1000rpm for 5 minutes, discard the supernatant, wash cells with PBS once. Use cell genome genome action kit to manipulate genome. Gene obtain amplification primers: LA Taq PCR Reaction system： Touchdown PCR Reaction conditions： PCR products are separated by electrophoresis using 1% agarose gel . Gel extraction Kit is used to recycle positive band.S1 nuclease digestion and recycled products separately t carrier connection sequence.5.1 S1 nuclease digestion S1 nuclease digestion: Recycled products are heated at 98∘C for 10 minutes, and then, cooled to room temperature, nuclease S1 (THERMO CAT.EN0321) enzymes at room temperature for 30 minutes, accordding with the commercial Kit instructions for specific methods. Harvest of CRISPR/Cas/Donor knockout group, LA Taq PCR amplification of target gene sequence, and S1 nuclease digestion with restriction enzymes BamH I restriction enzyme digestion.S1 nuclease could identify mutations annealed to form single stranded DNA convex Central and catalytic the break of DNA strand. Enzymes BamH I can cut off the homologous recombinant vector into the enzyme loci. Knockout group is located in less than 500bp Strip, being consistent with theoretical Strip locations. Figure 2:S1 nuclease digestion with restriction enzymes BamH I restriction enzyme digestion S1 nuclease has the function of degradation of single-stranded nucleic acid, releases single nucleotide 5' phosphoryl or oligonucleotides, but also as the incision is cut double-stranded DNA, gaps and mismatches, or ring-like structure caused by single strand region. CRISPR/Cas Sentinel-mediated gene knockout cell specific gene loci will be rendered to produce a random deletion and insertion.Total cells genome extraction and amplification of target gene fragments, through denaturation and annealing, the wild-type gene fragments and fragments of mutated hybrid annealing, mutation due to the cut, nicked, single strand region due to mismatch or a ring-like structure, S1 nuclease will double-stranded DNA cut at this point (Figure 2-6). Figure 3:S1 nuclease enzyme analysis principle    5.2 T carrier connection sequence: Gel extraction of electrophoresis sample stripes 773bp, recycling products are amplified by Taq PCR for the second LA, according with the commercial Kit instructions. PCR reaction conditions: predegenerated 95 ℃ for 5 minutes, 94℃ for 30 seconds, 42 ℃ for 30 seconds, and 72℃ for 30 seconds, 30 cycles, extension 72 ℃for 5 minutes. PCR reaction system: PCR products are separated by 1% agarose gel electrophoresis. 773bp positive band products are recycled using gel extraction Kit (TIANGEN CAT.DP209-2). PCR recovery products are connected with pMD19-T (TAKARA CAT.D102A) through a TA cloning vector, according with the commercial Kit instructions for specific methods.Translate DH5 Alpha cells according with the commercial Kit instructions for specific methods and incubate them at 37 ℃ for 16-hour . Monoclonal cells are picked and incubated in 4mL propagation LB medium, at 37 ℃, for 16-hours. DNA sequencing is perfomed to identify the actual sequence. That shows a cut has happened by sequencing because missing sequence has been found.April April: We start to design the g-RNA with high specificity. For working in the CHO, sequence of our tool kits hardly have similarity with the OMP of TMV. So we find the proper sequences and prove them. TAGAATTGATCAGAGGAAC AGGAACCGGATCTTATAAT GAGGAACCGGATCTTATAATCGG GTAGAATTGATCAGAGGAACCGG Gtacgcaccacgtgtgattacgg TACGCACCACGTGATTAMay We build up the parts of our toolkits. Restructure the PSB1C3 XbaI speI sites tgatttctggaattcgcggccgcttctaga actagtagcggccgctgcagtccggcaaa tgatttctggaattcgcggccgcttctaga TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGTCCCAGCTTTCTTGTACAAAGTTGGCATTA actagtagcggccgctgcagtccggcaaa actagtagcggccgctgcagtccggcaaa Use Gabc-F: tgatttctggaattcgcggccgcttctagaTGTACAAAAAAGCAGGCTTTAAAGG TM62 Gabc-R: CAAGAAATCGAAATACTTTCAAGTTACG Expend the gRNA to the following sequence TGATTTCTGGAATTCGCGGCCGCTTCTAGATGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTG Synthesis the first piece and the last piece GTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGNNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGTCCCAGCTTTCTTGTACAAAGTTGGCATTA actagtagcggccgctgcagtccggcaaa Gain: GA: tgatttctggaattcgcggccgcttctaga TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTAGAATTGATCAGAGGAACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGTCCCAGCTTTCTTGTACAAAGTTGGCATTA actagtagcggccgctgcagtccggcaaa GB: tgatttctggaattcgcggccgcttctaga TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGGAACCGGATCTTATAATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGTCCCAGCTTTCTTGTACAAAGTTGGCATTA actagtagcggccgctgcagtccggcaaa GC: tgatttctggaattcgcggccgcttctaga TGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGTACGCACCACGTGTGATTAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTAGTCCCAGCTTTCTTGTACAAAGTTGGCATTA actagtagcggccgctgcagtccggcaaa   Cells from the Knockout group (Figure 2-1a) had a fusiform morphology, grew in colonies and attached to the culture plate. A small number of cells had spherical shape and detached from culture surface when agitated. The negative control group (Figure 2-1 b) retained spherical shape and did not attach. This observation suggested the CRISPR/Cas system was functioning in the gene knockout experimental group, induced the recombination from homologous recombination donors, and conferred lca tolerance to transfected cells. 50ug/ml lca pressurized culture for 48 hours gRNA replacement module was constructed using PCR with expected length of 180bp, which was confirmed using 1% agrose gel-electrophoresis lane 1：marker 250；lane 2：grna module replacement Upstream homologous arm, downstream homologous arm, PCR electrophoresis lane 1：marker 250；lane 2：Upstream homologous arm; lane 3：downstream homologous arm The sequencing results of the homologous donor is consistent with expectation  UV quantification （260Å 280Å） of homologous donor DNA  Quantification/measurement of homologous donor plasmid   MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/noteboohumanpractice.html From 2014hs.igem.org MAINPROJECTBIOBRICKSNOTEBOOKSAFETYHUMAN PRACTICETEAMOUR PARTNERSCONTACT US ABSTRACT TOOKIT FUNCTION ADVANTAGE DESIGN BUILD UP PROVE BUILD UP EXAMINE Retrieved from "http://2014hs.igem.org/Team:Nanjing_NFLS/humanpractic1303biYOLOgistsCHAMPS Charter High School Van Nuys, CA 91405High School601BBa_K1303999http://2014HS.igem.org/Team:biYOLOgistsBBa_K1303000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166820140High SchoolLog in   Team:biYOLOgists From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:biYOLOgists/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:BiYOLOgists logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:BiYOLOgists team.png Your team picture Team biYOLOgists Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project Coming from a small crowded high school, which clashes together grown adults and pre-pubescent teenagers, we the bioYOLOgists are faced with the issue of overwhelming body odor every day. At first, we believed this to be due to the lack of deodorant on freshmen, but upon closer examination we realized the problem was the fact that the deodorants were failing. Antiperspirants block the healthy release of sweat, while other deodorants fade throughout the day. That’s why we decided to create a solution to the problem; a bacterial solution that is. The solution we came up with is deodorizing bacteria. Our bacteria creates a wintergreen smell when the cell dies. Salt is a natural antibacterial and is quite prominent in sweat. We want to create this bacteria so that when the user begins to sweat the bacteria will die, releasing the wintergreen scent to combat any body odor created by the wearer's sweat. The difference with our product, is that it would be applied to the outside of clothing around the body. This way it is a failsafe for when traditional deodorants stop working correctly, in addition to allowing areas other than where normal deodorants are applied to be coated and protected. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:biYOLOgists1257UCL AcademyThe UCL Academy Adelaide Road London NW3 3AQ United Kingdom http://www.uclacademy.co.ukHigh SchoolBio-purification of water to remove bio-toxins: Bio-IN, Bio-OUT.The project aims is to degrade microcystin, a toxic substance produced by cyanobacteria. When cyanobacteria die, the cell walls collapse, causing the release of microcystins into water. However, microcystins happen to be extremely stable, so this means they are able to resist common chemical breakdown, such as hydrolysis, at natural conditions. It also breaks down very slowly at high temperatures (40�C), so the best way to deal with the problem is to use a bacterium that can break it down. However they aren?t usually found in water, so this allows the toxin to ravage the aquatic ecosystem. Our idea is to make a genetically modified organism that can break down microcystins. So, we will modify an E.coli bacterium to float at the top of a water column, where the cyanobacteria are located and to detect and degrade microcystins.752BBa_K1257999http://2014HS.igem.org/Team:UCL_AcademyBBa_K1257000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161520140High SchoolLog in   Team:UCL Academy/HumanPractice From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ HUMAN PRACTICE ACTIVITIES Synthetic vs Natural Synbio Outreach Commercial algal growth containment Abstract Starting our project, we were interested in how algael growth is limited in commercial environments. We conducted research in two different environments - a zebrafish laboratory and a commercial aquarium. Following the news that consumer goods use unlabelled synthetic biology products, we investigated our community's response to the 'synthetic vs. natural' debate. We produced a short video of a dystopian fictional scenario to help our test group visualise an urgent situation. Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/HumanPracticeProject From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ CyanobusterProject IntroductionWe aim to reduce or even eradicate the harmful effects of algal blooms. To achieve this goal, we plan to engineer a genetically modified organism (GMO) that degrades Microcystin: a toxin produced by cyanobacteria [1]. Cyanobacteria are prokaryotic and due to their diverse genus, they are able to survive in many different aquatic environments from marine to estuarine water [2]. The toxin cyanobacteria produce, microcystin, is not only toxic to the immediate environment, but also to humans, domestic animals, and livestock [3]. Algal blooms are found mainly in regions with high concentrations of nitrates and phosphates in the body of water. High nitrates and phosphates result in increased algal growth, and hence increased toxin concentrations, such as microcystin, in the water. What functions does our GMO require? Our GMO needs to break down microcystin, remain buoyant at the level of algae, survive in harsh conditions and contain a kill switch for selective removal. We worked towards a four-module system, which we have described in detail below.Contents1 Breakdown of Microcystin2 Buoyancy3 Resistance to Harsh conditions4 Kill switch5 Bibliography Breakdown of MicrocystinMicrocystin is resistant to many common bacterial proteases due to its cyclical structure [4]. We selected the mlrA enzyme, which uses hydrolytic cleaving to linearize the structure of microcystin, and thus render microcystin more susceptible to degradation [5].When the GMO detects microcystin, the mlrA gene is expressed in response to the detection mechanism. The mlrA gene expression triggers the production of the mlrA enzyme, which degrades microcystin. In order to extract this gene we used 3 different organisms: Escherichia coli, Bacillus flexus, and Oceanibulbus indoliflex. By using more than one organism, we aim to expand the Registry of Standard Biological Parts.<p>We plan to express the mlrA gene after 2 different promoters:</p>Promoter 1: Lead sensitive promoter, part BBa_I721001. Research shows that high levels of lead are commonly found in algal blooms [6], and so this promoter will induce the mlrA gene to be expressed in presence of high levels of lead.Promoter 2: UV sensitive promoter, part BBa_I765001. When algal blooms occur, they occupy the surface of the water and block incoming sunlight. This reduces the amount of UV radiation in the body of water. We used a more complicated mechanism of the UV promoter that induces a repressor gene - part BBa_C0040 - when there are high levels of UV, which corresponds to little to no algal growth. The mlrA gene is inserted after a repressor-regulated promoter - part BBa_R0040 - which will continue inducing the mlrA until the repressor binds and prevents induction. This works as a reverse mechanism: if there is high UV, the repressor is expressed, which inhibits mlrA production. Buoyancy<p>Cyanobacteria grow on the surface of bodies of water. Therefore our GMO needs to float at the level with the highest concentration of toxins. We aim to engineer this process by introducing a buoyancy module. The production of gas permeable vesicles within the cell raises the partial pressure of the cell and causes the GMO to become buoyant.We planned to take the GVP gene cluster from the organism Bacillus Megaterium, based on the previous work of the 2012 UCL iGEM team. It contains 14 putative gens: gvp-A,-P,-Q,-B,-R,-N,-F,-G,-L,-S,-K,-J,-T and –U. The last 11 genes, in a 5.7-kb gene cluster, are the minimum genes required for gas vesicle synthesis and function in E. coli. Resistance to Harsh conditionsIn order to make our GMO resistant to microcystin, we inserted the IrrE gene found in Deinococcus radiodurans. The 2012 UCL iGEM team used the IrrE gene in their composite biobrick BBa_K729005. We placed the IrrE gene (BBa_K729001) after the same promoters we used in our degradation gene; the lead sensitive promoter, the UV sensitive promoter, and the repressor regulated promoter. We constructed the same circuit as for the degradation gene, however with the IrrE gene in place of the mlrA gene. Kill switchIn the case that Microcystin are able to enter our GMO, the metabolism of the cell would change and cause the bacterium to become unstable. In water, organisms have a high tendency to accept foreign DNA, and the effects of other organisms accepting our bacterium’s DNA may not be positive. In order to prevent this from occurring, we planned to implement a kill switch in our GMO. The kill switch we planned on using was used by Paris’ Bettencourt team, based on the wild type Colicin E2 operon, which has a system for degrading our GMO’s DNA. This would have been induced by genes/proteins that are activated by stress conditions, in this instance the entry of microcystin into our bacterium. Bibliography [1] Carmichael W.W. Health effects of toxin-producing cyanobacteria: the CyanoHABs. Hum. Ecolog. Risk Assess. 2001;7:1393–1407.[2] Dawson R.M. The toxicology of microcystins. Toxicon. 1998;36:953–962. [PubMed][3] Stewart I and Falconer IR (2008) "Cyanobacteria and cyanobacterial toxins" Pages 271–296 in Oceans and human health: risks and remedies from the seas, Eds: Walsh PJ, Smith SL and Fleming LE. Academic Press, ISBN 0-12-372584-4.[4] Harada K.I. Chemistry and detection for microcystins. In: Watanabe M.F., Harada K., Carmichael W.W., Fujiki H., editors. Toxic Microcystis. CRC Press; Boca Raton, MI USA: 1996. pp. 103–148.[5] Bourne D.G., Jones G.J., Blakeley R.L., Jones A., Negri A.P., Riddles P. Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin-LR. Appl. Environ. Microbiol. 1996;62:4086–4094. + Saito T., Okano K., Park H.D., Itayama T., Inamori Y., Neilan B.A., Burns B.P., Sugiura N. Detection and sequencing of the microcystin LR-degrading gene, mlrA, from new bacteria isolated from Japanese lakes. FEMS Microbiol. Lett. 2003;229:271–276.[6] García-Hernández J1, García-Rico L, Jara-Marini ME, Barraza-Guardado R, Hudson Weaver A. Concentrations of heavy metals in sediment and organisms during a harmful algal bloom (HAB) at Kun Kaak Bay, Sonora, Mexico.Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/Projec/Activity2 From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ HUMAN PRACTICE ACTIVITIES Synthetic vs Natural Synbio Outreach Commercial algal growth containment Science Week presentation to Foundation Level students at UCL Academy In addition to our other activities, we engaged the younger students in our school with our project. We gave a presentation to the foundation level students in UCL Academy. As they were young, we based the talk on synthetic biology as a whole, in relation to our project. We made the discussion highly interactive by showing videos we had made and asking questions, such as what they would make their own GMO do and who the GMO would benefit. We also involved two students from the audience to help us complete a small practical experiment: extracting DNA from blueberries and showing the younger students what DNA looks like outside of a cell.Our workshop was a very successful session and all the children were eager to answer questions and engage with the presentation.Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/HumanPractice/ActivityNotebook From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ Week 0 Week 1 Week 2 Week 3 Week 4 From researching and brainstorming to working in the lab, click on the week to see what we did!.Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/NotebooSafety From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ Safety comes first! Would any of your project ideas raise safety issues in terms of:Researcher safetyThere are a number of standard lab reagents that we require the use of in our project, that are harmful on contact. These include:Ethidium BromideMaterial Safety Data Sheet for Ethidium BromideAcute: Hazardous when ingested or inhaled, and is an irritant of the skin and eye. Chronic: In the long term exposure can have carcinogenic, mutagenic, and teratogenic effects, and can cause developmental toxicity.Environmental safetyAt the end of our project we are planning to release engineered E.coli into the rivers or other water bodies containing algae bloom. Our engineered organism will contain four gene network responsible for the following: degradation, resistance to the harsh conditions, buoyancy and the kill switch. In the water bodies where there is no possible physical containment of bacteria. This raises multiple concerns as to whether the use of living machines may integrate into and alter the local ecosystem, and potentially spread.Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?
One of our biobricks we are proposing to use a lead promoter. High levels of lead are found in algae bloom populations therefore we would like our biobrick to be able to correspond to the levels of lead.Did you document these issues in the Registry?None of the submitted biobricks have safety issues discussed above.How did you manage to handle the safety issue?As we did not clone the lead promoter we did not proceed onto characterisation part, therefore we did not carry out the characterisation of the biobrick. Safety issues are not applicable!Is there a local biosafety group, committee, or review board at your institution?Yes! We have the Departmental Genetic Modification Safety Officer (DGMSO) as well as the College Genetic Modification Safety Committee, who must approve of our experiments before we carry them out. Firstly, we complete risk assessments, which have to meet UCL Safety Rules. Secondly we had to be trained to do a particular experimental work. Finally, we must be supervised at all times by our supervisors.If yes, what does your local biosafety group think about your project?They are interested in our project, and they are fully supportive of our lab work and they make sure we follow all safety requirements.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?Biosafety is one of the key aspects of the iGEM competition. We are proposing an implementation of a kill switch which will prevent a transfer of GMOs DNA once it is no longer stable.Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/Safet/Activity1 From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ HUMAN PRACTICE ACTIVITIES Synthetic vs Natural Synbio Outreach Commercial algal growth containment Synthetic vs. Natural Investigation “The year is 2024, and algae toxicity is a growing problem. Abroad synthetic biology product Cyanobuster is widely used to contain algae blooms but it has not yet been approved by UK parliament. The GMO environmental law is up for review next month on the 28th of June… Which way would you vote?” Synthetic biology is becoming an increasing relevant tool in commercial biotechnology. In May, Ecover was denounced by the ETC group for using unlabelled synthetic biology ingredients in household products. This is reviewed by the NY times.To support our investigation, we produced a short dystopian film. The video was inspired by the iGEM 2012 Meeting of Young Minds.We presented a fictional scenario in which algae toxicity is increasingly dangerous, and the chemical solution is environmentally unfriendly. We conducted interviews with a questionnaire (results below).Bio-Fiction Film FestivalWe entered our short film into the Bio-Fiction film festival, a competition that will be held in Vienna on the 23rd to the 25th of October later this year. The competition features short films on all aspects of synthetic biology, including documentary films, animation, (science) fiction.QuestionnaireWe conducted interviews in our local community with questionnaires to collect qualitative and quantitative data in response to our video.Before showing the video, we asked:What does synthetic mean? What does natural mean?Do you know what synthetic biology is? What do you think it is?The majority of participants did not know what synthetic biology meant. We explained and discussed the concept of synthetic biology before asking two follow-up questions.Do you have a preference for synthetic or natural products?Why do you have a preference?We introduced the futuristic scenario before watching the video with the participants. After watching the video, we asked: What would you prefer: a GMO solution vs. chemical solution?Would you refuse to use synthetic products even if the natural products were toxic?The majority of participants would use synthetic products if the natural products were more environmentally damaging, and toxic like the chemical treatment of algae blooms.We then discussed the recent controversy over the use of synthetic biology ingredients in household products, and we asked:What concerns would you have using the product Cyanobuster? What information would you want on the packaging?Would you rather GMOs to be banned or labelled?In what products would the use of GMO be ok? Interestingly, a fair proportion of participants would still prefer GMO to be banned instead of labeled, despite a much smaller proportion refusing to use synthetic products even if the natural products were toxic.CIDEB-UANL CollaborationWe are collaborating with the Mexican team to collect an international response to our video in Mexico. Geographical location, country politics and culture play an important role in forming common understandings and public opinion. We are excited to compare the community’s opinion from a different continent to the response from our community. Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/HumanPractice/ActivityGALLERY From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ Having fun at the lab Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/GALLERParts From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ OUR PARTS! Parts submitted BBa_K1257001 BBa_K1257002Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/PartFAQ From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ FREQUENTLY ASKED QUESTIONS PROJECT GOALS1.How did your team come up with the idea of degrading microcystin produced by cyanobacteria?During our initial brainstorming sessions at the beginning of our project, we determined that environmental issues were to be the main focus of our work. After investigating a range of environmental issues, we came to the conclusion that underwater issues were becoming increasingly difficult to deal with and that it would be a good idea to centre our attention on a common algae problem which does not gain enough attention on a global scale.2.Realistically, does your project have potential to be used on an industrial scale?We are convinced that our project has enough potential to be used effectively around the world, including areas where algal blooms are prominent. This is demonstrated by the results we have obtained in our upscaling and bioreactor experiments. Although a number of challenges would need to be overcome to ensure the success of our product, we believe that a product with a function similar to that of Cyanobuster may have a place on the global market. CELL FUNCTION3. What safety precautions did you apply in order to ensure you were as successful as possible?As well as carrying out all of our investigations and experiments in the appropriate conditions, we applied a kill switch to ensure a greater degree of control over the production of microbes. This makes it possible to stop the production of a protein in order to increase the productivity of Cyanobuster. Moreover, we used an IRRE gene in order to ensure that the bacteria continue to perform in the harsh conditions present in some areas of algal bloom.4.Are there any substances that would impact the activity of your bacterium in degrading microcystin produced by cyanobacteria?The increased presents of nutrients, such as nitrates and phosphates, increases the presence of cyanobacteria in water columns, as demonstrated by our investigation involving different concentrations of the aforementioned substances This is largely due to poor farming practices, including the high use of fertilisers and the presence of livestock near water supplies.HUMAN PRACTICES5. What was the purpose of your human practice video?Our human practice video is branched into two parts. The first part of our video aimed to illustrate the effects of the increasing concentrations of cyanobacteria in water columns around the world, as well as its ability to harm ecosystems and society. The second part of our video focuses on giving the audience an idea of how Cyanobuster works and how it behaves in water columns containing algal blooms in the form of a television advert.6. How did your team generate publicity for your project?We spent a significant amount of our time generating a degree of publicity for Cyanobuster and our work. We created a Twitter account on behalf of UCL Academy, and also communicated a number of times with a team from Mexico where we discussed our progress. Moreover, we gave lectures to students at our school to outline the aims of our project, as well as creating a video to question whether synthetic biology is a sustainable solution for a given environmental problem.Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/FAFrom 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ Who you gonna call? CYANOBUSTERS! Scroll down to find more about our project! OUR PROJECTThe overall aim of our project to reduce or even eradicate the harmful effects of algal blooms. We aim to do this by making a GMO which can degrade Microcystin; a toxin produced by cyanobacteria. Microcystin is not only toxic to the immediate environment, but also to humans, domestic animals, and livestock. These algal blooms are mainly in regions where there is a high concentration of nitrogen and phosphorus in the water. This promotes more algal growth than the ecosystem can handle therefore there is an overpopulation of algae which can increase the concentration of toxins (such as Microcystin) in the water. Cyanobacteria are prokaryotic and due to their genus being very diverse, they’re able to occur in many different types of water such as marine and estuarine water. Top How does our project work? We have found three mlrA gene hmlrA in three different organisms, ''E.coli,'' ''Bacillus subtilis'', and ''Oceanibulbus Indolifex''. Microcystin is very resistant to many common bacterial proteases due to it’s cyclical structure. The mlrA gene codes for an enzyme, also called mlrA, which uses hydrolytic cleaving to make microcystin a linear molecule, and thus less resistant to the bacterial proteases. Top Who will our project help? Our project is aimed to help as many people and industries as possible. It could be used in domestic fish tanks, larger aquarium tanks, water reservoirs, local ponds, the uses are limitless! It’s more appropriate for use in areas in which having extensive filtration systems is not possible, and could possibly even replace the extensive filtration systems used in some places. Top Why did we choose this project? During our initial brainstorming sessions at the beginning of our project, we determined that environmental issues were to be the main focus of our work. After investigating a range of environmental issues, we came to the conclusion that water issues were becoming increasingly difficult to deal with and that it would be a good idea to centre our attention on a common algae problem which does not gain enough attention on a global scale. Top Retrieved from "http://2014hs.igem.org/Team:UCL_Academhttp://igem.org/wiki/images/6/60/Igemlogo_300px.png/Activity3 From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ HUMAN PRACTICE ACTIVITIES Synthetic vs Natural Synbio Outreach Commercial algal growth containment Commercial algal growth containmentAlgael growth is efficiently limited in commercial environments. Could these approaches apply to individual use, from fishtanks to local ponds? We conducted research on algal growth containment in two different environments - a zebrafish laboratory and a commercial aquarium. Visit to the UCL Zebra fish laboratoryThe 5000 tanks held at the UCL zebrafish lab all need to be kept free of algal growth. We visited the lab to find out more about their strategy. Tanks are emptied before cleaning as they are each hold about 1.5L only. The fish are fed through a solution that is put into their water rather than by the dry food you commonly purchase at pet stores. A small room next to the fish tanks contains the filtration system, a massive system including a UV chamber and several filters. This type of cleaning and filtration system is unfeasible on a household or natural lake scale. Therefore, our GMO would prove to be a more viable option for these types of settings.Meeting at the London Sea Life Aquarium We arranged a meeting at the London Sea Life Aquarium to investigate how they keep algal growth to a minimum in their tanks.Their main tips are:Tanks cleaned on the inside everyday to remove any algal growth (whilst the tanks are still full).ED Lighting rather than metal halide lights, as the latter create a lot of algal growth very quickly.Regular water changes; around 10 to 20 % of the water is removed and replaced by cleaner water; big water changes are difficult and expensive. Topped up with cleaned sea water. Professional salters to create that sea water, as it is artificial.Carbon filter in the tank which soaks up phosphates.Additional phosphate remover is often hung in a net bag in the water and replaced every few months.Denitrification filters grow a special type of bacteria to remove nitrates from the water.Fish food contains high levels of phosphates and nitrates; usually this isn’t a problem, but if you overfeed the fish too much of the food will rot and add more nitrates to the tank.This gave us the idea to test algal growth in varying phosphate and nitrate concentrations, leading to our algae experiments. Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/HumanPractice/ActivityTeam From 2014hs.igem.org HOME PROJECT ALGAE EXPERIMENTS PARTS HUMAN PRACTICE TEAM NOTEBOOK SAFETY GALLERY FAQ UCL Academy team UCL supervisors Mohammad HarigeI am 17 years old, from Persia and I study, Biology, Chemistry, Maths and Economics. I am striving to study medicine and hopefully become a neurologist.In my spare time I love to listen to music and play football. I am taking part in iGEM as it will allow me to acquire the skills I may need within the duration of medical school (if I get in).Ardit HashaniI’m from the small country of Kosovo in Eastern Europe. I study Biology, Chemistry, History and Mathematics at college. I am aspiring for a successful career in Chemistry after finishing university in England. I spend my free time playing football and I love listening to music. I am taking part in iGEM to improve and develop my practical skills, as well as gain a deep insight into the field of synthetic biology.Abigail DiazI'm part of the Wiki team (I spend more than enough time on the Internet already, anyway). I enjoy biology, chemistry, and procrastination. I wanted to be a part of iGEM to be able to get some extra hands-on experience in labs and working around biology, and to get some skills in different areas such as the human practices and wiki.Athena KourtiI’m 18 years old and aspire to study biochemistry specifying in molecular biology to become a geneticist. I currently study maths, biology, chemistry and psychology at A2 level and am involved in IGEM to gain experience and knowledge on what I plan to study as well as taking part in an interesting opportunity.Anna KrowitzI recently moved to London from South Africa. As well as science subjects I am studying A-level art. I think that the combination of art and science is exciting and I often find inspiration for my art projects in biology class. I have been involved mostly with designing the logos and presentation for the team. Jessica BalliaoI am currently studying biology, chemistry, mathematics and philosophy at AS level and am hoping to study medicine at university. In my spare time I visit galleries and museums religiously and make the most of what London has to offer. iGem for me was an amazing opportunity, the hands on experience and skills I gained in the labs are invaluable and has enriched my knowledge beyond the AS syllabus.Hashim SharifI am really interested in astrobiology and want to study it further at university. I'm really excited about the potential of synthetic biology to help us understand and colonise new planets. I've been a part of iGem for two years now and have learnt an awful lot from it that I hope to use in the future.Eniola SokaluI love all aspects of biology and synthetic biology has shown me the potential for solutions to all sorts of problems in the future. I particularly like being involved in the human practice aspect and communicating these ideas to others. I've also been involved in iGem for two years and have had a lot of fun being part of the team.Aurelija GrigonyteI am a graduating Biochemist, about to start a synthetic biology PhD. My key role is coordinating the team, and in the characterisation and construction of Biobricks. I am interested in iGEM as it combines both a new field of synthetic biology as well as interdisciplinary research.Josie FerreiraI am a biochemistry student about to graduate and start my PhD in Immunity and Infectious diseases.I think iGEM is amazing as it gives young people the ability to think beyond the scope of a classroom or lecture theatre and emphasizes the importance of creativity in the scientific world.Bethan WolfendenI am studying biochemistry, and responsible for coordinating the human practice aspect of iGEM as well as sponsorship.I love the potential of iGEM; by taking part in an undergradute-led research project, we get to truly experience the responsibility and independence of academic life.Miss Laura PriceHello, I’m Miss Price, head of biology at UCL Academy and this is the second year I’ve supervised the UCL Academy IGEM team. I am fascinated by the applications of synthetic biology and can’t wait to see what changes will be made to the world by these IGEM high school students in the future.Hadiza AutaI am a 3rd year PhD student in Biochemical Engineering Dept. at UCL. I focuse on the down stream processing of microalgae for biodiesel production. Due to the environmental hazards posed by fossil fuels, an alternative source of energy that is sustainable and can favourably compete with fossil energy is needed.I aim to use ultra-scale down engineering principles for rapid process development. By improving the technology of production of microalgae biofuels, we are hopeful that very soon algae will produce enough energy to fit the worlds demand.Desmond SchofieldI am an EngD student in the Department of Biochemical Engineering at UCL working on the production of a biotherapeutic protein for a novel cancer therapy. I enjoy interdisciplinary work and the application and commercialisation of scientific concepts. I am excited to be involved with iGEM highschool.Alex TemplarAlex is a third year EngD Biochemical Engineering student at UCL. His research focuses on improving PCR technology. He enjoys the spirit of iGEM and this is his third time mentoring an iGEM team.Darren NesbethDarren is a lecturer at Biochemical Engineering department at UCL. Darren teaches applied molecular and cellular biology to undergraduates and Masters students. He also spearheads UCL participation in the International Genetically Engineered Machines (iGEM) competition and serves as a competition judge. He initiated the collaboration between UCL and UCL Academy and acted as a supervisor for our iGEM team.Retrieved from "http://2014hs.igem.org/Team:UCL_Academy/Tea1299SKLBC-ChinaState Key Laboratory of Bio-ControlHigh SchoolWeIGEM+: A SNS-based Platform for iGEMWith the development of technology, the use of biobrick has been widen. However, it is not easy to find out the right one in thousands of biobricks. We have been dreaming a bioinformatics solution. Here comes the WeiGEM+, Wechat plus iGEM, which is a multi-functional public account that includes BioBrick search engine and iGEM articles. Biobrick search engine can be a useful tool for IGEM participants. With the Catalogs in the WeiGEM, users can search the ideal BioBricks by typing key characteristics in the text box. The science popularization part contains instant news on advanced science, including synthetic biology, life technology and brief introduction on previous works in iGEM and so on. Besides, human partice such as iGEM China meetup has been held by us in our region. With WeiGEM, synthetic biology can be spread to the global world and this can be a new way for leading science popularization.1427BBa_K1299999http://2014HS.igem.org/Team:SKLBC-ChinaBBa_K1299000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166220140High SchoolLog in   Team:SKLBC-China From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:SKLBC-Chinahttp://2014hs.igem.org/wiki/images/6/65/SKLBC-China2014-logo.png/Requirement From 2014hs.igem.org RequirementBack to Home     Register the team, have a great summer, and have fun attending the Jamboree.     During the whole of the participation, we have gained a lot while taking part in it. Having prepared carefully, we do understand quite a mass of new techniques and skills. Also, we are sure that we have had fun.    Create and share a description of the team's project via the iGEM wiki.     We have been busy working on our Wiki Project, in which we take efforts on its contents that had introduced our project to the audiences, showing our development and human practice, as well as the design of the wiki.    Present a Poster and Talk at the iGEM Jamboree. Moreover, we have already prepared for a Talk at the iGEM Jamboree.    Develop and make available via the The Registry of Software Tools an open source software tool that supports synthetic biology based on BioBrick standard biological parts (remember, the iGEM judges will be looking for substantial team-based software projects).     We have been dreaming of a project that help us to solve some issues that current situation of synthetic biology on biobrick searching with only sequence number is quite inconvenient, which has brought great difficulties to the scientific work, and Chinese citizens are lack of the understanding in biobricks. So we create WEiGEM+ which is a multifunctional public account that includes biobrick search engine, which is useful for users to search the ideal biobricks by typing key characteristics, and science popularization which contains a large number of articles, such as some introduction on previous works in iGEM, life technology, reviews on biological books or videos and so on.     Provide a detailed, draft specification for the next version of your software tool.    In addition to the supplement of articles, WEiGEM+ will be accomplished with a new Artificial Intelligence Function, with the automatic recognition of human voice and literal structures. The expansion of the platform, based on our users' feedbacks, is also on the schedule, which allows us to utilize WEiGEM+ on WhatsApp and Facebook, as well as other social platforms.    Provide a demonstration of their software either as a textual or video tutorial made available on their wiki. This tutorial should explain all the features of the tool as well as provide sample input and output as appropriate.     In order to make our users with a knowledge of WEiGEM+, we have exhibit a brief tutorial on our Wiki. Showing fundamental functions of WEiGEM+, the Tutorial uses videos and screenshots in details, with brief narrations in caption. Users can access http://2014hs.igem.org/Team:SKLBC-China/Tutorial for the Tutorial.     Have another team utilize the software developed by your team. You must clearly show how your software was used and the results that were obtained.    In our project, WEiGEM+, we provided an easy-going approach of searching for BioBricks and other necessary resources and supports. The Users of iGEM could input their keywords into the text box, and WEiGEM+ will make automatic inquiries in our databases. Once a matching result has been found, WEiGEM+ will give our users a feedback in the dialog box. In the box, there will be the related BioBricks and other supportive articles, which are all based on our user’s input. In a preliminary test, the Team SYSU_China, with requirement for enquiries, use our Search Functions to share Information on BioBrick conveniently.    Outline and detail how your software effects Human Practices in Synthetic Biology. Such topics include: safety, security, ethics, or ownership, sharing, and innovation.     In the WEiGEM+ project, we supplied a convenient system that will automatically push news and opinions on Synthetic Biology to users' devices. Through this system, our users can possess the access to a well-operated knowledge that maintained by our well-expertise editors. These articles, based on reliable and trustworthy sources, will provide our users a new experience with a new, innovative way that convinces them of the safety of Synthetic Biology. In addition, because of our convenient sharing routine, our users, who like our articles, could instantly share them via Weibo, Twitter, Facebook, or WhatsApp, which we think will spread our thoughts broader, convincing more people to understand Synthetic Biology and its applications. Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Requiremen/Student From 2014hs.igem.org TeamBack To HomeStudentTeacherPartnership Ica Riluci(Yuhua Wei)  Our INTELLIGENT webpage coder and art designer. Benny(Yuzhou Tong)  Our OUTSTANGDING writer, organizer and art designer. Wendy(Baiwei Huang)  Our GREAT organizer, writer and presenter. Tommy(Zhengkun Zhang)  Our EXECELLENT writer, organizer. Ivy(Haoying Huang)  Our AWESOME writer, organizer, publisher who is utterly enthusiastic. Sophie Yip(Shangshang Ye)  Our OMNIPOTENT writer, organizer and presenter. Icenowy(Xingda Zheng)  Our CHIEF coder, who is the genius of writing searching engines code. Daisy(Shu Dan)  Our EXTRAODINARY presenter and writer. Lillian(Yingyue Li)  Our CUTE art designer. Charlie(Chenglin Qu)  Our MARVELOUS writer, organizer and presenter. Peter(Junlin Zeng)  Our SPLENDIFEROUS presenter, writer and organizer. Zun(Zun Wang)  Our AMAZING organizer and writer. Team Participated in training Shaofan Wang, Junbin Xiao, Jingzhong Chen, Yifeng Zou, Yue Su, Hanming Li, Jiacheng Liu, Xiaoyu yang, Haojing He, Shenghao Wang, Tiantian Chen, Guoping Zhang, Qian Wang, Wenjie Xu, Xinyao Mo, Yue Pang, Xiaofei Jin, Yunyu Tang, Zhen Chen, Chenduo Tao, Xinyue Chen, Weicong Lin, Youming Guo, Xinhaoyun Wan, Zhe Jiang Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Studen/Teacher From 2014hs.igem.org TeamBack To HomeStudentTeacherPartnership Teacher Teacher Team James (Jun-Zhi WEN), TEAM LeaderWiser together, wiser the world!About James in iGEM-HS Johnson (Nie PENG), Project Manager Richard (Yong-Qiang ZHANG), Technology Teacher Serious (Yan ZHUANG), Operation Teacher Ying LU, Operation Teacher Hang LIU, Operation Teacher Lily (Li LI), Academic Teacher People who helped us Si-min LIU, Yi DAI, Yan HUANG, Guo-Pei HE, Tong ZHENG, Zhe-Fan XIE, Si-Min ZHANG, Beck HU, Gary YANG Advisor Liang-Hu QU Ling-Yan LIU Gui-Hui ZHENG Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Teache/Partnership From 2014hs.igem.org TeamBack To HomeStudentTeacherPartnership Supporter State Key Laboratory of Biocontrol(SunYat-senUniversity) WeChat Tencent Consulate General of United States Guangzhou China SME | Created in China Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Partnershi/Documents From 2014hs.igem.org DocumentsBack to HomeTutorialNotebook Documents Tutorial Notebook Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Document/Project From 2014hs.igem.org See Our ProjectBack to Home     Synthetic biology has been developing so quickly recently. It's a burgeoning field and has tremendous prospects for development, which benefits the society a lot and will change people's life greatly. However, people don't know much about it. They may know some words like recombinant DNA technologies, but actually they don't really understand it. They even think negative about it and avoid it.     Meanwhile, it's an era of intelligence. We need a more beautiful and convenient way to deal with about 20000 Biobricks,especially on mobile terminals. To satisfied these needs, we come across an idea: make a brand new searchi engine, which can both make searching much easier and can popularize synthetic biology. It's no longer a searching engine, we, SKLBC-China team, are designers, named it WeiGem+.     WeChat is the largest social system network in China. It's like WhatsApp, but with more functions and is compatible for iOS, Android system and PC. It would be our pleasure if you could install the application on http://www.wechat.com/en/download.html and follow us to have a taste of our work by scan our QR code. » «     Our work is consisted of two parts: the advanced search engine, and the push service platform,WEiGEM+. » «     For the first part, we provide the services of Biobricks searching in the of catalog searching and keyword searching.     Nowadays, there have been more than 20,000 Biobricks, but there hasn't a very convenient way to search for the Biobricks for mobile terminals, which has been a big problem.     According to this situation, we made a searching engine, with the Biobricks data from iGEM official organization.     We provide the services of Biobricks searching in the ways of catalog searching and keywords searching. » «     We didn't put this searching engine into a separated app, but made it link with the Wechat platform. WeChat is a Chinese app that has more functions than WhatsApp, and it can run on iOS, Android and PC.     All the data are storage on our server, so there's not huge data on user's device. Besides, there is an answering robot, when users input the keywords, the robot will give the feedback of searching results to the users.     After user begin a talk with the subscription account, the robot will launch a guiding massage, and the user can choose to use catalog searching (for Biobricks), keywords searching (for Biobricks), or article searching. Input "C", there will be a catalog, divide the Biobricks into different groups of types, such as DNA or Terminators. Moreover, user can type keywords to find the Biobricks linked to it, for instance, typing "purple", you will get 3 results:" M33355"," K1033906"," K1033919".     Next is push service of science articles. They can be harshly divided into 3 parts: iGEM projects, Recommendations and Focus Issues. » « iGEM projects: in this part, we write introductions to the best researches in a simple and understandable way, both in Chinese and English. The teams' wikis are affiliated and recommended by them. In this way, iGEM as well as Synthetic Biology is understandable, more straightforwardly. Recommendations: this is where we push excellent and fascinating science books, movies and speeches etc. All kinds of interesting resources can be found here, some of which are closely connected to articles in other two parts. The most compelling member would be GAMES. PLAY AND SAVE THE WORLD HERE! Thus, our followers can get more background knowledge and further understanding. Focus Issues: this part is about popular controversies, like Genetic Modified Food and health rumors. We present both for and against sides of the issues. In crowds of brainwash, we provide objective views. Hopefully, followers' critical thinking and elaborative faculties will be developed. We also build a search engine for theses articles. Enter "'#'+Keywords" to search Related Papers about a specific topic (Eg: #water) » « Q: Why we chose WeChat instead of developing an application or a website? A: We actually thought about them, but since Social Network System (SNS) has become a part of everyone's life nowadays, we regarded SNS as a better choice for its great convenience, sharing and propelling functions. In addition, WeChat, with over 600,000,000 users, is proved to amplify our influence. Now we have more than 500 followers and 300 selected articles only after 2 months work. With our effort, the number and influence will surely continue to explode in the future. Q: How do we spread our WEiGEM+? A: We have held lots of activities in schools, using our most compelling part-GAMES. Pictures: map of Guangzhou with our flags. » « Furthermore, we make close communications with other teams. We hosted a meet-up on 1st June, 2014, inviting teams in China. » « Needless to say, people who is impressed by our job would share WEiGEM+. » « You are unusual and have other special problems or insights, right? Feel free to contact us by sending an e-mail: JamesRNA(AT)gmail.com DO NOT hesitate to try our POWERFUL SEARCH ENGINE and WEiGEM+!! Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Projec/Team From 2014hs.igem.org TeamBack To HomeStudentTeacherPartnership     SKLBC_China team,sponsored by one of the State Key Laboratory for Bio Control in SYSU, an the first high school team in Guangzhou, China, is set up with students who are interested in synthetic biology and genetic engineer. Students from the key high schools in China including Guangdong Experimental High School,Guangya High School, Guangzhou No.6 High School and Zhixin High School, meet twice a week in SYSU to spread our ideas. Sparks caused by different inspirations from different teammates diversify our team work. With the belief of "Learning by Doing", we work in a "workshop" way. In our team, communication through different schools becomes not only a challenge but also a grand new try of an iGEM team. Our project mainly focused on the a social network system(Wechat) which combines a search engine and human practice project. We've held varieties kinds of activities in our schools, attracting other high school students interests in our project and iGEM. The special grouping way makes us unique! Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Tea/Notebook From 2014hs.igem.org NotebookBack to Home the first workshop welcome to join SKLBC-China Team! introduce ourselves be divided into 2 groups: Popularization Of Science Group and Academy Group build up our aim: a Biobricks Search Engines on wechat learn how to work in a team Academy Group  write the engine's code: Step1.Search Engines kernal  sort out all previous bioparts in iGEM competitions: Step1. 2014 HS teams bioparts Popularization Of Science Group  scheme to hold activities in some key high school Academy Group  write the engine's code: Step2. interface to wechat  sort out all previous bioparts in iGEM competitions: Step2. 2013 HS teams bioparts Popularization Of Science Group  finish the scheme of "Are you ready to welcome the unknown creatures?" Academy Group  test the engine's code  sort out all previous bioparts in iGEM competitions: Step3. 2013 Collegiate teams bioparts  design the WeiGEM platform: 5 parts: Tiny Message/Focus issue/About us/Video and Book/iGEM project Popularization Of Science Group  preparation of the activities Academy Group  test the engine's code  sort out all previous bioparts in iGEM competitions: Step4.2012 Collegiate teams bioparts  write and push WeiGEM content  write our wiki Popularization Of Science Group  hold an activities in Guangdong Experimental High School for a week  Name: "Are you ready to welcome the unknown creatures?"  *2 Groups amalgamated* Event in the Affiliated School of South China Normal University Event Name: Playing God play game foldit with the student in their Science Technology Festival spread the magic power of synthetic biology Preparation of iGEM China Meet-up  invite Gary Yang   (Gary Yang—business presentation coach and Ted speaking coach)  invite other iGEM teams: SYSU software team, SYSU China, Shenzhen_SFLS Children's Day: iGEM China Meet-up  Share experiment with other iGEM teams  Learn how to give speech in Gary Yang's class Workshop  be interviewed by a famous Guangzhou newspaper office  design the wiki home page Workshop  write a abstract and introduction of our WeiGEM+ Workshop  add more content in our WeiGEM+ resource platform Workshop  build up the frame of our presentation Workshop  make the presentation's PPT Getting a visa  get ready to the US! Presentation show  show our presentation to Bryon and Christina Workshop  complete our wiki page: Team, Project, Human Practice  improve our presentation Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Noteboo/Tutorial From 2014hs.igem.org DocumentsBack to HomeTutorialNotebook 1.Download and install Wechat in App Store or by google play. Versions for other platform are provided on http://www.wechat.com/en/download.html.   FAQ:http://www.wechat.com/en/faq.html 2.Sign up an accout. 3.Scan QR code to use our service: 4.Search a Biobrick by keywords. Eg:gfp. Just send @gfp to our WeiGEm+.   The result is like this: 5.View Biobrick catalog. Send c to WeiGEM+.   You can view the catalog of Biobrick and detailed information of the biobrick 6.Search articles on a topic. Eg: water. Just send #water to WeiGEM+. We will push articles related to the topic to you, Eg: water. Most articles are both in English and Chinese, a large percentage of our users are Chinese. 7.If you send the wrong command. We will give you a detailed instruction. Retrieved from "http://2014hs.igem.org/Team:SKLBC-China/Tutoria1273HTHS Trussville ALHewitt-Trussville High School Trussville, AL, 35173 www.trussvillecityschools.comHigh School701BBa_K1273999http://2014HS.igem.org/Team:HTHS_Trussville_ALBBa_K1273000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163520140High SchoolLog in   Team:HTHS Trussville AL From 2014hs.igem.org You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:HTHS Trussville AL team.png Your team picture Team HTHS_Trussville_AL Official Team Profile Contents 1 Team2 Notebook3 20134 2014 4.1 Results/Conclusions4.2 Safety4.3 Attributions4.4 Human Practices4.5 Fun! Team The Hewitt-Trussville High School team consists of seven students in the Biomedical Sciences Academy. Trussville, Alabama is rural community of approximately 20,000 people found eighteen miles northwest of Birmingham in Jefferson County, and Hewitt-Trussville High School has approximately 1300 students. Over the last four years, we have navigated our way through the Project Lead The Way biomedical sciences curriculum and worked together in labs. The seven members of our team are all 2014 seniors including Jessica Bacon, Darcy Echols, Nicole Hardesty, Nikki Newman, Sikandar Raza, Connor Staggs, and Chloe Wilks. We also have an amazing mentor from the Hudson-Alpha Institute of Biotechnology, Dr. Bob Zahorchak. But, most importantly, we want to save the endangered snail populations in the Cahaba River by developing a cheap and efficent phosphate detection plasmid for the Alabama Department of Environmental Managaement to use. Due to rising use of chemical based fertilizers, the runoff of harmful chemicals such as phosphate (PO43-) and nitrate (NO3-) into public water sources has increased. This accumulation of chemicals in streams and lakes is harmful to the environment. PO43- runoff in rivers is detrimental to aquatic life forms such as the Leptoxis compacta, a gastropod that was believed to have been extinct in 2000; however, in May of 2011 the Leptoxis compacta was rediscovered in the Cahaba River. PO43- is a food source for algae and as the levels of PO43- increase, the number of algae blooms increase and cover the surface of the water. This blocks sunlight so the energy cannot get to the bottom of the river. Currently tests are chemical in nature and specific to only certain forms of PO43- (testing only organic phosphate or orthophosphate). This research revolves around the creation of a biological plasmid to test for all forms of PO43- in a sample of water. Using a shuttle vector, the plasmid is first grown in E. coli, and then transferred into a specific type of yeast called S. cerevaise, which contains an outer sensor for PO43- .The sensor tests for the presence of PO43- because it is a food source for the yeast. If PO43- is present, then the yeast uses it for energy development; however, if no phosphate is present in the environment, then the sensor sends a cascading signal to a protein called Pho4, which binds to a gene called Pho5 to initiate the phosphate starvation cycle. This mechanism allows the yeast to produce its own phosphate. The plasmid that is inserted into S. cerevaise contains the genetic sequence for the Pho5 promoter that when activated will turn on a Red Florescent Protein (RFP). The Pho5 promoter will be removed from one plasmid using the restriction enzymes EcoRI and BamHI. An adaptor will then be used to convert the sticky end produced by BamHI into a second compatible EcoRI sticky end. After performing Polymerase Chain Reaction, this fragment of DNA will then be inserted into a new plasmid using 3A assembly. The new plasmid will already posses the RFP gene and antibiotic resistance which will be used to make competent cells. The recombinant plasmid will then be grown in E. coli before being shuttled into the yeast cells. The Pho5 promoter in the new plasmid will then be able to receive the Pho4 protein if it is initiated when PO43- is not present. When the Pho4 is bound to the Pho5 on the plasmid, the RFP gene will be activated. The RFP will cause the yeast to turn bright red, signaling that there is no PO43- present; if the yeast does not turn bright red, then PO43- is present in the environment. Therefore the plasmid serves as a qualitative and quantitative method to test for the presence of PO43- in a water sample, which in turn creates a biological mechanism that is not hazardous to monitor levels of PO43- . Notebook 2013 FOR ALL FIGURES RELATING TO PROJECT SEE THIS LINK: http://2014hs.igem.org/File:All_Figures_Corresponding_to_Wiki_PDF.pdf********************** August 26, 2013: 1. Determine if the Pho Sensor is the one the group wants to use 2. Find the yeast plasmid vector to put the sequence into 3. Determine the restriction enzyme needed to cut the plasmid (and put the sequence in) 4. Get RFP (Red Fluorescent Protein) DNA sequence 5. Find and determine if the inverter DNA sequence will work 6. Gibson Assembly 7. Put plasmid through PCR 8. Test with electrophoresis 9. Cut sequences out of gels if working 10. Inject plasmid into yeast 11. Grow Yeast 12. Test Yeast 13. Develop standardization test for red color 14. Water quality test September 17, 2013 To Do’s: 1. Download new software (Tinkercell and GENtle) 2. Redo Gantt Chart 3. Redraw plasmid 4. Start list of potential questions and problems 5. Finish scholarly article reviews 6. Put DNA sequences into software Find Pho5 sequence and promoter Find RFP sequence Find Plasmid Verify origin of replication Verify antibiotic resistance antibiotic vs. antifungal 7. Discuss restriction enzymes -See Figure 1 September 23, 2013: -First skype meeting with Dr. Zahorchak -Remove inverter from the plasmid -See Figure 2 and 3 October 2, 2013: To Do’s Before Visit to Hudson Alpha: 1. Fix the Gantt Chart 2. Make a flow chart of the process 3. Need software for tomorrow so we can look at it for plasmid creation. 4. Need to be able to discuss the following topics on complex levels: Restriction Enzymes Plasmids PCR Competent Cells What to use in place of antibiotics? What is a promoter and ribosome binding site? 5. Look through the following on parts registry Plasmids Yeast Parts October 3, 2013: Questions: 1) Is phosphate consistent throughout a river or are there different concentrations at certain spots? 2) Orthophosphate vs. Polyphosphate 3) Pho 89, Pho84”p”, and Pho87/Pho90 4) Are there more sensors than just those? 5) Target the transmitters so there could be a more colored result for the phosphate levels. 6) Will the stored phosphate in the vacuoles affect our plasmid? 7) Pho84 and excess amounts of phosphate 8) Could we turn off/bypass Pho4 9) Increase CDK? 10) How does the algae know that phosphate is in the water? Possible back up? Answers Dr. Zahorchak: Ortho PO4-2 vs. Poly PO4-2 OH | Ortho: 2PO4-2 attached to each other (bases sugar)--OH--P=O | OH Why trying to engineer these systems? ⇇ Detect PO4-2 (if it is present) fertilizer has a lot of PO4-2 Check latest ADEM reports for water quality How to detect levels? Determine sensitivity (down the road) What level will it switch on/off Qualitative data first Stored PO4-2 in a cell vacuole? Does that affect the test? Yes Think of a way to grow the yeast so that the cell can’t store subsistence in the vacuole (not a major priority right now) Make both programs compatible to use Pick One GENtle Wait to measure different levels of color Increase CDK? Do not want to mess with this Learn more about Yeast Transformation Lab? Either make your own kit or buy competent cells (pros/cons) Competent Cells Using antifungal to kill yeast that do not accept the plasmid Check biobricks for the yeast and vector Check for yeast iGEM projects from before/previous years iGEM material=free Make a list of what we need Include sequences What is this? Restriction Enzymes Notes from Dr. Zahorchak: Pho5 regulates phosphotase What enzyme(s) are included in the pathway? What if one of the sites you need is internal to Pho5? Find Pho5 promoter (BLast) Might need to find the exon. Need the entire Pho5 promoter Figure out if everything is on the RFP after Pho5 gene is fused Answers from Dr. Zahorchak (continued): Chemical test Converts PO4 to ortho Would our test look for orthophosphate or all forms? Test strips ($6) only test for ortho Chemical test ($45) converts all forms of phosphate into orthophosphate then tests Goals Before Christmas: 1)Finish Research 2)plug everything into GENtle and run simulations 3)materials 4)methods (plasmid methods) 5)Begin Plasmid construction October 9, 2013: Meeting and Discussion with Dr. Zahorchak: -Plasmid→ Antifungal DNA, Pho5, RFP DNA, Terminator Sequence -Restriction Enzyme Site -Gentle→ Plasmid, Pho5, Antifungal, RFP, entire plasmid -Why would we add RFP before Pho5? -Antifungal Agents -Amphotericin B -Aculeacin -Mulundocondin -Tunicamycin -Fluconazole -Itraconazole -Ketoconazole -Miconazole -Flucytosine -Terbinafine -Amcrofine -See Figure 4 October 16, 2013: Pho5 sequence found RFP sequence found Antifungal found RFP: GGCATTTTTT GTCATTTTTG GATGCAGATG ATTACTGGCA TCCAAAAAAA ACTAGAATTA CAACTATCAT TTATTAATGA TGAAAACTTG GATTTTTTAG GTTCAACGTG TTCCATTGGT GAGAAAAATA ACCAAGAAAT TAACCAAGGA ATTAAAAAAG AACATTTAAA ATTAAAAATA ATTTCATTTA ACATGATGTT GTTTAAGAAT TATTTCCAGA CTCCAGCTGT CATTATGAAA AGAGATATTT TTATTCCATT TAATGAGAAT CAGCGTTTTT CAGAGGACTA CATGTCATGG CTTGTTATCG TTTATAATAA AAAAACAAAT GTGGATTAAT ATATGGAAGG GATTTGGTTT TTCTCGATAA ATTTAACTTT GGAGTGTCAG GGTTGAGTGG TAATTTATGG TTGATGGAGA AGTGGGAGTT AAAAAATATA TTTAACTTCT TGTTGAAAGG TAAAATAATG GCAGTGCCTG CGATCTTGTT TTCTTTGATA AAATATGAAA GAAGATGCGC TTTAACAAAG AAAAATAAAG GTAAGGGTAA TAAATAATGA AGATCTCAAT AATAGGGAAC ACAGCAAATG CTATGATTTT GTTTAGATTG GATTTAATAA AAACACTAAC CAAGAAAGGG ATTTCAGTCT ATGCTTTTGC TACTGACTAT AATGATTCAT CCAAGGAAAT AATAAAAAAA GCAGGCGCCA TTCCTGTTGA TTATAATTTA AGTCGCAGTG GTATTAACCT TGCTGGTGAT TTATGGAATA CTTACTTATT AAGTAAAAAA CTAAAGAAGA TAAAACCAGA TGCTATTTTA TCTTTTTTTT CAAAGCCCTC TATCTTTGGA TCGTTGGCTG GTATTTTTTC AGGCGTTAAA AATAATACAG CTATGCTTGA GGGGTTAGGT TTTTTATTTA CAGAGCAGCC ACATGGAACT CCGTTAAAAA CAAAGTTACT TAAAAATATC CAGGTTCTCC TGTATAAAAT AATATTTCCA CATATCAACT CATTAATACT CCTTAACAAG GATGATTATC ATGATTTGAT AGATAAATAC AAAATAAAAT TAAAATCTTG CCATATTCTT GGTGGCATTG GTTTAGATAT GAATAATTAC TGTAAAAGCA CGCCACCAA AAATGAAATA TCATTCATTT TTATAGCTCG TTTGCTAGCA GAAAAAGGAG TCAATGAGTT TGTGCTTGCC GCAAAAAAAA TAAAAAAAAC ACATCCCAAT GTTGAATTTA TTATACTTGG CGCTATAGAT AAGGAAAACC CCGGAGGGTT ATCTGAATCT GACGTAGATA CTTTAATTAA ATCAGGAGTT ATTTCTTATC CCGGATTTGT TTGTAATGTG GCTGATTGGA TTGAAAAATC AAGCGTATTT GTTCTTCCTT CCTATTATCG AGAGGGAGTT CCTCGTAGTA CACAAGAAGC GATGGCTATG GGGAGGCCGA TTTTAACTAC TAATTTACCA GGCTGCAAAG AAACAATTAT TGATGGTGTG AATGGATATG TTGTAAAAAA ATGGTCACAT GAAGATCTTG CAGAAAAAAT GCTGAAGTTA ATTAATAATC CTGAAAAAAT AATCAGTATG GGAGAAGAAA GTTATAAGTT AGCAAGAGAA AGATTCGATG CAAATGTAAA TAATGTAAAG TTATTAAAAA TACTAGGGAT TCCTGATTA TAAACGAAAA GCGGCTCTGA TTCATTCGGA ACTAAGAAC TATCTCAATA GGAGCTAAAT TCATGACCTT ACCCAGCCAT ATCGAT Pho5 Promoter: GATCCGAAAG TTGTATTCAA CAGAATGCG CAAATATGTC AACGTATTTG GAAGTCATCT TATGTGCGCT GCTTTAATGT TTTCTCATGT AAGCGGACGT CGTCTATAAA CTTCAAACGA AGGTAAAAGG TTCATAGCGC TTTTTCTTTG TCTGCACAAA GAAATATATA TTAAATTAGC ACGTTTTCGC ATAGAACGCA ACTGCACAAT GCCAAAAAAA GTAAAAGTGA TTAAAAGAGT TAATTGAATA GGCAATCTCT AAATGAATCG ATACAACCTT GGCACTCACA CGTGGGACTA GCACAGACTA AATTTATGAT TCTGGTCCCT GTTTTCGAAG AGATCGCACA TGCCAAATTA TCAAATTGGT CACCTTACTT GGCAAGGCAT ATACCCATTT GGGATAAGGG TAAACATCTT TGAATTGTCG AAATGAAATG TATATAAGCG CTGATGTTT GCTAAGTCGA GGTTAGTATG GCTTCATCTC TCATGAGAAT AAGAACAACA ACAAATAGAG CAAGCAAADD CGAGATTACC AATGTTTAAA TCTGTTGTTT ATTCAATTT AGCCGCTTCT TTGGCCAATG CAGGTACCAT TCCCTTAGGC AAACTAGCCG ATG Anti-fungal Resistant Gene: TTAATTCATA ATCTAAGATT CAACTCATTA ACAATGTCTC CAGTTCAATT AGCAGAAAAA AATTACGAAA GAGATGAACA ATTCACTAAA GCTTTACATG GTGAATCTTA TAAAAAAACT GGGTTATCAG CTTTAATAGC TAAATCTAAA GATGTTGCTT CTGTTGCTGC TGAGGGTTAT TTCAAACATT GGGATGGTGG TATTTCTAAA GATGATGAAG AGAAAAGATT GAATGATTAT TCCCAATTGA CTCATCATTA TTATAATTTA GTCACTGACT TTTATGAATA TGGTTGGGT TCTTCATTCC ATTTTTCAAG ATATTATAAA GGTGAAGCTT TTAGACAAGC TACTGCTAGA CATGAACATT TCTTGGCCA TAAAATGAAT CTTAATGAAA ACATGAAAGT TTTAGATGTT GGTTGTGGTG TAGGTGGTCC TGGTAGAGAA ATCACAAGAT TTACTGATTG TGAAATTGTT GGATTAAATA ATAATGATTA TCAAATTGAA AGAGCTAATC ATTATGCTAA AAAATACCAT TTAGATACTA AATTATCTTA TGTTAAAGGT GATTTTATGC AAATGGATTT TGAACCAGAA TCATTCGATG CTGTTTATGC CATTGAAGCT ACCGTTCATG CTCCAGTTTT AGAAGGAGTT TATTCAGAAA TTTATAAAGT TTTGAAACCA GGTGGTATTT TCGGTGTTTA TGAATGGGTC ATGACTGATA AATACGATGA AACTAATGAA GAACATCCGTA AAATTGCTTA TGGTATTGAA GTCGGTGATG GTATTCCAAA AATGTATTCT CGTAAAGTTG CTGAACAAGC TTTGAAAAAT GTTGGATTTG AAATTGAATA TCAAAAAGAT TTGGCTGATG TTGATGATGA AATTCCTTGG TATTATCCAT TAAGTGGTGA TTTGAAATTT TGTCAAACTT TTGGTGATTA TTTGACTGTT TTCAGAACTT CAAGAATTGG TAGATTCATT ACTACTGAAT CAGTTGGTTT AATGGAAAAA ATTGGTTTAG CTCCAAAAGG TTCTAAACAA GTTACTCATG CTTTAGAAGA TGCTCTGTT AATTTAGTTG AAGGTGGTAG ACAAAAATTG TTTACTCCAA TGATGTTGTA CGTTGTTAGA AAACCATTAG AAAAGAAAGA TTAATGGGGC TTGACAAACA ACAAGTAAAC AGGGTGAGTT TATGTTGGGG GTGTTCAATT C October 23, 2013 Pho5 not made in high phosphate? -See Figure 5 Low Phosphate -See Figure 6 See Figure 7: Starvation Pathway The yeast already makes RFP so it would already be red. Grow it in high Phosphate Don’t worry about fusing RFP to Pho5 -See Figure 8 Stop codons/terminator sequence: Biobricks site Where does RFP come from? Jellyfish? Make sure codon sequence for RFP works in yeast November 4, 2013 Use shuttle vector and grow in E.coli Needs origin of replication E. coli does not produce protein that regulates Pho5 Compatible origin for yeast and E.coli So, no phosphate in E.coli Construct plasmid in vector Put in E.coli→ verify by PCR. Does it work? Plasmid has gene to make enzyme so that transformants will grow. Select Transformants Uracil metabolism Check out white/red colonies Find a selectable marker on plasmid Get the right yeast strain November 5, 2013 1) ORI E.coli 2) ORI Yeast 3) Pho5 Promoter 4) RFP 5) Ampicillin Resistant -See Figure 9 Meetings with Dr. Zahorchak Plasmid each time/change Notes on findings/changes November 6, 2013 To Do: 1) Verify that plasmid ORI sites for yeast and E.coli are present 2) If yeast is present match yeast type to ORI type 3) Find restriction enzyme that will make sticky end cut (after Pho5 promoter) that works with RFP, but only cuts beginning of RFP. 4) Continue research for plasmid November 12, 2013 Possible plasmid found pJRL2-PHO5prVYFP Already contains Pho5 promoter, but will need RFP and antifungal genes inserted. Insertion will most likely be done with Gibson Assembly -See Figure 10 Talk with Dr. Zahorchak: Decided it best to not use this plasmid as the final construct Best to create our own Instead this plasmid will be used for the Pho5 promoter Cut Pho5 promoter out using restriction enzymes BamHI EcoRI Adapters will be used to make the Pho5 compatible with iGEM standards (since RFP will come from iGEM database) Continue looking for a suitable plasmid November 13, 2013 Old Plasmid: -See Figure 11 Must have in new plasmid: ORI- E.coli ORI- Yeast Ampicillin resistance Pho5 promoter RFP Possible New Plasmid: -See Figure 11 To Do’s: Look at shuttle vectors on iGEM Relook at Pho5 promoter Methods: Ligation Competent Cells Conjugation November 15, 2013 Plasmid search still ongoing, but after a talk with Dr. Zahorchak what to look for is more clear. pUC ORI= E.coli ORI 2u origin= Yeast ORI Amp. marker Selection marker for yeast Decided to use a yeast antibiotic, such as G418, instead of an antifungal Questions to Answer: 1) What is the DNA sequence of each part? How will each part be obtained? If using PCR for parts what will be the primer sequence? 2) Sequences have all been found. Most parts will come from the registry and others synthesized. The parts that require PCR will have primers ordered with them. Primers come with parts from registry. 3) How will the pieces be put together? 4) Further research has shown that 3A Assembly is more effective than Gibson so 3A will now be used. Once a plasmid is found, adapters will be designed to link all parts for insertion. 5) How do you assure that the parts will assemble in the correct order? 6) The adapters will be designed to ensure the parts go where desired. 7) How will sequence orders be verified? 8) Horizontal gel electrophoresis will be run against a control. November 18, 2013 Plasmid found Contains both ORI’s, G418 (URA3 gene), and Amp iGEM compatible/registry compatible Also contains an RFP Pho5 will be inserted Research on G418 Aminoglycoside antibiotic Blocks polypeptide synthesis by inhibiting the elongation step in cells. Recommended concentration in yeast cells is 500-1000 mg/ml Resistance is confirmed by the neo gene from Tn5 encoding an aminoglycoside 3’-phosphotransferase ATP 3’11 -See Figure 12 November 25, 2013: Discussion with Dr. Zahorchak Since Pho5 is being cut from another vector, it will need to be PCRed. Primers will be worked on at a later date Most likely in person to be checked. Final plasmid will be first grown in E.coli because the transformation is easier making checking for the construct easier. The final plasmid will be then transfigured to yeast for final product and final testign. Need to make sure the promoter will control the RFP. Check end sequence for compatibility. COMPATIBLE OVERHANGS ARE NEEDED. December 16, 2013: Adapters: Very small piece of DNA with one restriction site on each end. Convert BamHI restriction enzyme site on Pho5 promoter, to and EcoRI site for insertion to plasmid (from database) and attachment to RFP. BamHI: -See Figure 13 EcoRI: -See figure 14 Decided on adapter sequence -See Figure 15 Order sequence from New England Biolabs December 20, 2013 Create methods for: Plasmid extraction 3A assembly Adding adapters Ligation Transformation Transfiguration PCR Create supply list 2014 January 15, 2014 Plasmid problem: The desired part from the registry was never submitted/unavailable Possible Plans: Use blank shuttle vector (contains E.coli ORI and Amp) and insert everything (Pho5 promoter, RFP, and G418) except yeast ORI Addition of yeast ORI exceed our current means so it would have to be done in the future Search for a new plasmid that contains the basics (E. coli and yeast ORI, Amp resistance, and preferably G418) E-mail original creator to request a copy of the plasmid. January 16, 2014 The Plan: Continue working as before and expect to us a blank shuttle vector. Yeast ORI can be added in the future by another group if need be While working, do brief research on any available plasmids with both ORI’s January 21, 2014 A new plasmid has been found! It contains a yeast and E.coli ORI, Amp, and G418 (KanMX). Only Pho5 and the RFP will have to be added, but all new adapter will have to be created. Dr. Z was able to find a colleague that had this plasmid on hand; he will deliver it to us in E.coli in order to start the process. Insert parts after SacI restriction sight See Figure 15* January 27, 2014 New adapter sequence have been created for Pho5 and RFP Adapter A: Attached to the 5’ end of the Pho5 promoter and will be attached to the plasmid pUG6 See Figure 16* Adapter B: Attached to plasmid pUG6 See Figure 17* Adapter C: Attached to the 3’ end of the Pho5 promoter and the 5’ end of the RFP (where RFP and Pho5 attach) See Figure 18* Adapter D: Attached to the 3’ end of the RFP and the 5’ end of Adapter B See Figure 19* Plasmid→A→Pho5→C→RFP→D→B→Plasmid February 3, 2014 Plasmid in E. coli and extraction methods have been received Process will begin after the next conference with Dr. Zahorchak to ensure everything is present See Figure 20* Febraury 6, 2014 Brief plan overview: Extract plasmid and run gel Cut RFP from travel vector Gel Add adapters Connect with Pho5 Cut Pho5 from plasmid Gel Add adapters Gel Connect with RFP Insert into plasmid Gel/PCR Transfer into E.coli Transfigure into yeast Final tests February 10, 2014 Extraction process has begun 3 different method being used A single colony of E. coli was mixed with the solution and shaken at room temp for 24hrs; Another was in an incubator for 24 hours; Another was heated and shaken. February 17, 2014 A buffer is missing as well as ampicillin from plasmid prep kit Snow days invalidated experiment so it must be redone Will be started after a meeting with Dr. Zahorchak to pick up missing items February 18, 2014 Meeting with Dr. Zahorchak a success. All necessary items are present and accounted for. The extraction process has been started once again Two samples (E.coli colonies) were incubated for 24hrs while in the solution then underwent standard protocol. Another two were shaken at room temperature for 24hrs before undergoing standard protocol. Different methods were used for the prep to see which were the most effective. February 19, 2014 Extraction has been finished. All checkpoints have been met so far. A gel will be run in the morning to check accuracy. February 20, 2014 Analyzed the gel with LoggerPro Extraction was a success Samples shaken at room temperature for 24hrs were the most successful .5% and 1.0% error showing the successful extraction of the plasmid from E. coli Next Steps: Once supplies arrive- Extract RFP and Pho5 Attach adapters Attach Pho5 and RFP Insert into plasmid Insert into E. coli Transform into yeast Note these are very general and not step by step methods March 1, 2014 Regional Science Fair at UAB Honorable Mention in Biology May 9, 2014 iGEM Tests with Dr. Zahorchak 1.5 agarose gels Plasmid Prep: Pho5 pUG6 Only 1 Pho5→ incubator tube No pUG6 worked (Spin columns were supposed to be taken out because it dried up the samples) Low melt gel Mixed incubator and shaken samples 1 restriction enzyme did not work (which one?) Band at 10,000 base pairs→ linearized Needs to be two bands (one at 500 base pairs one at 9,500) Order new restriction enzyme Pho5→ prep, cut once, run gel, next enzyme, run gel Verifies both enzymes work If works, low melt gel and purify Next Week Plasmid Preps Loading dye, flash gels, buffers, plates Order adaptors and restriction enzymes Anneal adapters Gels May 12, 2014 pUG6 cut at SacI: See Figure 22* Pho5 cut 5’ end at EcoRI: See Figure 23* cut 3’ end at BamHI: See Figure 24* RFP cut 5’ end at EcoRI: See Figure 25* cut 3’ end at PstI: *See Figure 26* See Figure 27* May 21, 2014 1.Gel Lanes (1) 2.pUG6 I1 3.Empty 4.pUG6 I2 5.Empty 6.pUG6 S1 7.Empty 8.pUG6 S2 9.Empty 10.Empty 11.DNA Standard Ladder 12.Empty 13.Pho5 I2 14.Empty 15.Pho5 S2 16.Empty 17.Pho5 S3 18.Empty 19.Pho5 I3 20.Empty 21.Empty 250 volts➼222 volts 1.5% agarose gel 0.5xTAE Gel Lanes (2) Pho5 I2+I3 Empty Pho5 S2+S3 Empty Empty DNA Standard Ladder 222 volts 1.5% low melt agarose gel 0.5xTAE To Do’s Today SacI digest of pUG6 EcoRI/BamHI digest of pJRL2 (Pho5 plasmid) EcoRI digest of Adapter A Gel Lanes (3) pUG6 1 pUG6 2 Pho5 1 Pho5 2 A B C D Small DNA Ladder Large DNA Ladder 1 cut of sample 2 microliters of dye 1.5% agarose gel 0.5x TAE Gel Lanes (4) Empty Empty Empty A Empty C Empty D Empty Small DNA Ladder Empty B Empty Empty Empty Empty Empty Empty Empty Empty 1 microliter of dye 5 microliters of adapter samples Gel Lanes (5) Empty A Empty A Empty C Empty D Empty Small DNA Ladder Empty B Empty Empty Empty Empty Empty Empty Empty Empty Flash Gel Lanes (1) DNA Ladder Empty A (annealed 5/8) C (annealed 5/8) A (annealed 5/19) C (annealed 5/19) pUG6 1 (extracted 5/20) pUG6 2 (extracted 5/20) Pho5 1 (extracted 5/20) Pho5 2 (extracted 5/20) Empty Empty Empty Flash Gel Lanes (2) DNA Ladder Empty pUG6 digest 1 (5/21) pUG6 digest 2 (5/21) Pho5 digest 1 (5/21) Pho5 digest 2 (5/21) A (annealed 5/8) C (annealed 5/8) A (annealed 5/20) C (annealed 5/20) Empty Empty Empty June 10, 2014 Flash Gel Lanes DNA Ladder Empty Pho5 1 Final Empty Pho5 Double digest Empty Pho5 2 Final Empty Pho5 2 Single Empty pUG6 Final Empty Empty Gel Lanes DNA Ladder Empty Pho5 1 Digest Empty Empty Pho5 2 Digest Empty Empty Empty Empty Empty Empty Empty June 18, 2014 Final Chemical Phosphate Test Results Cahaba River on Roper Road (6/16)- 2ppm Lake Purdy (6/15)- 1ppm Cahaba River in Bibb County (6/16)- <1ppm Dauphin Island (6/13)- 1ppm Cahaba River behind Hewitt Trussville High School (6/18)- <1ppm Run off pond behind Hewitt Trussville High School (6/18)- <1ppm Cahaba River on Grants Mill Road (6/14)- <1ppm Cahaba River behind Old Jr. High (6/14)- <1ppm Cahaba River behind Little League Baseball Fields (6/14)- 1ppm Cahaba River at the Trussville Bridge (6/14)- <1ppm Mobile Bay (6/13)- <1ppm Flash Gel Lanes (1) DNA Ladder Standard (8 microliters) Empty Pho5 plasmid prior to being cut pUG6 (I1) pUG6 (I2) pUG6 (S1) pUG6 (S2) Pho5 2 Pho5 cut June 19, 2014 Flash Gel Lanes (2) Empty DNA Ladder Standard (8 microliters) Pho5 cut with EcoRI and BamHI June 20, 2014 Single Digestion: Reaction 1 Pho5 2 (1 microliter) Distilled Water (7 microliters) EcoRI (1 microliter) EcoRI Reaction Buffer (1 microliter) Incubate for 1 hour Place in dry bath at 65C for 10-15mins Single Digestion: Reaction 2 Reaction 1 post incubation and dry bath (10 microliters) BamHI (1 microliter) NEB Buffer 3.1 (5 microliters) Distilled Water (34 microliters) Incubate for 1 hour Flash Gel Lanes (1) Empty DNA Standard Ladder (8 microliters) Digestion (5 microliters) 4-13. Empty Low Melt Gel Lanes (1) Empty Empty DNA Ladder Standard (8 microliters) Digestion (45 microliters) Results/Conclusions Over the past semester, we were able to take a years worth of research and begin experimenting. We were able to work through and learn from our mistakes to help make our project a success. Though the plasmid may not be finished, by reaching the final step of ligation, we are able to make plans to proceed forward in the future. Safety Q1. Ans. · E.Coli was used in this experiment as a rapid growth mechanism before the shuttle vector was palced into yeast. E.Coli is a biosafety level 1 organism, as natonal stnadards of this organism characterizes this organism as not known to consistently cause disease in healthy adults. By this standard it is recommended for those coming in contact that they where lab coats, eye protection, and gloves. · If project goes according to plan, team member, the public, and the environment will face no risks. Proper lab equipment, such as an autoclave, and protective-wear, such as gloves and lab coats, will be essential to allow no risk. Through lab safety training every year in high school showing lab-safety procedures and handling of various apparatus. · If proper lab-safety procedures are not followed, then the E.Coli has the potential to enter a physical opening in a group member, which will place the group member at risk for illness. If this interaction occurs, the group member will be transported to the hospital for a check-up with his/her doctor to ensure no potential health risk. Q2. Ans. · No parts were submitted to the registry. Q3. Ans. · The Trussville City Schools Board of Education is our local biosafety group, and they are in support of the materials and methods that use the bio-hazardous organisms. Q4. Ans. For future teams, opening registration earlier, as well as sending out kits earlier would be helpful for a quicker and more accurate route to success. Attributions The lab work as well as research was split up evenly amongst group members. Dr. Zahorchak and Mr. Walters served only as mentors and guidance, listening to our ideas, but never placing their own upon us. They gave us space to work and perform our project but were never truly, physically involved in any lab work or research. Human Practices Our project will allow a safer way to test for phosphate levels. Current tests include harmful, poisonous chemicals. This test will also make the public aware of the runoff that they are placing in their streams, harming the aquatic life. Fun! Our team spent many hours working hard on research and notes at Starbucks, so naturally scones and coffee were our favorite snack. Retrieved from "http://2014hs.igem.org/Team:HTHS_Trussville_AL2014hs.igem.org/wiki/images/a/a1/HTHS_Trussville_AL_logo.png1264BHSF-BeijingBeijing No.4 High School A2 Xihuangchenggen Beijie, Xicheng District, Beijing China http://www.bhsf.cnHigh SchoolFormaldehyde bio-monitorAs an important industrial chemical, formaldehyde is widely used to manufacture building materials and household products. Continuous exposure to formaldehyde can cause adverse health effects, such as eye, nose, throat and skin irritation, chronic respiratory symptoms, or even cancer. The BHSF_Beijing team has developed a new system for sensitive detection of formaldehyde exists in our living environments.1601BBa_K1264999http://2014HS.igem.org/Team:BHSF-BeijingBBa_K1264000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162420140High SchoolLog in   Team:BHSF-Beijing From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:BHSF-Beijing/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:BHSF-Beijing logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:BHSF-Beijing team.png Your team picture Team BHSF-Beijing Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project As an important industrial chemical, formaldehyde is used to make building materials and many household products, etc.. Continuous exposure to formaldehyde can cause adverse health effects. To solve this problem, we have come to an idea of reforming the harmless E.coil with synthetic biology techniques and make them have the ability to degrade formaldehyde exists in our living environments. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:BHSF-Beijing1260Shenzhen SFLSShenzhen Foreign Languages Schcool Shenzhen, Guangdong, China http://www.sfls.net.cnHigh SchoolLǘdagunrOur project is named as Lǘdagunr, whose chinese meaning is a kind of traditional snacks from China. Aflatoxin is a highly toxic substance which exists on grains. The most poisonous kind of aflatoxin is Aflatoxin B1 (AFB1). Since it is difficult to degrade the toxin, we decided to use ScFv (single chain variable region fragments), an antibody, to neutralize it. We used ScFv to neutralize AFB1 because it has been used before and its molecular modification is easy. We used ScFv, SH3 ligand and 6*his-tag to construct fusion protein so that it can combine with SH3 domain. In this way we can use cells to recover the ScFv that have been expressed out to reduce the antibody-antigen complex that exists on grains. We anchored SH3 domain on E.coli's cell membrane LGT to recover the ScFv. Keywords: AflatoxinB1(AFB1),ScFv, SH3 ligand, SH3 domain, LGT2014BBa_K1260999http://2014HS.igem.org/Team:Shenzhen_SFLSBBa_K1260000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161820140High SchoolLog in   Team:Shenzhen SFLS From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SFLS1255CIDEB-UANL MexicoCIDEB (Centro de Investigacion y Desarrollo de Educacion Bilingue) Monterrey, NL, Mexico www.cideb.uanl.mxHigh SchoolE. CARUiGEM CIDEB 2014 team?s project is a bio-filter of sodium ions in salt water. In order to work and test the different processes of the project, it was divided into four modules, which also made up the acronym of our project?s name: Capture, Aroma, Resistance, and Union. Capture module is in charge of recollecting sodium ions in the water due to the new NhaS gen registered by the team. Aroma module is with the help of salicylic acid, responsible of producing a Winter Green odor that acts as a reporter if the bio-filter is working. Resistance module allows the E. coli to survive in the salty environment of the water, and this allows the bio-filter to works. Finally, the Union module allow the bacterium to joins to a silica pearl, which facilitates the removal of the bacteria from the water.1862BBa_K1255999http://2014HS.igem.org/Team:CIDEB-UANL_MexicoBBa_K1255000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161120140High SchoolLog in   Team:CIDEB-UANL Mexico/labwork discussions From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Results Interpretation Here is the interpretation of the results that were obtained on all the experiments Capture module - [Go to Results] Ligation of NhaS and pSB1C3 The ligation transformed in E. coli of the NhaS module produced red and white colonies when we expected only red colonies (bacteria expressing the RFP). We did not know the reason of the unexpected result so we designed an experiment with the UV light promoter. The NhaS module was proved in the experiment with the Petri Dishes in the UV camera. The red bacteria was already red (meaning that the RFP expression already started) before being exposed to the UV camera at 302nm. The promoter pUV 1765001 is activated by the UV exposition under 360 nm as the iGEM team Colombia_Israel 2007 reported. (iGEM Colombia_Isreal, 2006) The UV promoter does not have a reported wavelength interval. The unexpected result obtained can mean that the promoter is so sensible to the UV light that the normal UV radiation is enough to activate it without the need of being exposed to the UV camera. In the normal light the UV rays are present in different ammount as it is presented in the Global Solar UV Index publication. (World Health Organization, 2002) In the part description is reported that the UV promoter gets active and the protein must be expressed after 10 minutes of exposure in the UV camera, but after 2 hours there was no change in the white colonies so the RFP was not expressed there. After doing the experiment we obtained that the red colonies continued with the RFP expression and the white colonies did not changed in color. As we expected to activate the RFP expression in the white colonies, this means that the time of the exposure was not enough to activate the UV promoter or the promoter did not work in the conditions we thought it would work. To see if the promoter was already activated we did another experiment. The experiment with NaCl in different concentrations in Petri Dishes showed that the bacteria grew in a medium with high NaCl concentration. The control group with non-modified bacteria did not grow because it did not contain the Nhas insert so it was not able to survive in a saline medium. The white bacteria did actually grow but they did not expressed the RFP, but the fact that they did grow means that they have the NaCl resistance and the insert is inside them. As the white and red colonies are supposed to come from the same ligation and to contain the same genetic information we need to prove that the insert was inside them. In order to prove this we sent samples of DNA to be sequenced to the DNA Synthesis and Sequentiation Biotechnology Institute Unit (USSDNA in Spanish), from the UNAM. The primer used was in the complementary reverse chain, so the sequences are in the 3’ to 5’ direction. We did an analysis of the sequences obtained by aligning them with the BLAST Software. The RFP sequence used in the alignment was the following (in 5' to 3' direction): Atggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaa cggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaac tgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggt tccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggttt caaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgc aagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatg cagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaagg tgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctaca tggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccac aacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaata acgctgatagtgctagtgtagatcgctaa It was aligned with the sequences obtained from the samples Nhas white bacteria and Nhas red bacteria. NhaS sequence from white colonies (in 3' to 5' direction of the complementary reverse) TAAATAAAAAGTTTTTTCTAATGCGTTTCTTCTCCTACAACCGAAAACACCGGGTCAGTGAGCGAGGA ACCTGCATAACGCGAAGCACGCTTTTCCGCAAGAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTT TTTGCCGGACTGCAGCGGCCGCTACTAGTATTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCA CCGGTGGAGTGACTACCTTCAGCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGT GATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGG TTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGA GCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGG ACCGTCGGACGGGAAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGG AGTCCTGGGTAACGGTAACAACACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCC GGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTGTTTAACGTAAGCTTTGGAACCGTACTG GAACTGCGGGGACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGG TCTGGGTACCTTCGTACGGACGACCTTCACCTTCACCTTCGATTTTCGAACTCGTGACCGTTAACGGA ACCTTTCCATACATGACCATGTTCTCTCGTCTGATTAGCATCGTGAGCCTGATTCTGTCCTTCTACTT CGCTTACAAATACCGTTATCGTGTGATTAACGCGGTGCTGGGCCGTCGCTGGCTGCGTAAAGTTATTA TCGGTTTTGCCATGCAGATTCCGATGATTCGTGACCGTATGCTGGGTAGCGTTCTGCAAAGTAACCGT CCGCAAAATGTGTAA NhaS sequence from red colonies (in 3' to 5' direction of the complementary reverse) AAAGTGTCCACCCCGTACGACCGAGCGGAGCGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTA ATCTTTTCGGCTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGC CGCTACTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGT TCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGC CTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACGACCTTCA GCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGT TTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGT GACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGAGCACCGTCTTCCGGGTACATA CGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGGAAGTTGGT ACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAA CACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAG TCCGGGATGTCAGCCGGGTGTTTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGAACAGGATG TCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGGTCTCGGGTACCTTCGAACG GACGACCTTCACCTTCACCCTTCAATTTTCAAACTCGTGACCGTAAACGGAACCTTTCCATACAACTT TGAAAACGCATGAAACTCATTTGAATAACGTCTTCCGGAAGAAAGCCCAATCTAAGTATTTTCTCCCT CTTTTCTCATATAAATGTGATGAATATTTGATCTATCCGCCCTCCAACAACTTTCCCACAACAATCAT GTATCGAAATTCCTGTTATACGACACTATAAAGATGGTATAAAAAGCCCGTGGAGGGGGCGTGACCA Report The report obtained from the analysis with the NhaS in red colonies is the following: Image 1. Report generated by the software BLAST with the alignment of the RFP original sequence and the NhaS in red colonies sequence. The RFP original sequence has a length of 709 bp. The match started at position 3 and ended at position 708, this means that almost all the RFP is present in the sample sequenced as we expected because the colonies were red. The report obtained from the analysis with the NhaS in white colonies is the following: Image 2. Report generated by the software BLAST with the alignment of the RFP original sequence and the NhaS in white colonies sequence. The match ends at the 709 position from the original RFP sequence, but it did not start from the position 1 or 3, it starts at position 50. This means that there are 49 nucleotides that did not match with the original RFP sequence. This can be a possible cause in the problem with the RFP expression in white colonies, a mutation in the region of the RFP. We also made an analysis with the Ribosome Binding Site (RBS) sequence: Image 3. Report generated by the software BLAST with the alignment of the RBS original sequence and the NhaS in white (which presented 1 match) and red (which presented 2 matches) colonies sequence. In the red colonies there where two matches, which is the complete sequence but in two parts: from 1 to 7 and from 6 to 12 positions. In the white colonies there was only one match, this means that the RBS sequence was not found there. If the RBS previous to the RFP has a problem, the mRNA cannot bind in the ribosome, and is not able to be translated. Image 4. The region of the Biobrick at which a possible mutation could had happened. With these results we can infer that the BioBrick works find and expresses the NhaS (because both of them survive in a high NaCl concentrated medium) but it stops being translated in the RBS or in the RFP region causing the colonies to be white instead of red but being able to survive in a medium with high NaCl concentration. The question is if the problem is caused by a mutation, When did it happen? The ligation transformed contained a DNA obtained from a digestion done the May 16th in the 3rd week registered in the Notebook. This digestion was exposed to the UV light camera at 302nm for about 5 minutes. After the digestion it was ligated and purified. Later it was transformed in E. coli. The chance of occurring a mutation of this insert was 1) before the miniPrep, inside the cell or 2) due to the UV radiation after the transformation. The UV radiation at 312nm can cause a damage in the DNA sample and to reduce the succes in transormation in E. coli (Gründemann, 1996). There are also several types of mutagenesis due to the UV radiation inside the cell (Ikehata & Ono, 2011) that can have occurred before the transformation to some samples of the plasmid. The red bacteria had the original DNA and the white bacteria had the mutated DNA. Return to the Top UV experimentation In this experiment it was expected the white bacteria to express the RFP protein to be equal as the red colonies. But after the experiment was done there was not a change and we can conclude this is because the white transformed bacteria had a mutation on the RBS region for the RFP gene leading to grown colonies without the RFP (red color) and according to the Department of Biochemistry, University of Turku, Finland when the ribosome-binding site (RBS) sequence, AGGAAA, was altered to AAGAAA, PPase production decreased to 19% of the wild-type. Surprisingly, when the RBS sequence was mutated to the consensus RBS sequence, AGGAGG, the intracellular levels of both ppa mRNA and PPase decreased drastically(Department of Biochemistry, 1991) meaning that a mutation in the RBS region leads to a malfunction of the gene and a decrease of the production of it.. The implications of these results are discussed.. This explains why we obtained two different transformed bacteria: the white and the red ones. About the red colonies it can be stated that the expression of the RFP protein is due to the sensitiveness of the UV promoter, it means that the promoter is so sensitive that even though the plates were inoculated with no light they showed the red color. The interpretation is that there is still UV radiation that does not belong to visible light spectrum according to the Global Solar UV Index publication (World Health Organization, 2002) so the promoter was activated with the UV radiation present in the lab. When the red bacteria were grown in a petri dish some white colonies grown as well along with the red bacteria, it can be concluded that two things happened: 1) At the moment of lifting bacteria from the petri dish to the tube some white bacterium came in the micropipette and were streaked in the petri dish with the red bacteria. 2) Also there is certain predisposition to mutation of the RBS of the GFP so white bacteria does not express RFP because of the mutation that occurred in the purification. Viability test of the NhaS gene containing bacteria in salt - [Go to Results] The first time the experiment one was performed, bacteria transformed with the capture plasmid were inoculated in Petri dishes with different concentrations of salt. All the bacteria transformed with NhaS in pSB1C3 (Red and White) lived in the 15% saline medium. This means that these bacteria have the resistance and support a medium with high NaCl concentration. All the control bacteria exposed to any concentration of salt died. This result supports the fact that the transformed bacteria with Nhas are more able to survive in a high NaCl concentrated medium. It means that this group (not transformed bacteria) did not have resistance to a medium with a high NaCl concentration. All the control bacteria inoculated only in LB medium (without salt) lived, as we expected because they were inoculated in normal conditions. As in the experiment 1 was confirmed that the bacteria survived in a medium with LB and salt, in the experiment 2 the bacteria was inoculated with salt and without LB medium. The bacteria did not survived and this can be explained by the fact that the medium contains nutrients and substances the bacteria needs to survive. In the experiment #3 after analyzing the data in tables, the results were positive, but inconclusive. With "positive but inconclusive" it is meant to say by this, is that the colonies with NhaS only displayed a higher number of colonies in the medium, while the other groups showed a higher death toll among bacteria expressed in less colonies. With inconclusive it is meant to say some colonies were contaminated with other groups from the same experimented bacteria and in some of these contaminated groups, only the contaminant (NhaS) survived while the other bacteria died. Only the NhaS only and control bacteria displayed a correlation in the data showed as a result of the experiments, so this same experiment is planned to be repeated to obtain better, more viable results in order to finally test the effectiveness of the nhas protein production. Aroma module - Characterization The 3 sequences obtained from the DNA Synthesis and Sequentiation Biotechnology Institute Unit (Go to sequences) were aligned with the MEGA software and the complete original sequence was found in the 3 samples. This indicates that the BioBrick was cloned in E. coli and that the part was actually there. Image 5. Alignment of the 3 sequenced samples, with the original sequence of the Aroma gene, done with the MEGA software Qualitative experiment People had different opinions while smelling the different Petri dishes with the aroma transformed bacteria. All of them used different words but at the end most of them remitted to the same meaning. The MIT team documented that the odor was produced when it was added 2mM of salicylic acid, so it was expected that the Petri dishes did not smell so much, but the results indicated that there was a mayor odor at 10mM than at 2mM (experiment previously done). In the samples of 20 mM, both of the bacteria that was grown under 32 ºC had a little smell, which in theory, should not had happened because the riboswitch should be a loop at that temperature. One posible explanation is that the riboswitch is very sensitive to heat, and it could be activated during the time in which the Petri dishes were outside the incubator to perform the experiment and with the heat of the hands of the people who smelled it. Finally, all of the samples that had a concentration of 30 mM of salicylic acid smelled like rotten food. By this, it can be inferred that all the bacteria in the Petri Dishes could not withstand the condition that particular concentration of salicylic acid, therefore they died, because “growth in a subinhibitory concentration of salicylic acid resulted in a significant reduction in the number of bacterial cells and a reduction in the rate of the number of bacteria increasing during logarithmic growth” (Bandara MB, et. al. 2006) Return to the Top Bibliography/References ● Bandara MB, et al. (2006, October). Salicylic acid reduces the product... [Invest Ophthalmol Vis Sci. 2006] - PubMed ●Department of Biochemistry, University of Turku, Finland. (1991). Characterization of the 5' flanking region of the Escherichia coli ppa gene encoding inorganic pyrophosphatase: mutations in the ribosome-binding site decrease the level of ppa mRNA. 06/20/2014, of NCBI from http://www.who.int/uv/publications/en/GlobalUVI.pdf ● iGEM Colombian Team 2007 (2007, October 26). Part:BBa I765001. Retrieved June 15, 2014, from http://parts.igem.org/Part:BBa_I765001 ● iGEM Colombian Team 2006 (2006). A Microbial Biosensor Device for Iron Detection under UV irradiation. Retrieved June 15, 2014, from http://2007.igem.org/wiki/index.php/IGEM_2006_Project ● NCBI. Retrieved June 13, 2014, from http://www.ncbi.nlm.nih.gov/pubmed/17003439 Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANL Retrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_discussionsteam From 2014hs.igem.org Students Advisors Instructors Assistants Fun Students Angelina G. Miranda Lara   Age: 17 -Nickname: Angie Hobbies: Singing, reading, sleeping (lots) and yes, talk through a Walkie. What do you want to study? Medicine :)  Why iGEM? Because I love anything related to science and I love learning something new. I think biology is the most interesting science so why not?   Diego Iván Valadez Lozano    Age: 18  Nickname: Vala  Hobbies: Eat, watch movies and play videogames.  What do you want to study? Computer Sciences  Why iGEM?  Because I became interested in the applications of this science and I wanted to learn the work in a molecular lab.   Arnulfo A. Peña    Age: 18  Nickname: 18+ (18 mas), papá pato.   Hobbies: Dance, play videogames, practice martial arts, listening to music and being very curious  What do you want to study? I want biotechnology or nanotechnology  Why iGEM?  Because it is a very interesting competence for it involves a lot of biology and also the implementation of new ways of scientific manipulation in order to create something better and improve the actual processes as well. I think that the area of biotechnology doesn't have a limit and that inspired me to join this team.   Antonio Cárdenas Hernández    Age: 17  Nickname: Toño, no Tony  Hobbies: Reading, play video games, play basketball, but mostly: Be Weird, BE SAFER!!!  What do you want to study? Medical Biotechnology and Neurology  Why iGEM? iGEM is a big opportunity to demonstrate and to acquire more knowledge in diverse areas, including biology, laboratory and math, social sciences; and also it represents a big challenge: I’ll never regret for taking it!!!    Odalisse Ibarra    Age: 16  Nickname: Ody  Hobbies: skateboarding, watch movies (fantasy drama films, science fiction psychological thriller films), reading, listening to my favorite music, go to the cinema *alone*  What do you want to study?Mechatronics(?) Medicine(?) Architecture(?) my idea of studying at this moment is unclear, but any of the above are my interests.  Why iGEM? Because I had never been in a disciplinary project that has significant and real applications. Moreover, I’ve always had preferences in science.   Jesus Delgadillo    Age: 16  Nickname: Yisus  Hobbies: Reading novels; drawing machines; cooking and then eating; Leave some boogie on the dance floor; Watch movies in foreing languages (some with subtitles); make people laugh.  What do you want to study? I don't know what will I study; maybe something related with arts and humanities.  Why iGEM? Because the lab procedures are really interesting for me; iGEM is a big project which comprehends many activities, people and skills.   Jorge Luis Hernandez Ayala    Age: 17  Nickname: Choche  Hobbies:Blogging, reading, growing trees and annoying my cats  What do you want to study?: Medicine?, Biotechnology?, Aeronautics engineering?; I still don’t know yet, but I know it will be something related to science and its applications.  Why iGEM? Because it is the most interesting project that I have been involved, and I love practical applications for the biology and doing things in the lab.   Adrian Alejandro Rodríguez Rodríguez    Age: 17  Nickname: Adribubu  Hobbies: Listen to music, watching movies, sleeping, running, reading, hanging out with my friends, meeting new people.  What do you want to study? Biotechology  Why iGEM? Because I like working in the lab, and for me iGEM is a very interesting project where I can develop my skills and learn more about science nowadays.   Ingrid Melissa Chávez de la Portilla    Age: 17  Nickname: Mely  Hobbies: Hanging out with friends, listening to music, doing gymnastics and going to church  What do you want to study? Medicine.  Why iGEM? Because I like what it is done and it seems interesting to me. I like Biology, so I decided to try it out.   Joel Abdias Aguilar Orta    Age: 17  Nickname: Jlouz or Chol (a variation of Schöll)  Hobbies: Listening to music all day, cooking, sleeping, going where the wind takes me.  What do you want to study? Industrial and/or chemical sciences.  Why iGEM? Because it is an opportunity for me to develop knowledge in another branch of sciences. Also because, “why not?”   Raúl de la Garza    Age: 18  Nickname: Raúl  Hobbies: Playing drums, listening to music and being in the lab.  What do you want to study? Biotechnology  Why iGEM? (Why not?) I love exact sciences and therefore, like synthetic biology.   Dulce Valdez    Age: 16  Nickname: Dulce de leche  Hobbies: Hugging people and cooking brownies  What do you want to study? Biotechnology  Why iGEM? Because when I heard about Synthetic Biology I just fell in love, and I wanted to try something new and make new friends   Fernanda Puente    Age: 17  Nickname: Nanda  Hobbies: Playing video games, reading, listening to music, eating tortillas and being a Safer.  What do you want to study? Biotechnology  Why iGEM? Because I’m very interested in biology and genetics and iGEM pretty much sums it up.   Eva Quintana    Age: 17  Nickname: EVA (Wall-E <3) or YOLO  Hobbies: Dancing Ballet, listening to music, reading books, being me.  What do you want to study? Biotechnology  Why iGEM? Because I can learn a lot of things, I have lots of fun and I love science.   Irina Romero Guerrero    Age: 17  Nickname: :C  Hobbies: Reading, listening to music, eating.  What do you want to study? Biotechnology.  Why iGEM? Because I like biology, and I love to learn and discover new things.   Lisandro Hiracheta Torres    Age:16  Nickname: Lisandro  Hobbies: listening to music, playing videogames, sleeping and painting  What do you want to study? Medicine  Why iGEM? It was a great opportunity to know I can work with a group, and it was a challenge where I wanted to test myself   Andrés Marcelo Jiménez González    Age: 16  Nickname: Copernicus  Hobbies: Solving equations, reading algebra books, watching movies about statistics, and dreaming about math.  What do you want to study?  Actuarial Science  Why iGEM? I consider it a really interesting project, in which the projects can be later be use on real applications.   Marina Emilio Aguirre    Age: 17  Nickname: Tortuga /Turtle  Hobbies: Reading, playing basketball, painting, eating chocolate (it is my favourite), sleeping (well, it is my favourite too!), drawing, I think anything.  What do you want to study?: Astrophysics ;D  Why iGEM? I don’t really know, I just love any science and it looked pretty interesting and great. Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/teateam assistants From 2014hs.igem.org Students Advisors Instructors Assistants Fun Assistants   Alexandra Lab Work   Alan Lab Work Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/team_assistanproject parts From 2014hs.igem.org Description Capture Module Aroma Module Resistance Module Union Module Parts Parts New Parts Submitted Name Part Type Description Length BBa_K1255000 nhaS Coding Putative sodium binding protein that we, iGEM CIDEB 2014, are going to introduce to iGEM for the first time. 207 bp. BBa_K1255001 BSMT1 opt. Coding Wintergreen-odor enzyme generator, used to allow the production of methyl salicylate, when it is induced by salicilyc acid. This year, the team optimized the sequence for Escherichia coli making a new biobrick. 1074 bp. BBa_K1255002 pUV + RBS + nhaS + RBS + RFP Device NhaS with an RFP reporter induced by an UV promoter. 1041 bp. BBa_K1255003 pConst + RNA Thermoswitch + BSMT1 Device BSMT1 opt. activated with a constitutive promotor, regulated a RNA thermoswitch 1206 bp. Parts Used Name Part Type Description Length BBa_K729001 IrrE Coding Protein that changes the bacteria’s metabolism and allows bacteria to survive to extreme conditions, some examples could be high UV rays exposition, or high salt concentration levels in an aquatic environmen, oxidative or thermal shock. 933 bp. BBa_K888000 L2 Coding L2 protein of adhesion to silica 819 bp. BBa_K888001 AIDA-I translocator domain Coding A signal peptide which is cleaved during transport trough the inner membrane 1041 bp. BBa_K888005 AIDA-I Signal Peptide Coding When this part is coupled with a passenger attached to AIDA-I translocator domain (K888001), it is posible to express functional proteins in the outer membrane of E. coli. 147 bp. BBa_I765001 pUV. Promotor UV Promoter from iGEM Colombian Team 2007. Tim ITB_Indonesia 2013 proved that this part worked for them. 76 bp. BBa_J23119 pConst Promotor Contitutive promoter. 35 bp. BBa_B0034 RBS RBS Very common Ribosome Binding Site, based on Elowitz repressilator. . 12 bp. BBa_K115017 Riboswitch RBS A RNA thermometer, used for temperature post-transcriptional regulation (thermo sensor), and is designed to initiate transcription around 32°C. 83 pb. BBa_E1010 RFP Coding Red Fluorescent Protein. 706 bp. Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/project_parproject aroma From 2014hs.igem.org Description Capture Module Aroma Module Resistance Module Union Module Parts Aroma Module Since the beginning of iGEM project, the use of fluorescent reporters has been used in each one of the proposed projects in previous years, trying to test the theoretical presence of other proteins in E. coli. For our iGEM 2014 project, this module proposed to promote the usage of aroma reporters, instead of fluorescent ones.DescriptionIn the module, WinterGreen is the most important part. Normally, it catalyzes the conversion of salicylic acid into methyl salicylate. The protein is excreted by the bacteria and when salicylic acid is added to the medium, a chemical reaction takes place and produces methyl salicylate, which is responsible for the wintergreen odor.But in this module, it will be regulated by temperature with the use of the RNA thermometer. When adding salicylic acid to the bacteria in a 32° Celsius environment, the production of the WinterGreen protein will begin.Figure 1. Production of Wintergreen OdorDeviceOriginally, WinterGreen was thought to be the reporter of the Capture module, but in order to prove the function of this gene and also the one in charge of the capture of sodium ions (NhaS), we decided to separate the full device into the actual modules of Capture and Aroma, as seen in the figure below.Figure 2. The device originally thought, and the 2 modules derived from it.This device is composed by the following parts (see figure 3): a constitutive promoter (1), a RNA thermometer (3); used to regulate the WinterGreen-odor protein production through temperature, a Wintergreen-odor enzyme generator, used to allow the production of methyl salicylate, induced by salicylic acid (2); and a terminator (4). All of these parts are ligated by an 8-bp scar (TACTAGAG).Figure 3. Aroma ModuleThese 4 genetic parts form the Aroma device of the project. The full device's length is 1,251bp (including restriction sites).Parts of the module IMAGE CODE DESCRIPTION   BBa_J23119A   In the specific case of our aroma module, it will help the bacteria to continuously transcribe the WinterGreen gene in order to allow the bacteria to continuously produce the aroma. This promoter has a length of 35bp. BBa_K115017 A RNA thermometer, used for temperature post-transcriptional regulation (thermo sensor), and is designed to initiate transcription around 32°C. BBa_K1255001   Produces a transferase to convert salicylic acid into methyl salicylate (WinterGreen odor). The wintergreen odor generator requires of 2mM of salicylic acid to produce methyl salicylate. BSMT1 (BBa_J45004), WinterGreen Odor Generator original name, was created by MIT 2006. This year, the team optimized the sequence for Escherichia coli. The new biobrick has a length of 1,074bp. BBa_B1002   Part made of 6bp, responsible for transcription stop. The terminator stops the production of methyl salicylate. JustificationDifferent to fluorescent reporters, this module was made in order to (in the future) perform as an aroma reporter and also to test the correct function of the bacteria, for its future usage as a new reporter and functional part (CDS). It is desired to use this part in the project to replace the red fluorescent protein (RFP) in the Capture module. But it was preferable to test it apart to demonstrate its effectiveness. Similarly, this piece is also helpful when the biofilter is assembled, because when performing the filtration by silica, WinterGreen can demonstrate the presence of bacteria in the beads. The team added the RNA thermometer in the device for regulating the production of the aroma. Another reason for selecting the RNA thermometer as a regulator was to continue the CIDEB UANL 2013 work with it.Other teams that used RNA thermometer and BSMT1 Team Part Used Use TuDelft 2008: RNA themometer   Temperature-sensing bacteria that changes color at different temperatures; as a temperature reporter system inlarge-scale fermentations, or as a temperature-inducible protein production system.   Victoria BC 2009:   RNA themometer   NAND logic gate using the ribo-key/ribo-lock system designed by Berkeley 2006 team , producing RFP except when the cells are grown in the presence of both arabinose and IPTG, also coupling fluorescent outputs with the ribo-thermometers made by TUDelft 2008 team.   CIDEB UANL 2013:   RNA themometer   Production of Vip3ca3, which acts as a pesticide protein, regulated by specific temperatures in order to avoid overproduction and it will show activity against target organisms Coleoptera and Lepidoptera..   MIT 2006:   BSMT1 This device produces methyl salicylate in the presence of salicylic acid. Methyl salicylate smells strongly of mint (wintergreen). Production of methyl salicylate was verified both by scent and by gas chromatography: E. coli with no WGD did not produce methyl salicylate when SA was added to the medium, while E. coli with the WGD did produce methyl salicylate when SA was added to the medium.. Project Zoom inBibliography/References● Huang, H. (2006, August 30). Part:BBa_B1002. Retrieved August 30, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_B1002.● iGEM2006_Berkeley. (2006). Part:BBa_J23100. Retrieved August 30, 2014, from http://parts.igem.org/Part:BBa_J23100.● iGEM2006_MIT. (2006). Part:BBa_J45004. Retrieved August 30, 2014, from http://parts.igem.org/Part:BBa_J45004. ● iGEM CIDEB Team. (2013). iGEM CIDEB UANL 2013. Retrieved on March 31th, 2014. http://2013hs.igem.org/Team:CIDEB-UANL_Mexico/Project. ● iGEM08_TUDelft. (2008). Part:BBa_K115017. Retrieved August 30, 2014, from http://parts.igem.org/Part:BBa_K115017. ● MIT IGEM Team. (2006). MIT 2006. Retrieved on March 31th, 2014, from: http://2006.igem.org/wiki/index.php/MIT_2006. ● TUDelft iGEM Team. (2008). TUDelft 2008. Retrieved on March 31th, 2014, from: http://2008.igem.org/Team:TUDelft. ● VictoriaBC. (2009). VictoriaBC 2009. Retrieved on March 31th, 2014, from: http://2009.igem.org/Team:VictoriaBC. ● Zubieta, Chole et al. (2003). Structural Basis for Substrate Recognition in the Salicylic Acid Carboxyl Methyltransferase Family. Manuscript submitted for publication. Retrieved from www.plantcell.org. Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/project_aromhp race From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame Race for Science Image 1. Race for Science logo. As it is said: “Mens sana in corpore sano” (healthy mind in a healthy body), we, as a team, believe that everyone should enjoy a healthy life, in which there is a balance between study and physical activity.So after doing the “Explosion”, once people knew what we do in iGEM, we wanted to expand the knowledge and interest of people about our project. That's why we decided to organize this "Carrera por la Ciencia" (Race for Science), which included a circuit of about 4 kilometers long, and at the end, a fair was placed, with entertaining games related to the project in a way that people could spend time together as a group and learn at the same time. Thus, the event gave us the ability to combine the information about our project and synthetic biology, with physical exercise. This last concept is particularly important for us, especially in Mexico, where the rates of obesity and sedentary lifestyle are disturbing. Objectives Image 2. Obesity chart among different countries. According to an article from the United Nations Food and Agricultural Organization (FAO), Mexico is considered as the world's fattest country with a 32.8 percent adult obesity rate, surpassing United States' 31.8 obesity rate. About 70 percent of Mexican adults are considered to be overweight.Monterrey is an industrial city where most people lead stressful and sedentary lifestyles. We, as a part of this society, are too familiar with the dangers of obesity and the prevalent lack of healthy diet and exercise habits. This, combined with the need to develop our project in the Human Practices area, is the reason why we felt motivated to organize this Race for Science. The main purposes of the Race for Science were: • To spread information about the project within both the academic and non-academic community, in a fun, innovating and accessible way • To promote physical activities that have a beneficial impact on human health and thus, help cultivate a balance between body and mind The event was promoted through a personal invitation to the people in our high school, and they were suggested to invite their family members and/or friends to join them. Posters were also made with all the information about the event; they were placed in different parts of the school and simultaneously posted digitally on the official page of the iGEM CIDEB team on Facebook. 1st Stage of the Race: Physical Activity The race was held within the “Mederos” campus of the UANL. The attendees were asked to arrive at 6:30 a.m., so that the event could start at 7:00 a.m. We welcomed about 600 participants, ranging in age from 7 years old to third age.At 7:00 am a warm-up session was performed. The session was developed with the help of a professional, who lead a dance for about 30 minutes, turning conventional exercises into something fun. The mascot of our team had a very active participation in the warm-up, which encouraged the people to do the exercises properly and enthusiastically. We then proceeded to take the participants to the start area. The women’s group was the first one to start running, followed 5 minutes later by the men’s group.The start of the route was the main entrance to our school. Then, the road stretched by the campus’ internal streets to the Faculty of Economics, and then the participants resumed their route in the opposite direction of the streets to return to the entrance of the school. Image 3. Images of people during the race. 2nd Stage of the Race: Project informationAlong the route, there were people on the sidewalks with informative signs about different synthetic biology fun facts, general information about the project, about the impact that our project would have on society, etc... At the halfway point, several people could be found giving away small bags of water to hydrate the runners.At this point, we had already accomplished one of the main objectives of the race (to promote physical activity). The next step was encouraging people to learn more about our project (and have some fun while doing so). At the end of the route, the participants returned to the starting point (the parking of the school). In this place, there were different modules with information relevant to our project. Each module included an allusive game to every action of the bacterium (Capture, Union, Resistance and Aroma), so that people could learn in an easy and interactive way. Thus, while participants took a break, they could observe and receive a brief explanation of our project, and they could play and get coupons which could be exchanged for prizes. Image 4. People participating in the project games.Impact Two weeks before, the students learned from the DNA WEEK what was our project about, so they made comments such as “Hi E.CARU!” to our costume, or “Is this from iGEM?” and “How can I get in?” to our team members. Even in the game time, when looking at it, some of them were able to recognize easily the parts referring to our project. (Each game was created to give a reference about our project, its modules and their function). People, who were not students and hadn’t received those talks, were asking about the iGEM team and the project that we are working on. They asked how does it work, how did we do it, what will it be able to do, and more. They were very interested; even though they did not want to play because they were older, they were paying attention while the younger ones were playing and listened to the explanation that the team members gave.The race was a complete success. We had received supportive comments during it, and we realized that people were interested in what we are doing. The assistants gained knowledge about our project while having a good time, spending time with their families and friends. Gallery 1. Images of the race.Opinions after the raceBibliography/References United Nations Food and Agricultural Organization (FAO). (2008). The state of food and agriculture. Retrieved from: http://www.fao.org/docrep/018/i3300e/i3300e.pdfReturn to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_racmath resistance From 2014hs.igem.org Overview Capture Aroma Resistance Union Resistance Module The resistance module is based in the use of IrrE. IrrE is a protein which is not affected by external factors during its transcription or in its translation, so established parameters were needed to use. \begin{equation}\large \frac{d\left [ mRNA \right ]}{dt}=\alpha_{1}-d_{1}\left [ mRNA \right ]\end{equation}The parameters for translation and transcription rate from Singapore 2008 iGEM team were used, as well as the transcription and translation speeds carried out by E. coli; assuming a transcription speed of 70nt/s and a translation speed of 40aa/s. They were used in the equations below with the IrrE gene length (986nt) and protein length (311aa) respectively.\begin{equation}\large \alpha_{1}=\frac{transcription speed}{gen lenght (nt)} \end{equation}\begin{equation}\large \alpha_{2}=\frac{translation speed}{protein lenght (aa)}\end{equation}\begin{equation}\large \alpha_{1}=\frac{(70)(60)}{986}=4.25\end{equation}\begin{equation}\large \alpha_{2}=\frac{(40)(60)}{311}=7.7\end{equation}After, it was needed to use the parameters for degradation rates of proteins and mRNAs obtained from Beijing PKU 2009 iGEM team:\begin{equation}\large d_{1}=\frac{1}{half-life(min)}+\frac{1}{30min}\end{equation}\begin{equation}\large d_{2}=\frac{1}{half-life(min)}+\frac{1}{30min} \end{equation}Since IrrE half-life has not been determined yet, it was decided to research about homologous proteins with the same function as IrrE (transcriptional factor), and according to Dibden and Green (2005) the average half-life for transcriptional factors in E. coli is 45 min. They tested FNR proteins (transcriptional factors) through thermo-induciblefnr expression observing that their half-life was 45min average. Later, was used the information from Selinger’s team (2003) to determine the mRNA degradation. They carried several experiments for finding average mRNA half-life in E. coli. They used mRNAs about 1100nt concluding they have an average half-life of 5min. Using the previous relation was found mRNA's half-life from IrrE which was about 4.45min.\begin{equation}\large \frac{d[P]}{dt} = \alpha_{2} \cdot[mRNA] - d_{2}[P] - f_{post(p)}\end{equation}With the previous information the degradation rates for both transcription and translation of IrrE were found.\begin{equation}\large d_{1}=\frac{1}{4.5}+\frac{1}{30}=0.25\end{equation}\begin{equation}\large d_{2}=\frac{1}{45}+\frac{1}{30}=0.055\end{equation}For the simulation the team used Simbiology® program, by plugging in the previously calculated data from the equations to find the amount of proteins E. coli would produce at certain time. The following graph was the result of the simulation:But for translation there was another factor taken in consideration, the “fpost(p)” which were the post-translational variables affecting the production of the functional protein.\begin{equation}\large \frac{dpi}{dt}=Ti-Dpmi- f_{post (p)}\end{equation}After researching, it was found that IrrE needs to have a positive charge to be functional, accepting one Zn2+ ion from E.coli (Vujicic, 2009). Vujicic’s team developed the structure of IrrE deducing it should have three domains; one specific is the zinc-binding site where the Zn2+ ion binds to make IrrE positive. IrrE positive charge is what makes possible its binding to a substrate forming a substrate complex, but the substrate is unknown. For that reason it was only taken as a “fpost(p)” variable the Zn2+ binding because the data for finding the affinity between IrrE and the unknown substrate was not possible to determine.Since it was not obtained the data fom the affinity between IrrE and Zn2+ it was needed to find the data from homologous proteins. In fact, proteins with zinc binding domains were found to have an affinity between 0.1 and 0.2 in E. coli (Vorackova, 2012). We used 0.15 as average in the equation for association constant which is defined as the following:\begin{equation}\large Ka=\frac{\left [ C \right ]}{\left [ S \right ]\left [ E \right ]}\end{equation}Where “[C]” is the complex formed, “[S]” is the substrate and “[E]” is the enzyme, ligand or ion. Substituting for IrrE it is expressed as below:\begin{equation}\large Ka=\frac{\left [ C \right ]}{\left [ IrrE \right ]\left [ Zn+ \right ]}0.15=\frac{\left [ C \right ]}{\left [ IrrE \right ]\left [ Zn+ \right ]}\end{equation}With the association constant, Simbiology® was used to model the functional IrrE production. The results were the following:When both graphs were compared, (Graph 1 and Graph 2) it was concluded that not all the IrrE production was functional . Actually, from the total amount produced (about 2400), only about 1700 are functional . So it demonstrates that the rate at which Zn2+ ions binds to IrrE is slower than the rate at which IrrE is produced, leaving a nonfunctional IrrE which is later degraded.Bibliography/References● DIBDEN, David J. G. (2005). In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states. Microbiology, 4063-4070.● SELINGER, Douglas R. M. (2003). Global RNA Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript Degradation. Genome Research, 216-223.● VORACKOVA Irena, S. S. (2011). Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography. Protein Expression and Purification, 88-95.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/math_resistanchp From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame Race for Science More than 600 people learned about our project, while doing excercise. DNA Week Fun activities were held to teach CIDEB students, about synthetic biology. Explosion The explosion, consisted on spreading iGEM information all over our high school, so everyone knows about our project. Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hproject union From 2014hs.igem.org Description Capture Module Aroma Module Resistance Module Union Module Parts Union Module After E.CARU performs its other tasks, in order to remove E. coli from the salty water for making it useful, the ability for binding to silica was introduced to the bacteria. Using this ability, it would be easy to clean the water free of bacterium through a biological filter.DescriptionL2The gene L2 encodes for a protein that is able to attach to silica. Taniguchi et al. reported in 2007 that the L2 ribosomal protein from E. coli strongly binds to silica surfaces, even up to 200 times tighter than poliarginine tags commonly used for protein purification. In their work, Taniguchi et al. (2007) constructed a fusion protein containing L2 and green fluorescent protein (GFP) which kept attached to a silica surface even after been washed for 24 hours with a buffer containing 1 M NaCl (Figure 1). UANL Mexico 2012 did not have this piece in stock, so we decided to synthetize L2 along with a peptide signal.Figure 1. Proteins absorbed to a silica slide and washed for 24 hours a)GFP b) L2-GFP fusion c) R9-GFP fusion. Taken from Taniguchi (2007)AIDAAIDA-I is an E. coli membrane protein with a passenger domain of 76 kDa exposed to the extracellular space and a transmembrane beta-barrel domain of 45 kDa; the latter has been used to express functional proteins in the cell-membrane of up to 65 kDa (van Bloois et al., 2011). Furthermore passengers coupled to AIDA-I have been reported to reach an expression level of more than 100,000 copies per cell in the outer membrane (Jose and Meyer, 2007). Figure 2. Schematic representation of AIDA-I carrier proteinHow do L2 and AIDA act together?As AIDA is a membrane protein producer and L2 produces a protein for making possible the attachment to silica, the team decided to make a fusion protein, so that the protein produced by L2 can be translocated to the membrane of the bacteria with the help of AIDA, and in that way, binding the E.CARU to silica pearls for being filtered.Why is it important to use BgIII and BamHI to link L2 and AIDA-I?It was needed to join both proteins in order to make a fusion protein, but SpeI and XbaI could not be used to join them because the reading frame would change, coding for a completely different protein. In order to avoid such problem, BgIII and BamHI were used instead, which could join AIDA and L2 without changing the reading frame. The scar produced between BamHI and BgIII, as it is shown in Figure 3, is formed by six bases, respecting the reading frame from both proteins in order to synthetize the correct protein.Figure 3. Example of a ligation using BamHI and BgIIIDeviceInitially, L2+AIDA and IrrE, protein for giving resistance to E. coli to adverse conditions (Resistance module), were joined together in only one circuit, but we needed to separate them because L2+AIDA has not been tested and it could affect the production of IrrE (see Figure 4.) as well as for testing each module alone.Figure 4. Circuit for our project and for testing resistance and union modules. The union circuit consists mainly of a constitutive promoter, an RBS, a peptide signal attached to L2 and this attached to AIDA by a scar and a terminator. The device is designed this way in order to produce a protein that helps the bacteria to bind to silica.Figure 5. Union DeviceParts of the module IMAGE CODE DESCRIPTION BBa_J23119 The J23119 is the most effective and common constitutive promoter used. It has a length of 35bp. BBa­_B0034 This specific RBS is based on Elowitz repressilator. It has a length of 12bp. BBa_K888000 L2. This CDS gives the property for binding silica and glass surfaces to E. coli, it has a length of 819 bp. BBa_K888001 AIDA-I is synthetized as a 132 kDa pre-protein featuring a signal peptide which is cleaved during transport trough the inner membrane, a 78 kDa adhesin (passenger) domain, and a 45 kDa translocator. This autotransporter has a large capability in translocating relatively large passengers from 12-65 kDa by showing a N-terminal type of fusion. Coupled with a passenger domain and a signal peptide (K888005), it is possible to express functional proteins in the outer membrane of E. coli. It has a length of 1482 bp.   BBa_K888005 When this part is coupled with a passenger attached to AIDA-I translocator domain (K888001), it is possible to express functional proteins in the outer membrane of E. coli. The signal peptide is naturally cleaved during transport trough the inner membrane (Li et al. 2007; van Bloois et al. 2011).It has a length of 147 bp. BBa_B1002 Part made of 6bp, responsible for stopping transcription. JustificationsAlthough E. coli could acquire the ability to bind to silica, a biological filter was designed to remove bacteria from water. The team´s proposed biofilter is shown next:Figure 6. The team's proposed biofilter model Silica was chosen as the ideal material because it is cheap and it is commonly found and also because the team wanted to give this material a new use. It was also chosen in order to finish the UANL Mexico 2012 team work. They created a circuit to make E. coli bind to silica, but as they did not prove it, we wanted to determine if it is functional.AIDA-1 allows the expression of proteins larger than small peptides in the outer membrane, which is why it is the best option to use with L2. AIDA-I was obtained by PCR by UANL Mexico 2012, so we decided use their piece for our project, as it was easy for the team to obtain it because both of the teams are in the same city.Other teams that used itUANL México 2012: They proposed the fusion protein for using it to binding silica after detect and capture arsenic acid in groundwater, and in that way removed the pollutant arsenic acid from the water, as part of water bioremediation. However, their project was not finished.Union Module Zoom InBibliography/References● Antiquity. (2003). Part:BBa_B0034. Retrieved March 30th, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034.● iGEM2006_Berkeley. (2006). Part:BBa_J23119. Retrieved April 30, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_J23119.● iGEM12_UANL_Mty-Mexico. (2012). Part BBa_K888000. Retrieved March 29th, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_K888000. ● iGEM12_UANL_Mty-Mexico. (2012). Part BBa_K888001. Retrieved March 29th, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_K888001. ● iGEM12_UANL_Mty-Mexico. (2012) Part BBa_K888005. Retrieved March 29th,2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_K888005. ● UANL Mexico. (2012). Recovery module. Retrieved March 28th,2014, from http://2012.igem.org/Team:UANL_Mty-Mexico/Project/recovery. Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/project_uniomath overview From 2014hs.igem.org Overview Capture Aroma Resistance Union Overview When Biology meets MathIn an iGEM project, mathematical models are necessary to predict the behavior of a biological machine, representing the quantitative relations between two or more variables involved in the function and expression of a gene or a set of genes in organisms like E. coli. Our team decided to use a deterministic model to simulate and represent the function of the four modules, assuming that the variables (mRNA and protein concentrations) adopt a continuous behavior and obey kinetic rules that can be represented with constant values.This type of mathematical model is used to include variables that considerate both, the gene expression and the physiological cycles (chemical process, transport of proteins, etc.). By using traditional differential equations, the description was able to be constructed and it also permitted the analysis of the behavior of mRNAs and protein production. The focus was to determine through a mathematical model the ideal behavior of the four modules independently.CaptureAromaResistance UnionDeterministic Modelling: Equations and ParametersAs it was previously stated, it was necessary to work with different equations focused in the production and degradation rate of mRNAs and proteins, according to the length of the genes in each module. This was performed in order to obtain both, the concentration rates of mRNA and protein based on system (gene) length and protein length respectively.Equations● mRNAGenerally, to describe the amount of mRNA produced over t time, the equation implemented is shown below:\begin{equation}\large \frac{d[mRNA]}{dt} = \alpha_{1} \cdot f_{y} - d_{1}[mRNA]\end{equation}Where: “α1” means the transcription rate of a given gene; “fy” represents a regulatory function (if there is) that can activate or inhibit the system; and “d1 [mRNA]” the degradation rate of the mRNA produced.● ProteinThe same happens with the protein production, but differs in the formula because it also involves a post-translational variable:\begin{equation}\large \frac{d[P]}{dt} = \alpha_{2} \cdot[mRNA] - d_{2}[P] - f_{post}\end{equation}Where: “α2 [mRNA]” means the translation rate of a protein based on the amount of mRNA available; “d2[P]” represents the degradation rate of that protein; and “fpost" the post-translational variables which affect the production of the final (functional) protein.ParametersTo determine both the transcription (1) and translation (2) rates, the parameters from Singapore 2008 team at which E. coli carries out transcription and translation were used ; as well as the degradation rates from PKU Beijing 2009 team. The parameters from Singapore 2008 were used, assuming a transcription speed of 70nt/s and a translation speed of 40aa/s. The speeds were multiplied by 60 because minutes were used as units in the simulations of the modules. It was determined that E. coli's division time was 30min, based on PKU Beijing 2009 team.\begin{equation}\large \alpha_{1} = \frac{transcription speed}{gene length (nt)}\end{equation}\begin{equation}\large \alpha_{2} = \frac{translation speed}{protein length (aa)}\end{equation}\begin{equation}\large \alpha_{1} = \frac{70 \frac {nt}{s} \cdot(60)}{gene length (nt)}\end{equation}\begin{equation}\large \alpha_{2} = \frac{40 \frac {aa}{s} \cdot(60)}{protein length (aa)}\end{equation}\begin{equation}\large d_{1} = \frac{1}{half-life(min)} + \frac{1}{division time (min)} \end{equation}\begin{equation}\large d_{2} = \frac{1}{half-life(min)} + \frac{1}{division time (min)} \end{equation}\begin{equation}\large d_{1} = \frac{1}{half-life(min)} + \frac{1}{30min} \end{equation}\begin{equation}\large d_{2} = \frac{1}{half-life(min)} + \frac{1}{30min}\end{equation}Bibliography/References● BERGANT, Martina N. M. (2010). Modification of Human Papillomavirus Minor Capsid Protein L2 by Sumoylation Journal of Virology, 11585-11589.● DIBDEN, David, J. G. (2005). In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states. Microbiology, 4063-4070.● HARE, James K. T. (1991). Mechanisms of plasma membrane protein degradation: Recycling proteins are degraded more rapidly than those confined to the cell surface. PNAS, 5902-5906.● iGEM CIDEB UANL. (2012). Modelling: Equations.Retrieved from: http://2012hs.igem.org/Team:CIDEB-UANL_Mexico/Math/Overview.● NTU Singapore. (2008). Modelling: Parameters. Retrieved from: http://2008.igem.org/Team:NTU-Singapore/Modelling/Parameter.● PKU Beijing. (2009).Modelling: Parameters. Retrieved from: http://2009.igem.org/Team:PKU_Beijing/Modeling/Parameters.● SELINGER, Douglas R. M. (2003). Global RNA Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript Degradation. Genome Research, 216-223.● VORACKOVA Irena, S. S. (2011). Purification of proteins containing zinc finger domains using Immobilized Metal Ion Affinity Chromatography. Protein Expression and Purification, 88-95.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/math_overviehp videogame From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame Videogame IGEM CIDEB 2014 decided to develop a videogame that represented this year’s project. In the game, our mascot, E.CARU, is being used in a biotechnology lab to remove sodium ions in three different mediums. Each medium has a different concentration of a saline solution; the objective of E.CARU is removing sodium ions without absorbing the chlorine ones.By Capturing sodium ions and filling its respective tube, you pass to the next medium, or level, and a bonus function to eliminate all the chlorine ions from the medium (Carbonation-Calcination Reaction) becomes available. If any chlorine ions are captured by accident, nothing happens, because of the Resistance module; but if too much chlorine is captured, E.CARU can’t continue capturing sodium ions and the game is lost.The game was programmed so that sodium and chlorine ions appear on the screen randomly. If you don’t capture sodium ions as they fall through the screen, they disappear; but all chlorine ions stay moving on your screen, unless you eliminate them using the CCR function or click and capture them.ObjectiveIGEM CIDEB 2014 decided to develop a videogame that represented this year’s project, with the objective that people could learn about it in a fun and entertaining way.The objective of this game is to make people undersatnd our project in an easy way, by playing, in the game the main objective is to catch all the sodium ions, which is what our bacteria makes, and avoid catching the chlorine ions, because these ions are not catched by our bacteria in the project.How to playThe instructions for playing the videogame are:-Try catch the soduim ions.-Avoid catching the chlorine ions.-Try to catch as many sodium ions as you can to advance to the next level.-Fill up your progress bar to activate CCR.-Click the CCR button to eliminate chlorine ions in the medium.-Advance as many levels as you can.-You can pause the game whenever you want.Download the gameThis game was developed using “Game Maker” which allows the programmer, to make his or her own game without using a specific programming language.Link to download this game for Windows: click here.If the previous link does not work: click here.Image 1. Screen-captures from the game.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_videogamproject capture From 2014hs.igem.org Description Capture Module Aroma Module Resistance Module Union Module Parts Capture Module As a step to desalinize water, iGEM CIDEB 2014's project intended to capture sodium ions from saline water using a protein named NhaS produced from the nhaS gene expression. This gene was obtained from a 1994 patent by Krulwich & Ivey and no further information was found about the gene in other sources. A lot of research of related information and predictions about the NhaS protein in different sites had to be made in order to obtain more information about it and being able to work with and include it in the project.DescriptionNhaS is a putative protein from Bacillus firmus that is characterized by its ability to bind and sequestering sodium ions, with a calculated weight of 7100 Daltons and a pH of 12. It “can enhance the Na+ -resistance of antiporter- deficient strains by increasing the availability of Na+ to the integral membrane antiporters on the cytoplasmic side of the membrane and by sequestering Na+ while rate-limiting efflux mechanisms catalyze extrusion of the cation.” (Krulwich & Ivey, 1994)Research by Krulwich and Ivey (1994) supports that in its origin bacteria, NhaS works as a regulatory of pH in protein's homeostasis because it makes the cytoplasmic pH more acidic than the external medium, usually basic. Essentially, NhaS performs three different functions; (1) capturing sodium ions, (2) regulating pH, and (3) enhancing the resistance of bacteria to high saline conditions.Figure 1. Patent US 5346815 A shows extracts of the E. coli EP432 transformed with pGEM (fig. 4A) and pGRVH (fig. 4B). pGEM is a control plasmid and pGRVH is a plasmid with the nhaS gene. Those are crude extracts shown by the effect of putting the bacteria to an SFBI excitation, which is a sodium-sensitive molecule used to measure intracellular Na+. Resuming, it shows in basic draws that the protein is expressed in E. coli and in what quantity according to the excitation level where it is exposed.What would happen with Cl- ions?After the removal of sodium ions from saltwater, it is needed to remove Cl- ions as well in order to complete the desalination process. E. CARU only captures Na+ ions, leaving Cl- ions in the water medium; but since Cl is a diatomic molecule (meaning it cannot be alone in normal conditions), it joins another Cl molecule, forming Cl2.Normally, Cl2 is in a gaseous state at normal conditions, so what would remain after E. CARU takes Na+ ions from water is a mixture of Cl2 gas and water molecules. In order to remove it from water it is possible to use a method involving the separation of a gas from a liquid based in the boiling points of each component in the mixture. Cl2 gas has a boiling point of -34.6°C and water has a boiling point of 100°C. By cooling the mixture at -34.6°C, the gas would evaporate, separating itself from water. (Bentor, 2014).Yet still, there is an important factor to consider: Cl2 gas is toxic, but as it is 2.5 times heavier than air (CFC StarTec LLC, 2007), it would stay in water at room temperature. For this reason, before cooling the Cl2 gas in order to take it away, it is necessary to take safety measurements. The one proposed by the team is the usage of an Atomic Absorption Spectroscopy (see Figure 2). Atomic Absorption Spectrometry (AAS) is an analytical technique that measures the concentrations of elements. Atomic absorption is so sensitive that it can measure down to parts per billion of a gram (µg dm–3). The technique uses light wavelengths specifically absorbed by an element that correspond to the energies needed to excite electrons from one energy level to a higher one. (Royal Society of Chemistry).Figure 2. Atomic Absorption Spectroscopy dispositive.The use of an Atomic Absorption Spectrometry is a way to remove the Cl2 gas from water and measure it to prevent its escape. Then, the stored Cl2 gas can be used to sterilize drinking water, to disinfect swimming pools and to be part in the manufacture of many consumer products; such as paper, dyestuffs, textiles, petroleum products, medicines, antiseptics, insecticides, foodstuffs, solvents, paints, and plastics (especially PVC). It can also be used to produce bleaches, chlorates, chloroform, carbon tetrachloride and bromine. A further substantial use for this element is in organic chemistry, both as an oxidizing agent and in substitution reactions (Emsley, 2011). Research on NhaSIt is important to be familiarized with what it is being worked with, and since this putative protein has never been used at iGEM before, it was done a lot of research on it. The composition and form of a protein show relevant data about its actions and functions, that it is why it was investigated NhaS’ predicted type. It was found in the modelling tool 3D-JIGSAW from Cancer Research UK' site that its possible protein or peptide type would be helix, coil or strand (Figure 3). Also, it was found in the Raptor X protein modelling site a predicted structure of the protein. (Figure 4)Figure 3. Interactive 3D-JIGSAW's result that indicates the predicted protein type of NhaS.Figure 4. Predicted protein structural results from Raptor X.Research was done in different sources. The previous information was confirmed in Predict Protein site. As shown in Figure 5 and Figure 6.Figure 5. Results given by Predict Protein showing the secondary structure composition and solvent accessibility of the putative NhaS protein.Figure 6. Results given by Predict Protein showing the predicted precise structure of the NhaS protein. As it can be apprecciated, the results from all sources match, so, based on the previous information it was concluded that NhaS is most possible to be of the helix type. Being aware of the secondary structure of proteins is relevant, since hence, the protein folding mechanism can be taken into consideration.According to Krulwich and Ivey (1994), the location of the protein is in the cytoplasmic side of the membrane, however, when the research was made, it was found out that the protein is predicted to be highly non-cytoplasmic (Stockholm Bioinformatics Center SBC Phobius, 2014), as shown in Figure 7.Figure 7. Predicted protein overview results from Stockholm Bioinformatics Center SBC Phobius.This makes an antithesis of theories. Also as it is presented in Figure 5, NhaS is predicted to be exposed and buried, which accords with Figure 6, that show how the amino acids sequence of Nhas is divided many times in exposed and hidden. As nobody has described exactly how is NhaS and where it is placed inside E. coli, the team, based on all the modeling above, came out with a hypothesis in which NhaS would cross two times the membrane as it is shown in the Figure 7, having two parts exposed, the beginning loop and the final helix with loop, and an inner part, consisting in the two big helixes as transmembrane and the little loop in the middle of the helixes in the cytoplasmic side. The determinant factor was the structure of NhaS predicted by Raptor X (Figure 4) that is very similar to the transmembrane proteins and ion channels, which are in similar positions, as NhaS in our hypothesis, inside the bacteria.Figure 8.“iGEM CIDEB 2014's prediction about the location of the NhaS protein”This prediction was used for the understanding and explaining of the module, as well as for designing different animations.Further information about NhaS and the experimental results made by the team about it can be found in its parts registry section, as well as the results section in this wiki.DeviceThe proposed circuit for the module is as follows: An UV promoter, an RBS; the nhaS gene, another RBS, a WinterGreen odor reporter (BSMT1 Opt.) and a terminator. However, for practical and experimental reasons explained in the next paragraph, it was decided that nhaS and BSMT1 Opt. would be tested separately, leading to both Capture and Aroma modules. As mentioned before, in the ideal project it is proposed an odor Wintergreen (BSMT1 Opt.) reporter for this module in order to know if there is any production and expression of NhaS, but since BSMT1 Opt. wasn't proved yet and NhaS is a putative protein it was decided to test NhaS physically with RFP, which is a simple and common reporter, in order to observe functional results in the tests for this specific segment of the project, obtaining conclusions about both genes, and being able to put them together in the ideal device.Further information about the odor reporter can be found in the Aroma module in this wiki.Figure 9. Project's ideal device and how it was divided for its respective tests.Capture module's parts description IMAGE CODE DESCRIPTION BBa_I765001 UV Promoter from iGEM Colombian Team 2007. Its length is 76bp. Tim ITB_Indonesia 2013 proved that this part worked for them. BBa_B0034 Very common Ribosome Binding Site, based on Elowitz repressilator. Its length is 12bp.   BBa_1255000   nhaS is a putative gene that produces the NhaS protein. iGEM CIDEB 2014 introduced it to iGEM for the first time. Its length is 207bp. BBa_K1255000 Wintergreen-odor enzyme generator, used to allow the production of methyl salicylate, when it is induced by salicilyc acid. Its length is 1074bp. BBa_B1002 Part made of 6pb responsible for stopping transcription. JustificationsUV promoter as an initiator for the systemThe NhaS’ production and functionability was regulated using a promoter from iGEM Colombian Team 2007 that is regulated by UV irradiation and does not causes mutations in the bacteria. This type of promoter was chosen in order to have a control about the NhaS action in the project, so it would only be activated under UV rays, and therefore, it could be decided at which time the NhaS gene was going to be expressed. There were chosen UV rays as the initiator of the expression because they are present in normal conditions, and in case the biofilter was used in a water treatment plant, as it is intended as a future projection for the project, it could be simpler and cheaper to activate the system with sunlight, since the mentioned promoter is activated under 360 wavelengths of light spectrum. As an extra, the activation of the system without the use of reactants that could pollute the water is one of the advantages of its use.Other teams that used pUV and NhaSIn the case of pUVBefore choosing the BBa_I765001 UV promoter the team reviewed the experiences by other participants with it, with the intention of assuring the maximum possible success rate if used in this module. The following table shows information about its usage with other teams. Team Use NYMU_Taiwan 2012: An UV induced promoter and a CDS of a testosterone-making gene.   ITB_Indonesia 2013:   pUV and mCherry (BBa_J06702) to detect DNA damage.   Colombia_Israel 2007:   Expression of the UV promoter in presence of UV irradiation light lead to the expression of the EYFP reporter. In the case of nhaSThere are no teams that used this gene in the past. IGEM CIDEB 2014 was the first team that synthesized, characterized, tested and registered it in the parts registry. Future usageThe team considers NhaS to be useful for the scientific community in general, in addition to iGEM. NhaS, as mentioned before, is a putative protein. Knowing specific results about its functions could be of great use for future projects. The potential for future usage of this gene is great. As mentioned in the patent from Krulwich and Ivey (1994), the gene encoding NhaS can be introduced into cells to produce desalination bioreactors, can be introduced into plants as a transgene to produce plants that are resistant to sodium, may be used for treatments involving NNa+ /K+ ATPase disorders, e.g., in heart disease, and may be introduced parenterally, preferably orally, to bind to and sequester dietary sodium. The possibilities are wide and promising.Capture Module Zoom InBibliography/References● Antiquity. (2013, January 31). Part: BBa_B0034. Retrieved May 1st, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034● Bentor, Y. (18 de Jun de 2014). Chlorine. Obtained from Chemicalelement: http://www.chemicalelements.com/elements/cl.html● BMM Cancer Research UK. Interactive 3D Jigsaw. Retrieved May 1st, 2014, from bmm.cancerresearchuk.org● CFC StarTec LLC. (24 de Dec de 2007). Chlorine Cl2. Obtained from CFC StarTec LLC: http://www.c-f-c.com/specgas_products/chlorine.htm● Colombia Israel Team. (2007). Part: BBa_I765001. Retrieved April 1st, 2014, from Part-BBa_I765001● Emsley, J. (2011). Chlorine. Obtained from Periodic table: http://www.rsc.org/periodic-table/element/17/www.matvalue.com● IGEM Colombian Team. (2007). UV promoter. Retrieved April 1st, 2014, from http://parts.igem.org/Part:BBa_I765001● Jianzhu Ma, Sheng Wang, Feng Zhao, and Jinbo Xu. Protein threading using context-specific alignment potential. Bioinformatics (Proceedings of ISMB 2013), Vol. 29, Issue 13, pp. i257-i265. From http://bioinformatics.oxfordjournals.org/content/29/13/i257.full/● Jian Peng and Jinbo Xu. A multiple-template approach to protein threading. PROTEINS, 2011 Jun;79(6):1930-9. doi: 10.1002/prot.23016. Epub 2011 Apr 4. From http://www.ncbi.nlm.nih.gov/pubmed/21465564/● Jian Peng and Jinbo Xu. RaptorX: exploiting structure information for protein alignment by statistical inference. PROTEINS, 2011, Vol 79, Issue S10, pp. 161-171. From ● Knight Lab. (2006). Part: BBa_B1002. Retrieved from http://parts.igem.org/Part:BBa_B1002● KRULWICH Terry, IVEY Mark.(1994) Sodium ion binding proteins. Retrieved April 1st, 2014, from http://www.google.com.mx/patents/US5346815● Morten Källberg, Haipeng Wang, Sheng Wang, Jian Peng, Zhiyong Wang, Hui Lu, and Jinbo Xu. Template-based protein structure modeling using the RaptorX web server. Nature Protocols 7, 1511-1522, 2012. From http://www.nature.com/nprot/journal/v7/n8/full/nprot.2012.085.html/● Predict Protein. Request ID: 473277. Retrieved May 3rd, 2014, from https://www.predictprotein.org● Raptor X (2014). Protein Structure and Function Prediction: Result for job NhaS. Retrieved June 18th, 2014, from http://raptorx.uchicago.edu/StructurePrediction/myjobs/30991450_67869/● Royal Society of Chemistry. (s.f.). Atomic absorption spechtrometry. London: Burlington House.● Stockholm Bioinformatics Center SBC (2014). Phobius. Retrieved from http://phobius.sbc.su.se Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/project_captursafety questions From 2014hs.igem.org Safety Questions Posters Lab Safety Cards Memory Game Risk Analysis Safety Questions 1. Would any of your project ideas raise safety issues in terms of:● researcher safety,● public safety, or● environmental safety▪ IGEM CIDEB 2014’s Project does not raise any safety issues in either researcher, public or environmental safety. The chassis used is Escherichia coli K12 DH5α, which belongs to the Risk Group 1, meaning that the organism is unlikely to cause human or animal disease. According to the WHO, “E. coli K12 is a non-pathogenic strain that cannot permanently colonize the gut of healthy humans or animals.”The following table shows the main parts used in the project, as well as safety related information: Gene Used in Organism from which is derived Risk group level NhaS Capture module Bacillus Firmus OF4 1, according to German TRBA (Technical Rules for Biological Agents). BSMT1 Opt. Aroma module Petunia x hybrida It can’t be classified in a risk group since it comes from a plant, however, it is only used to produce a fresh aroma and it can be used in a Level 1 laboratory IRRE Resistance module Deinococcus radiodurans 1, according to German TRBA. L2 and AIDA Union module Escherichia coli 1, according to WHO If the project goes according to plan neither team members, publics or environment could or would be harmed, since the project acts in water and the bacteria used would not survive normal environmental conditions.The most dangerous substances used while working in the laboratory were the following (You can see further information by clicking on their names, and see more substances used and their respective information in the risk analysis section in this wiki):● Ethidium bromide ● NaOH ● Acetic acid ● Methyl salicylate However, this substances, as well as the laboratory equipment and the biological parts, were used with extreme caution and following the safety guidelines for each one.The whole team received a training laboratory workshop given by our instructors and advisors before the project started. We learned the different laboratory protocols needed for the project and safety measurements for working on it. Besides, all members were given a laboratory manual including all the protocols that could be used.If the project does not go according to plan and organisms or parts were released there would be no real danger either for team members, publics or environment since the chassis would not bear the conditions outside of the laboratory.2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes,● Did you document these issues in the Registry?●How did you manage to handle the safety issue?●How could other teams learn from your experience?▪ No, most of the parts used in the project were taken from the parts registry, assuring its safety, and the ones that we built all proceed from organisms from risk group 1 levels.However, any detail related to safety, if existent, was included in the specific page of the part or device submitted.3. Is there a local biosafety group, committee, or review board at your institution?● If yes, what does your local biosafety group think about your project?● If no, which specific biosafety rules or guidelines do you have to consider in your country?▪ There is a group in our school that is in charge of the different laboratories and their safety. This group was in frequent contact with the team, ensuring that the safety guidelines were being fulfilled. They approved our project.The team worked with organisms and parts of BSL 1 in a level 1 laboratory, and safety was respected at all times. The project is in compliance with national regulations and university requirements.The following is an official national document containing biosafety information:-Biosafety law of Genetically Modified Organisms4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?▪ If the E. CARU project succeeded and became widely used it could only be managed by industries or qualified people, for the organism is delicate and the project is regulated by certain aspects, like UV and temperature.In case something unexpected happen, a safety measurement the team thought of was incrementing the UV needed for activating one of the modules in our project, this would result in the bacteria dying.Also, most of the devices are activated by regulated promoters, assuring the system would activate only on certain conditions controlled by us.Future teams could consider similar ideas on mind. Ideas like this are simple and easy to perform in iGEM projects and contribute to the project’s safety as a whole.Bibliography/References● Cámara de Diputados del H. Congreso de la Unión. (2005). Ley de Bioseguridad de Organismos Genéticamente Modificados From http://www.diputados.gob.mx/LeyesBiblio/pdf/LBOGM.pdf● Committee for Biological Agents; GermanyTechnical Rules for Biological Agents. From: http://www.baua.de/de/Themen-von-A-Z/Biologische-Arbeitsstoffe/TRBA/pdf/TRBA-466.pdf?__blob=publicationFile&v=6Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/safety_queshp cinvestav From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame CINVESTAV Conference Image 1. The team at the end of the presentation.On Friday March 7th, 40 Chilean teachers visited our high school. They were not very familiar with what the iGEM competition is all about, so we invited them to a conference in the Auditorium were we gave a presentation including an iGEM introduction, its principal areas, our team background and the explanation of our project, enclosing also our previous ideas, with the intention of letting people from other countries know about iGEM, specially about our team and what we are doing. DescriptionIn a PowerPoint presentation, we covered the most related topics about iGEM. We started with an introduction of what the iGEM competition is and what it is about, we also included the basics of iGEM’s history, mentioning how the high school division was formed.We explained how this competition is multidisciplinary and showed all the characteristics that an IGEM team has, therefore, we explained to the teachers the different parts of the project like safety, dry lab, human practices, and more.Then, we wanted to show them how we finally got to our project; including a brief description of all the ideas we had during the team meetings until deciding the final one. Afterwards, we included a complete description of our project and the purpose we had for it. Finally, a brief explanation of the previous teams in our high school was included. We also had a short time of questions and answers, and it was very encouraging that they gave us supporting comments about us and the presentation. This was a great feedback for us.Image 2.Chilean teachers attending to the presentation.ImpactWe applied surveys with 5 different questions to each teacher and registered the results:Graph 1. Graph showing the results of the surveys. The questions above showed us that 80% of the teachers that received our presentation considered that our project is an idea that can be very useful for the society.With this survey it was also shown that most teachers qualified our presentation between 4 and 5. This means that what we tried to explain was very clear for them.Also rating between 5 and 4, teachers considered very complete and understandable our concepts in the presentation.97% of the teachers considered synthethic biology as a good path to develop solutions for actual issues in the world, showing they were in favor of the usage of synthethic biology (see Chart 1).Finally, we wanted to have a general overview of the presentation and decided to include in the answers different adjectives so the teachers could choose. The most chosen option was “Interesting”, which is a positive feedback for us, a minor of 3% chose “Confused" (see Chart 2).Chart 1 Results from one of the questions.Chart 2 More results from the surveysWe consider this activity had great repercussions in both the teachers and us, because they learned about a very interesting international project and took that information to Chile, their country. Probably they are going to spread the information we gave them and maybe there could be more Chilean iGEM teams in the future.As for us, it helped us to hear the perspective of teachers about our project, and as we mentioned, it was very encouraging to hear their positive thoughts and feedback to improve ourselves.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_cinvestaconclusions From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Conclusions Capture moduleAfter all the experiments and results interpretations, it was concluded that even if the ligation of nhaS and RFP with pSB1C3 came out with two types of bacteria (red and white), both of them had the nhaS gene functioning. The reasons of why there were red and white colonies, first, is that the Petri dishes where the transformed bacteria were inoculated with the gene, were not covered with aluminum. This meant that the UV promotor was activated with normal light and activated the production of nhaS and RFP. The second reason is that during the purification process, the RBS and the first 50 nucleotides of the RFP region mutated because of the exposure to UV irradiation, a necessary step in the process. Because of this, bacteria transformed with the ligation of mutated fragments would not express the RFP gene, and therefore they appeared white.The mutation of the RBS before RFP causing white bacteria and the functionality presence of nhaS in both types of colonies was proved in the sequencing and in the experiments of viability in salt.As it was reported in the patent, the nhaS gene gave certain resistance to salt but there was not any exact percentage of the resistance. There were performed diverse experiments in order to know the maximum salt concentration bacteria transformed with the gene would survive. The concentrations where the bacteria survived were from 1% up to 15% of NaCl in the medium. It can be concluded that the bacteria survived in different NaCl concentrations up to 15%, while not transformed bacteria can survive in a 1% NaCl medium, but not in higher NaCl concentrated mediums. Another conclusion obtained from the experiments is that the bacteria with NhaS can survive in a high NaCl concnetrated medium only if it has its corresponding nutrients, because in a medium containing NaCl only it dies. We already know that the bacteria survives in a high NaCl medium, but the experiments that prove whether the bacteria captures sodium ions or not will be performed the weekend of June 21th and 22th. The results will be shown in the Jamboree presentation.In summary, nhaS is a gene that produce a multitask protein with a lot of advantages, not only because it is pretty little and do not represent a large genetic charge to the bacteria, but also it gives many abilities to it:●Give resistance to salty environments up to 15% of NaCl concentration●Captures ion sodium ions●Regulates the pH of the cell (this is the reason of the resistance it gives)In the case of the E.CARU project, those characteristics where used with the purpose of remove sodium ions in order to desalinize water, but nhaS can be used in many different forms and in other aspects of biotechnology.Aroma moduleIn the Aroma module tests, the medium with 10mM of salicylic acid was the one that produced the most intense odor. If the quantity is increased to 30m the odor is not reported. This indicates that the plasmid was actually in the bacteria and that the riboswitch worked at 35°C. In the case of the plaque that had the odor even if it was incubated at 29 ºC, it is concluded that this happened because the riboswitch is very sensitive to heat, and it was activated during the little time at which it was outside the incubator. In the case of the bacterias exposed to salicylic acid at 30mM, they presented a very similar odor despite of other conditions. This means that the 30mM concentration of salicylic acid could eliminate bateria or could affect the enzymatic reaction of the WinterGreen enzyme by saturating it. As an extra, at first, the experiments where performed in test tubes. After obtaining an aroma result, the team did the same experiment in Petri dishes. It was appreciated that the odor was more intense in Petri dishes than in test tubes.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_conclFrom 2014hs.igem.org Students Team 4 modules Project Race for Science Human Practices Posters Safety We are iGEM CIDEB 2014 team, from CIDEB high school in Monterrey, Nuevo Leon, Mexico. This year, our proyect is E. CARU, a bio-filter that captures sodium ions and remove them from water, in order to make a cleaner water. In this wiki, you can learn from our proyect, our activities, and our experiments. Learn about the four modules from our proyect to understand how E. CARU works, and learn the benefits from our biofilter. Learn More See our work inside the lab! Including our construction plan, lab notebook and project experiments. Learn More See the results of our biobricks constructions, minipreps, digestions, purifactions, ligations, characterizations and experiments. Learn More More than 600 people, learning about our project and iGEM, while exercising. Learn More An entertaining way to learn the basics of our project from your computer. Download it! See the work in the mathematical model to predict our project's behavior. Learn More Analysis and measurements of the possible risk of our project and its control. Learn More Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexichttp://2014hs.igem.org/wiki/images/7/72/Logo_dos_y_asicideb2014.pngsafety From 2014hs.igem.org Safety Questions Posters Lab Safety Cards Memory Game Risk Analysis Safety Questions Read our answers to the iGEM's safety questionnaire, and how is our project safe for the environment and people. Posters Read about our activities, that were specifically orgainized to transmit our project's safety precautions to the CIDEB students. Risk Analysis Read about how the safety measurements in the lab were applied, in order to guarantee that our project does not represent any risk. Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/safethp explosion From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame iGEM Motley With the purpose of obtaining information of how our High School and the public in general sees iGEM and our iGEM CIDEB project we created our own iGEM motley. According to the divulgation objective, the group has developed activities with the goal of showing an overview of synthetic biology, as an initiative and entertaining visual recognition, a motley was elaborated. The importance and objective of the motley is to generate interest among people, causing them to come and see paraphernalia explaining our project (posters, bracelets bearing an explanation of our project, ando more.) There are two main activities where the motley was used; the first one is called DNA Week – it took place inside a high school, and consisted of several activities that promoted synthetic biology. The second activity was called "Race for Science", and consisted in a series of modules concerning synthetic biology and its different areas.Image 1. Motley during the "Race for Science".Image 2. Motley participating during the different iGEM activities. iGEM Explosion Objective and DescriptionWhen we started our project, we noticed that the students, teachers and administrative employees in our high school, were not involved in our project and in the iGEM competition as we wanted them to be. For this reason, we decided to make the iGEM “Explosion” to inform everyone in our high school about iGEM and stimulate their curiosity.Image 3. Different logos used during the iGEM explosion.Our activities were all focused in giving them a little bit of information about us in a creative way.For this “Explosion”, we designed several posters with images related to iGEM and some more with information about our project. After this, on the first day after vacations, we placed these posters all over our high school building in commonly visited areas. With this, we could take the students' attention and make them look what the posters said, inciting curiosity towards iGEM.Image 4. Messages showed to the students on the iGEM explosion.We also made a Facebook page and Twitter account with the purpose of uploading pictures of the team working, several science related images, information related to our project and updates of our activities in laboratory, so people could see all of our progress in an easy way.Image 5. Different social webs of the team.We wanted everyone to have something personal and related to us, so we made bracelets and sold them. Both of the designs we made had the caption “iGEM” on them, the blue bracelets also had some little figures representing the 4 modules or our project and a tiny E.CARU with its representative snorkel. The other design was either given in green and white (iGEM colors) or purple and yellow (our High School colors), they looked like the DNA structure and were hand-made by us!Image 6. Bracelets sold during iGEM explosion.ImpactThe posters gained the attention of the people who were walking nearby. Teachers and students stopped and after reading them they continued their way. Now people had the opportunity of learning what our project is about just by walking through the high school halls.Image 7. Posters used during iGEM explosion.With the purpose of motivating second semester students, we planned and exposed an informative conference for the people who were interested in being part of next year’s iGEM CIDEB team. We introduced them the idea that our project should continue in our high school. With activities like “Explosion”, we caught their attention because they could find out what an iGEM project is, what it consists of, and how exciting it can be. The conference was in our High School Auditorium. When the set hour arrived, students were already inside; they showed real interest of being part of this, because they discovered what iGEM is about. Image 8. Students interested in forming part of the new iGEM CIDEB 2015 team. Project Zoom In Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_explosioachievements From 2014hs.igem.org Achievements During the project, iGEM CIDEB 2014 acquired the following achievements:Dry Lab Cloned BioBricks in E. coli and characterized them: -> BBa_K1255003 which gives our bacteria the ability to produce an odor. -> BBa_K1255002 which gives our bacteria the ability capture Na ions. Characterized a part from another team: ●From MIT 2006:-> BSMT1 (BBa_J45004) which gives our bacteria the ability to produce an odor. ●From Tu Delft 2008:-> RNA Thermomether: BBa_K115017 which gives our Aroma module the ability to be regulated by temperature. ●From Colombia-Israel 2007:-> UV Promoter: BBa_I765001 which lets our Capture module the ability to be regulated by UV light. Created a completely new part in the registry. -> nhaS gene, which gives our bacteria the ability to bind to Sodium ions: BBa_K1255000 Finished a construction of the project from another team. ->From the UANL 2012 team: - AIDA-I Signal peptide: BBa_K888005- L2 protein of adhesion to silica: BBa_K888000- AIDA-I Translocator domain: BBa_K888001 Used an already existent part from the registry. -> Which gives our bacteria the resistance to a saline environment: IrrE (BBa_K729001) from UCL 2012.Lab Work Obtained the experimental results that were needed for the correct function of an important part of our project. We had a control over our experiments, being regulated by heat (Aroma module) and UV light (Capture module). All the lab work was performed following the correct methods and the safety protocols.Math Model Mathematically modeled all the four modules of our project: -> Capture -> Aroma -> Resistance -> Union Graphs were done with the correct information from the model.Human Practices Achieved that all students from our high school knew what iGEM is and the explanation of this year’s project. Explosion & DNA Week Gave a presentation to 40 foreign teachers in which it was explained what iGEM is and our project. CINVESTAV Organized a 4k race for students and general public to promote: health, science and iGEM’s work. Race 4 Science Collaborated with the HS teams UCL Academy and GenetiX Tec CCM. Collaborations Collaborated with the College team Evry. Collaborations Created a videogame to recreate the main function of our project. Videogame Participated in an event with all the iGEM teams from the state. Enlace CientíficoSafety Performed a complete Risk Analysis from our project. Risk Analysis Designed a contention method for our bacteria. Risk AnalysisReturn to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/achievementeam instructors From 2014hs.igem.org Students Advisors Instructors Assistants Fun Instructors   Mr. Heber Torres     Miss. Jannet Salinas   Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/team_instructomath union From 2014hs.igem.org Overview Capture Aroma Resistance Union Union Module The union module is based on the use of a fusion protein composed by L2 and AIDA. L2+AIDA is a protein which is not affected by external factors during its transcription or in its translation, so the established parameters were needed to use:\begin{equation}\large \frac{d\left [ mRNA \right ]}{dt}= \alpha_{1}-d_{1}\left [ mRNA \right ]\end{equation}The parameters for translation and transcription rates from Singapore 2008 iGEM team were used, as well as the speeds at which E. coli carried out the transcription and translation, assuming a transcription speed of 70nt/s and a translation speed of 40aa/s. These data were used in the equations below with the L2+AIDA gene length (2620nt) and protein length (856aa) respectively:\begin{equation}\large \alpha_{1} = \frac{transcription speed}{gene length (nt)}\end{equation}\begin{equation}\large \alpha_{2} = \frac{translation speed}{protein length (aa)}\end{equation}\begin{equation}\large \alpha_{1} = \frac{(70)(60)}{2620} = 1.6 \end{equation}\begin{equation} \large \alpha_{2} = \frac{(40)(60)}{856} = 2.8\end{equation}After, it was needed to use the parameters for degradation rates of proteins and mRNAs obtained from Beijing PKU 2009 iGEM team:\begin{equation}\large d_{1} = \frac{1}{half-life(min)} + \frac{1}{30min}\end{equation}\begin{equation}\large d_{2} = \frac{1}{half-life(min)} + \frac{1}{30min} \end{equation}Since it was a fusion protein, a research was made to find out the half-life of each protein. The half-life of membrane proteins range between 2 to 20 hours in E. coli (Hare, 1991), and as AIDA-I is a membrane protein its half-life must be between that range, but it was not obtained the specific AIDA’s half-life. L2’s half-life was assumed to be 7.8 hours (Bergant, 2010). Bergant’s team made a test with a homologous protein, but found in the minor capsid of the Human Papillomavirus (HPV). Although the function of the L2 strand in HPV is viral and in E. coli is ribosomal, both share similar structures and sequences. Once it was decided to use the half-life from the homologous L2, it was used as the half-life for the fusion protein because it was between the range of AIDA-I and also because it was the lower half-life, assuming when E. coli starts L2 degradation, the whole protein would be degraded.Later, was used the information from Selinger’s team (2003) to determine the mRNA degradation. They performed several experiments for finding the average mRNA half-life in E. coli. They used mRNAs about 1100nt concluding they have an average half-life of 5min. Using the previous relation was found mRNA’s half-life from L2+AIDA which was about 11.9min.\begin{equation}\large HL = \frac{1100(nt)}{5 min}\end{equation}With all these information, the degradation rates for both transcription and translation of L2+AIDA were found:\begin{equation}\large d_{1} = \frac{1}{11.9} + \frac{1}{30} = 0.11\end{equation}\begin{equation}\large d_{2} = \frac{1}{468} + \frac{1}{30} = 0.035\end{equation}Simbiology® was used for the simulation. The previous data from the equations were used to calculate the amount of proteins E. coli would produce at certain time. The results obtained are shown in the following graph:For translation there was another factor that was needed to be taken into consideration, the “fpost”, which were the post-translational variables affecting the production of the functional protein:\begin{equation}\large \frac{d[P]}{dt} = \alpha_{2} \cdot[mRNA] - d_{2}[P] - f_{post}\end{equation}Since the fusion protein needs to be expressed in E. coli’s membrane, it was needed to find the average velocity at which E. coli exports proteins to its membrane. The process by which bacteria exports its proteins are divided into three phases: the “breathing” (which happens between translation and the second phase), the second phase (which is protein movement), and translocation (in this phase the protein attaches to the membrane of the bacteria) (Peskin, 1991).With the previous data it was determined E. coli completes these phases in an average of 5 to 6 min, depending on the protein size (Driessen, 1990). The time used was 5.5min because L2+AIDA is not too big (2620nt).According to Ikeda and Kuroda (2011), L2 carries out an unfolding process to become functional . It was found that 50S ribosomal proteins L2, L3, L14, L23, L24, and L32, as well as the 30S ribosomal proteins, S12 and S18 are native premolten globules in their free forms, but adopted rigid well-folded conformations during the formation of a functional ribosome. They exhibit several amounts of ordered secondary structures; the unfolding of a protein molecule results in an essential increase in its hydrodynamic volume. For instance, there is a well-documented 15–20% increase in the hydrodynamic radius of globular proteins upon their transformation into the molten globule state (Unversky, 2002). Also, Unversky developed an equation used to determine unfolding rates shown next:\begin{equation}\large [H]boundary = \frac{[R]+1.51}{2.785}\end{equation}This equation gives the estimation of the "boundary" mean hydrophobicity value, “[H]boundary”, below which a polypeptide chain with a given net charge “[R]” will most probably be unfolded. Thus, sequences of natively unfolded proteins may be characterized by a low sequence complexity and/or high net charge coupled with low mean hydrophobicity (the values are specified for globular proteins). According to Ikeda and Kuroda (2011) the net charge “[R]” of L2 is 10.9, so it was substituted in the equation below:\begin{equation}\large [H]boundary = \frac{10.9 + 1.51}{2.785} = 4.45\end{equation}With the unfolding value and the rate of membrane transport in E. coli Simbiology® was used to model the functional L2+AIDA production. The results obtained were the following:Graph 2. Amount of functional and nonfunctional L2+AIDA protein When both graphs were compared (Graph 1 and Graph 2) it was concluded that although E. coli needs to transport L2+AIDA proteins to its membrane, the rate at which E. coli does it is slower than the fusion protein production, but something that was noticed (and was great) is that according to Simbiology®, almost all the proteins, once they are inserted in the membrane, unfold correctly leaving less than 25 nonfunctional proteins which are later degraded.Bibliography/References● BERGANT, Martina N. M. (2010). Modification of Human Papillomavirus Minor Capsid Protein L2 by Sumoylation. Journal of Virology, 11585-11589.● DRIESSEN, Arnold W. W. (1990). Proton transfer rate-limiting for translocation of precursor proteins by the Escherichia coli translocase. Biochemistry, 2471-2475.● HARE, James K. T. (1991). Mechanisms of plasma membrane protein degradation: Recycling proteins are degraded more rapidly than those confined to the cell surface. PNAS, 5902-5906.● IKEDA, Takeshi A. K. (2011). Why does the silica-binding protein "Si-tag" bind strongly to silica surfaces? Implications of conformational adaptation of the intrinsically disordered polypeptide to soli surfaces. Colloids and Surfaces, 359-363.● PESKIN, Charles S. S. (1991). What drives the translocation of proteins. Biophysics, 3770-3774.● SELINGER, Douglas R. M. (2003). Global RNA Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript Degradation. Genome Research, 216-223.● UVERSKY, V. (2002). Natively unfolded proteins: A point where biology waits for physics. Protein Science, 739-756.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/math_unioteam fun From 2014hs.igem.org Students Advisors Instructors Assistants Fun Just for fun In case you thought that iGEM was boring, here is a bunch of random stuff we did while we were taking breaks form working in our project: Mr. Walter the potato Lab Break with E.CARU Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/team_fuFrom 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Experiments See the methods the team followed to realize the experiments to each of the four modules of E. CARU Results See the final results of all the experiments done to the different modules of this year project. Conclusions Read the conclusions our team reach after the discussion of the results, and the final ideas of the project. Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labworresults From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Results Biobricks construction Experiments Biobricks construction- Return to the topHere are the results of how the team got the modules of the projectMiniprepsDigestionsPurificationLigationsCharacterizationMinipreps- Return to the topThe first step for building the four modules in a bio-brick format, with the vector pSB1C3 is to isolate the plasmid DNA from the bacteria through a mini prep.The next electrophoresis geles (Image 1) shows that the extraction of the DNA was performed correctly.At the beginning, it was planned to put the four modules in vectors with different antibiotic resistance (such as pSB1C3, pSB1T3, pSB1A3 and pSB1K3) in a single E. coli, but the team decided to first, insert all of the genes in pSB1C3 so they could be sent to the parts registry. Image 1. Electrophoresis geles showing the plasmid DNA gotten from mini preps of the bacteria transformed with pUC57-NhaS, pUC57-BSMT1 opt., pUC57-AIDA, pUC57-L2, pSB1C3-RFP, pSB1K3-RFP and pSB1A3-RFP.Digestions-Return to the topThe next step of obtaining the DNA is get only the needed fragment for the project, this would be accomplished through a digestion with the enzymes of the biobrick format: EcoRI and PstI (In the case of pSB1C3, nhaS and BSMT1 opt.). But in the case of the union module, there is a fusion protein, it means that the digestion of AIDA and L2 (parts of the fusion protein) is with other enzymes, the first with PstI and BglII, and the second with EcoRI and BamHI.In the digestion made in order to get the plasmid pSB1C3 and the gene NhaS (Image 2. right gel) both were differentiated from their respective insert (RFP) and plasmid (pUC57) by its length. The plasmid stays in the upper part of the gel while the insert stays below it.The same happened with the digestion of BSMT1 opt., AIDA and L2 (Image 2. left gel) the genes were differentiated by its length.Image 2.In the left gel: Gel of digestion of pSB1C3 (left black square) and NhaS (right black square). In the right gel: Gel of digestion of pSB1C3 (upper left square), AIDA, L2 and BMST1 opt. (In that order, the tree black squares of the right).Purification- Return to the top There is not need of make all the purification process of the fragments gotten in the digestion, easily the ligation can be done. But to make sure that only the pieces that were wanted to ligate, were together, it was done a purification. Then to confirm that there were only the fragments such as pSB1C3, BSTM1 opt. L2 and AIDA, it was made an electrophoresis gel:Image 3. Electrophoresis geles of digestion after purification process after. The "M" before the first well of the gel, stands for MarkLigations- Return to the topAfter the purification of the digestions was made, the fragments were ligated with their respective gene-plasmid. Then bacteria were transformed with those ligations and inoculated in a Petri dish obtaining the next result:Ligation of NhaS and pSB1C3 [Capture Module] - Return to the topIn the first transformation of the ligation of NhaS and pSB1C3 grew red (expressing RFP) and white bacteria. The Petri dish was not cover by aluminum after its inoculation. Image 4. NhaS first ligation with pSB1C3. Result of the transformation of the ligation between NhaS and pSB1C3.There was a second transformation of the same ligation, also it was inoculated in a Petri dish getting Image 5. as result. There were also present red and white bacteria. The Petri dish was not cover by aluminum after its inoculation.Image 5.NhaS first ligation with pSB1C3. Result of the transformation of the ligation between NhaS and pSB1C3. Result of ligation BSMT1 (optimized) and pSB1C3 [Aroma Module] - Return to the topThe main gene of the aroma module, BSMT1 opt. was ligated with pSB1C3, and then transformed in bacteria in order to be inoculated. In the resulting inoculation there were only white colonies of bacteria.Image 6.Colonies obtained from the transformation of the ligation BSMT1 opt and pSB1C3. Characterization- Return to the topCapture module characterizationOnce NhaS was in the pSB1C3 plasmid, it was needed to prove it, through a characterization; also there was the question of why were there in the ligation red and white bacteria, if all had the plasmid to chloramphenicol resistance (reason they lived). Which of the two types really had NhaS?. To know with which enzyme make the digestion, it was made a digital digestion of the plasmid and the insert getting the next result:Image 7. Virtual digestion of NhaS (yellow) +RFP (red) +pSB1C3 (purple) by the enzyme Arsl (blue), showing that its restriction site is repeated two times, one in NhaS and other in pSB1C3. The problem was that the enzyme that cuts NhaS and pSB1C3 was not available to the team, and it would take a long time to get it. To solve this problem, it was sent the DNA to be sequenced and then prove that the ligation actually occurred, and NhaS was inside pSB1C3. It was used a primer that is from 5' to 3' in the complementary chain:5´ATTACCGCCTTTGAGTGAGC 3'The result of the sequencing of the mini prep of the bacteria transformed with NhaS in pSB1C3 that showed the RFP production was the next (in 3' to 5' direction): 3' AAAGTGTCCACCCCGTACGACCGAGCGGAGCGAGTCAGTGAGCGAGGAAGCCTGCATAACGCGAAGTAATCTTTTCGGCTTAAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACGACCTTCAGCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGAGCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGGAAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAACACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTGTTTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGAACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGGTCTCGGGTACCTTCGAACGGACGACCTTCACCTTCACCCTTCAATTTTCAAACTCGTGACCGTAAACGGAACCTTTCCATACAACTTTGAAAACGCATGAAACTCATTTGAATAACGTCTTCCGGAAGAAAGCCCAATCTAAGTATTTTCTCCCTCTTTTCTCATATAAATGTGATGAATATTTGATCTATCCGCCCTCCAACAACTTTCCCACAACAATCATGTATCGAAATTCCTGTTATACGACACTATAAAGATGGTATAAAAAGCCCGTGGAGGGGGCGTGACCA 5'And this is the sequence obtained from the miniPrep of the white (non-RFP) transformed bacteria with NhaS:3' TAAATAAAAAGTTTTTTCTAATGCGTTTCTTCTCCTACAACCGAAAACACCGGGTCAGTGAGCGAGGAACCTGCATAACGCGAAGCACGCTTTTCCGCAAGAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACTACCTTCAGCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGAGCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGGAAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAACACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTGTTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGGTCTGGGTACCTTCGTACGGACGACCTTCACCTTCACCTTCGATTTTCGAACTCGTGACCGTTAACGGAACCTTTCCATACATGACCATGTTCTCTCGTCTGATTAGCATCGTGAGCCTGATTCTGTCCTTCTACTTCGCTTACAAATACCGTTATCGTGTGATTAACGCGGTGCTGGGCCGTCGCTGGCTGCGTAAAGTTATTATCGGTTTTGCCATGCAGATTCCGATGATTCGTGACCGTATGCTGGGTAGCGTTCTGCAAAGTAACCGTCCGCAAAATGTGTAA 5'Aroma module characterizationTo characterize the aroma module, the process of sequencing was made too3' TAAATAAAAAGTTTTTTCTAATGCGTTTCTTCTCCTACAACCGAAAACACCGGGTCAGTGAGCGAGGAACCTGCATAACGCGAAGCACGCTTTTCCGCAAGAAGAAAAAGGGCAGGGTGGTGACACCTTGCCCTTTTTTGCCGGACTGCAGCGGCCGCTACTAGTATTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCACCGGTGGAGTGACTACCTTCAGCACGTTCGTACTGTTCAACGATGGTGTAGTCTTCGTTGTGGGAGGTGATGTCCAGTTTGATGTCGGTTTTGTAAGCACCCGGCAGCTGAACCGGTTTTTTAGCCATGTAGGTGGTTTTAACTTCAGCGTCGTAGTGACCACCGTCTTTCAGTTTCAGACGCATTTTGATTTCACCTTTCAGAGCACCGTCTTCCGGGTACATACGTTCGGTGGAAGCTTCCCAACCCATGGTTTTTTTCTGCATAACCGGACCGTCGGACGGGAAGTTGGTACCACGCAGTTTAACTTTGTAGATGAACTCACCGTCTTGCAGGGAGGAGTCCTGGGTAACGGTAACAACACCACCGTCTTCGAAGTTCATAACACGTTCCCATTTGAAACCTTCCGGGAAGGACAGTTTCAGGTAGTCCGGGATGTCAGCCGGGTGTTTAACGTAAGCTTTGGAACCGTACTGGAACTGCGGGGACAGGATGTCCCAAGCGAACGGCAGCGGACCACCTTTGGTAACTTTCAGTTTAGCGGTCTGGGTACCTTCGTACGGACGACCTTCACCTTCACCTTCGATTTTCGAACTCGTGACCGTTAACGGAACCTTTCCATACATGACCATGTTCTCTCGTCTGATTAGCATCGTGAGCCTGATTCTGTCCTTCTACTTCGCTTACAAATACCGTTATCGTGTGATTAACGCGGTGCTGGGCCGTCGCTGGCTGCGTAAAGTTATTATCGGTTTTGCCATGCAGATTCCGATGATTCGTGACCGTATGCTGGGTAGCGTTCTGCAAAGTAACCGTCCGCAAAATGTGTAA 5'Experiments- Return to the topIt is needed to characterize and prove the modules, here are the results of the experiments made in order to accomplish it.CaptureAromaUV Experimentation - Return to the topDuring 2 hours the four Petri dishes were exposed to UV irradiation. None of them showed any change. Image 8.Petri Dish inoculated with NhaS in pSB1C3 exposed to UV irradiation at 302 nm. Before, no time being exposed (left) and after 2 hours of being exposed (right). Repetition UV Experimentation - Return to the topTwo Petri dishes inoculated by streak: One with NhaS Red and the other with NhaS White. Both exposed during 30 minutes to UV irradiation at 302 nm and none of them showed any change. With no time exposed to UV irradiation: Image 9.Nhas in pSB1C3 before being exposed to UV irradiation. After 30 minutes: Image 10.Nhas in pSB1C3 before being exposed to UV irradiation. Viability test of the NhaS gene containing bacteria in salt [Capture Module] - Return to the top Experiment #1Bacteria transformed with the capture plasmid were inoculated in Petri dishes with different concentrations of saltImage 11. All the 18 Petri dishes inoculated with NhaS Red in pSB1C3 of all the 9 used concentrations.Image 12. All the 18 Petri dishes inoculated with NhaS White in pSB1C3 of all the 9 used concentrations.Image 13. All the 18 Petri dishes inoculated with the Control bacteria of all the 9 used concentrations.All the bacteria containing the NhaS in pSB1C3 (Red and White) survived to a 10% concentration of salt. None of the control group lived in any concentration of salt.Image 14. Nine Petri dishes with the maximum concentration of salt (10%) used in this experiment. From up to bottom: NhaS Red in pSB1C3, NhaS White in pSB1C3 and the Control bacteria. The first time the experiment #1 was performed, transformed bacteria with the capture plasmid were inoculated in Petri dishes with different concentrations of salt, but this time the mayor concentration is higher (15%) All of the transformed bacteria with NhaS in pSB1C3 (Red and White) lived in the 15% saline medium. All the control bacteria exposed to any concentration of salt died.All the control bacteria inoculated only in LB medium (without salt) lived. Image 15. In the upper left part NhaS Red in pSB1C3 at 15% of salt. In the upper right part NhaS Whit in pSB1C3 at 15% of salt. In the lower left part the Control bacteria at 15% of salt. In the lower right part the Control bacteria with only LB medium (without salt) Experiment #2 - Return to the topIn the experiment #1, it was tested the hypothesis in which the NhaS transformed bacteria (red and white) survived to a saline environment with LB agar. Then it order to know if it would survive only in a saline medium, it was designed a second experiment where the bacteria was inoculated in erlenmeyer flasks with only salty water at different concentrations (1%, 2.5% 5%, 10% and 15%). None of all the inoculated erlenmeyer flasks was murky, it means that all of the bacteria was dead.Image 16. Erlenmeyer flasks inoculated with NhaS transformed red bacteria of all concentrations. From left to right: 15%, 10%, 5%, 2.3% and 1%.Experiment # 3In the experiment #3 the bacteria's hability to withstand salty environments was again tested, but this time the effect that RFP could possibly have in the expression in the NhaS gene. In this experiment, three groups of bacteria (NhaS+RFP, NhaS and a control group) were exposed to different salinity concentrations to test their resistance to salt. This resistance was given by the NhaS gene. A similar process as the one in the experiment one was followed in which bacteria was cultivated in mediums containing salt at different concentrations, and incubated for 24 hours to appreciate how were the colonies formed. In the experiment, the team had the chance to test the effectiveness of the NhaS-producing bacteria. The results of the experiments were measured in the amount of bacteria colonies on the petri dish. In some cases the amount of bacteria were uncountable, because they were spread all over the petri dish, forming a thin film. So it was measured an average size bacteria colony and it was compare with the size of the petri dish. So it was assume that a completely full petri dish has an approximately number of 2500 bacterial colonies. There were 3 different types of bacteria. The ones with NhaS that did not had RFP, the ones with Nhas and RFP, and the bacteria that were used as control (no RFP or NhaS). Each one of these were introduced into a different salt percentage solution. The percentage on which the bacteria were tested are 15%, 10%, 5%, 2.5% 1% of salt on the solution. Also another variable for the experiment was the amount of bacteria concentration within the solution. These concentrations are shown in ratios which are 1:10, 1:100 and 1:1000 of a solution with bacteria on the solution. The experiment were executed twice so it means that there are two results for each type of bacteria on a given concentration on a given salt percentage solution.   The bacteria’s resistance to salinity was expressed in the amount of colonies grown in a medium with a certain level of salinity due to the certain tolerance to salt given by the NhaS gene. After the experiment, the data obtained was plotted into tables for further processing and analysis.You can download the raw data from this experiment in here. Aroma Qualitative Experiments [Aroma Module] - Return to the topExperiment 1 - Test tubes AQUI S; Experiment 2 - Petri dishesRandom people were chosen to smell our bacteria, four people per each concentration. The experiment was performed with three different concentrations of salicylic acid, which were of 10 mM, 20 mM, and 30 mM. All of the samples contained salicylic acid. There was a controlled group grown bellow and above the 32 ºC and a group with transformed bacteria with the Aroma module, also grown bellow and above the 32 ºC; per each concentration. Those are the words that people used to repeat, or synonyms of what the said, because all the opinions were described in a different way. Controlled Group Below 32 ºCGroup Below 32 ºCControlled Group Above 32 ºCGroup Above 32 ºC10 mMCornRotten food but with a fresh scentRotten foodOintment or a very Fresh Scent20 mMRotten foodA little bit freshRotten foodHousehold product. (Fresh)30 mMRotten foodRotten foodRotten foodRotten foodImage 12. People smelling the different Petri dishes previously inoculated with the bacteria transformed with the aroma module in order to describe the odor they perceive. Qualitative Aroma experimentation in petri dishes Go to Results InterpretationsReturn to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_resulhp enlacecientifico From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame "Enlace Científico" Conference Objective and Description After an advance in science is achieved, the scientific community is obligated to share their discoveries. In the specific case of México, there are many programs and businesses eager to share those achievements.The company “Enlace Científico” sent an invitation to the CIDEB-UANL 2014 team to participate in a conference at the Facultad de Agronomía (Agronomy College) in collaboration with other institutions. This conference represented a great opportunity to create relationships with other institutions, encourage people to create their own iGEM teams and talk about our experiences, feelings and opportunities to grow through the competition.In order to achieve these objectives, the conference was planned and presented by the older students in the team, those who had participated at an iGEM competition before; we made a presentation which included the information of CIDEB-UANL 2013 including our goals, difficulties, experiences and most importantly, what happened with all members after finishing the project. We worked with a representative of IGEM-UANL 2014 team, a representative of TecMonterrey team and some others.When the conference began, the director of the company “Enlace Científico” presented each of the speakers and gave a general idea of what the iGEM competition was.Image 1. All participants in the expositions, including iGEM CIDEB UANL team members.After that, the first speaker was a representative from IGEM-UANL 2013. He gave a more detailed explanation about the iGEM competition and he showed details about the project of his team, his experiences and achieved goals. Consecutively, we showed our presentation; it contained 3 main stages: the first being "what the competition was", followed by the second "who were part of the CIDEB-UANL 2013 team and how were they distributed", the third goal was to describe which were the difficulties and goals achieved, and finally, the fourth goal was to speak about what happened after the competition.For the first stage we introduced the competition information and its history, about the first participants and the amount of teams participating today.Afterwards, we showed an image of the CIDEB-UANL 2013 team’s members and explained the way we were organized in order to achieve each important part: Math model, Human practices, Safety, etc.For the 3rd part, we explained the initial difficulties of the creation of the project, mostly about the ideas that were a possibility but didn’t evolve into the real project. The idealization of a viable project is the most difficult part of the entire work because most of the time an idea can be very difficult to achieve or another team could have done it already.Image 2. iGEM CIDEB UANL team members presenting in the exposition.Finally, we talked about what followed the project, in the case of the UANL-CIDEB 2013 team. As we all were high school students the general idea was that everyone would continue their education, each member took a different career according to their ideas and aims but before that we established that our project could be viable and could be used by one of the members or another person for future experiments. This is the most important part of the iGEM project, to think about the consequences of the project and try to improve something in the world.After our presentation, the rest of the groups explained their projects and goals through the competition.Impact The impact of this conference was showed by the students. First of all, there were various questions about the projects and the experiences obtained by participating in a project of this category. There were students that wanted to create a new team but planned to participate until 2015.It was an event during which we had the opportunity to share time with other iGEM teams from Mexico, talk about our projects and, at the same time, encourage students to join the iGEM community.Image 3. Students that attended to the exposition.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_enlacecientificsafety memorygame From 2014hs.igem.org Safety Questions Posters Lab Safety Cards Memory Game Risk Analysis Memory Game Objective and DescriptionObjective: This activity was done to accomplish three main targets of the Team: (1) to make people understand the importance of safety in the lab; (2) to make easier and faster the way people learn biohazard, risk and laboratory signs; and (3) to identify them and keep them in mind every time people work in the laboratory.Description: Team Safety from iGEM CIDEB 2014 designed and built two memory games in one single structure of a considerable size in order to use them in most activities that were done in the semester.The structure’s size was 1 meter of length, 1.22 meters of height and .71 meters of width. Each side of the cubes that hold the images measured 22cm.The first game, the main one, was used to give a fun and easy explanation of the warning signs in the laboratory by inviting people to match the signs with their corresponding meaning.The other one was about the project. It was used to explain the different modules and its objectives to the members of our school. The “2 in 1” memory game was also used in Human Practices’ activities, to obtain a maximum advantage out of it. Some activities were planned with both of this games, mainly competitions within 2 people trying to beat the time record other participant accomplished.DesignThe images used in both of the games were the following:(1)  Laboratory warning:(2)  Project related:PicturesImpactThe shape of the game and its size contributed to its attractiveness for the students and people in general. They would came closer to it just for curiosity and when the team mentioned them its purpose and explained the rules to them, they would become interested in participating actively, as well as asking questions about the signs and the project. This incremented their knowledge about the two aspects that were included in the memory game.All of the students had fun and learned something new about safety and the team after playing this game.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/safety_memorygammath capture From 2014hs.igem.org Overview Capture Aroma Resistance Union Capture Module The capture module is based in the use of NhaS, but NhaS was not the only gene expressed in the circuit (also RFP was used as a reporter). It was needed to consider this factor. The circuit is shown below:As it is shown in the circuit, the transcription rate is affected by Uv light. Uv promoters, according to Shuang Li’s team (2006), have less efficiency than constitutive promoters, which is approximately 60% of constitutive promoters.\begin{equation}\large \frac{d\left [ mRNA \right ]}{dt}=\alpha_{1}\cdot f_{y}-d1\left [ mRNA \right ]\end{equation}\begin{equation}\large f_{y}=0.6\end{equation}It was assumed that “fy”, is the regulatory function that can activate or inhibit the system of a given gene; it was assumed 0.6 of the percentage given by Shuang Li’s team. Uv rays activate the promoter, but it is not as efficient as constitutive promoters in the transcription process.The parameters for translation and transcription rates from Singapore 2008 team were used, as well as the transcription and translation speeds carried out by E. coli, assuming a transcription speed of 70nt/s and a translation speed of 40aa/s. They were used in the equations below with the NhaS and RFP gene (1019nt), NhaS with 69aa.It was not needed to model RFP because is not the purpose of the team, so this section is only focused in NhaS.\begin{equation}\large \alpha_{1} =\frac{transcription speed}{gene length (nt)}\end{equation}\begin{equation}\large \alpha_{2} =\frac{trasnlation speed}{protein length (aa)}\end{equation}\begin{equation}\large \alpha_{1}=\frac{(70)(60)}{1019}=4.12\end{equation}\begin{equation}\large \alpha_{2}(NhaS)=\frac{(40)(60)}{69}=34.78\end{equation}After, it was needed to use the parameters for degradation rates of proteins and mRNAs obtained from PKU Beijing 2009 team:\begin{equation}\large D_{i}= \frac{1}{half-life(min)} + \frac{1}{30min} \end{equation}\begin{equation}\large D_{p} = \frac{1}{half-life(min)} + \frac{1}{30min} \end{equation}However, since NhaS half-life has not been determined yet, it was decided to search for homologous proteins with the same function as NhaS. The team found that the half-life of membrane proteins produced in E. coli range between 2 to 20 hours (Hare, 1991) and as NhaS is a membrane protein, its half life should be between those values. At the end was decided to use 2 hours as NhaS half-life because of its relatively small size (69aa). Later, it was used the information from Selinger’s team (2003) to determine the mRNA degradation. They performed several experiments for finding the average mRNA half-life in E. coli. They used mRNAs about 1100nt concluding that they have an average half-life of 5min. Using the previous relation was found mRNA's half-life from NhaS and RFP gene was about 4.63min.\begin{equation}\large HL=\frac{1100(nt)}{5min}\end{equation}With the previous information the degradation rates for both transcription and translation of NhaS were found.\begin{equation}\large d_{1}= \frac{1}{4.63} + \frac{1}{30}=0.25 \end{equation}\begin{equation}\large d_{2}(NhaS)= \frac{1}{120} + \frac{1}{30}=0.041\end{equation}For the simulation, the team used Simbiology® by plugging in the previously calculated data from the equations to find the amount of proteins E. coli would produce at certain time. The next graph shows the results of the simulation (assuming E. coli is under UV rays which actives its promoter):But for translation, there was another factor taken in consideration, the “fpost” which were the post-translational variables affecting the production of the functional protein:\begin{equation}\large \frac{d[P]}{dt} = \alpha_{2} \cdot[mRNA] - d_{2}[P] - f_{post}\end{equation}Since NhaS protein needs to be expressed in E. coli's membrane, it was needed to find the average velocity at which E. coli exports its proteins. The process by which bacteria exports its proteins is divided into three phases, the “breathing” (which happens between translation and the second phase), the second phase (which is the protein movement to the membrane) and finally, the translocation (in this phase the protein attaches to the membrane of bacteria) (Peskin, 1991). With the previous data it was determined E. coli completes these phases in an average time of 5 to 6 min, depending on the protein size (Driessen, 1990). 5min were plugged into the data because NhaS protein is relatively small (69aa).With the rate of protein transport, Simbiology® was used to model the functional NhaS production. The results are shown in the next diagrams (assuming E. coli is under UV rays which actives its promoter):When both graphs were compared (Graph 1 and Graph 2) it was concluded that the amount of functional NhaS is really big. It was assumed that this happened because NhaS is only 69aa in length. Also, it was concluded that NhaS is functional when it is located in the membrane (by functional is meant that it has the potential to capture Na+ ions and be able to give resistance to saline environments) of E. coli. As is shown in Graph 2 the amount is of proteins ready to bind Na+ produced are about 6500.Bibliography/References● DRIESSEN, Arnold W. W. (1990). Proton transfer rate-limiting for translocation of precursor proteins by the Escherichia coli translocase. Biochemistry, 2471-2475.● HARE, James K. T. (1991). Mechanisms of plasma membrane protein degradation: Recycling proteins are degraded more rapidly than those confined to the cell surface. PNAS, 5902-5906.● LI, Shuang L. X. (2007). A set of UV-inducible autolytic vectors for high throughput screening. Journal of biotechnology, 647-652.● LONNGO, Diane J. H. (2006). Dynamics of single-cell gene expression. Molecular Systems Biology, 1-10.● PESKIN, Charles S. S. (1991). What drives the translocation of proteins. Biophysics, 3770-3774.● SELINGER, Douglas R. M. (2003). Global RNA Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript Degradation. Genome Research, 216-223. Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/math_capturhp collaborations From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame Collaborations Objective Collaborate with other iGEM teams and share information, knowledge and experiences, so we can receive new points of view and also help more iGEM competitors.GenetiX_Tec_CCMOn Monday June the 2nd we had a videoconference with “GenetiX_Tec_CCM” team, from “Tecnológico de Monterrey Campus Ciudad de México” high school. At the beginning, they were contacting one of our teammates and they asked if we could make a Skype Call, we of course accepted and scheduled it when both teams could concur. As we are both Mexican teams, we decided to help each other, also as a symbol of the national help between its habitants.On the videoconference both teams talked about how our projects were developing, if we had any problem with the BioBricks or tools needed for the project. We also talked about general information from our teams, like the number of members, how old we are, how we started in the iGEM Competition, and more. We also had an exchange of information about our projects and it was very interesting to know in what are they working on, how they did it or if they received some kind of capacitation or help from their institution, also telling them how these situations were in our team.This was a great experience for us; to talk to other students with the same age as us, which are working in their own project as we are doing in ours and share our great iGEM experience.Image 1. Skype conversation with GenetiX_Tec_CCM team.UCL_LondonOn Monday June the 9th, we had a Skype videoconference with the “Team:UCL” from London. At first, we scheduled the conference to discuss things about the usage of the IrrE gene. The conversation was about our project, but they also explained their project to us. It was very interesting listening to their project, and how they were developing it; their project was about removing ammonia from water and they were using the IrrE gene to give resistance to the bacteria and a kill switch to avoid uncontrolled spreading. We also spoke about the sponsors they have, and how many team members they were and vice-versa. The team offered us to sequence some pieces, unluckily; we had already sequenced everything that had to be sequenced.The collaboration ended up to be actually very nice and friendly, sharing our opinions about each other’s projects and talking about the teams was a great experience.Image 2. Skype conversation with UCL_London team. EVRY FranceOn Wednesday June the 11th, we shared a videoconference with the “Team: EVRY-Genople” from the college category, in order to continue with our teams collaboration activity. The reunion was scheduled from 12:00 to 12:40, in which we shared our projects and ideas, in order to improve them.In this conference we found out that both projects were very similar, because both worked in saline environments. Their project was about modifying sponges to absorb certain substances in a marine environment, and the piece that we were using for the resistance module: IrrE, would be very helpful in their further project development. In order to continue with this collaboration, we agreed to send the necessary information of our working genes when the experimentation was over.This conference was very rich in content, because is the first time in which we are able to be helpful to another team, besides we met different people, which was a great experience too, like the previous ones.Image 3. Skype conversation with EVRY France college team.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_collaborasafety posters From 2014hs.igem.org Safety Questions Posters Lab Safety Cards Memory Game Risk Analysis Posters about microbes Objective and description Objective: To increase student’s curiosity and knowledge about the microscopic world, as well as the transmission and risks that could happen, if contagion with different bacteria or viruse occurred, according to the risk group they belong.Description: We consider visual learning to be one of the most important ways that people use for acquiring interest and knowledge of things. The safety team from iGEM CIDEB 2014 decided to design and print 10 different 11 x 17 inches posters, including information like their risk group, type, symptoms if an infection occurred, affected organs and transmission of 10 different interesting bacteria or viruses.Each poster included a different bacteria or virus. The described organisms were the following:● B. Anthracis● Campylobacter jejuni● Clostridium tetani● Ebola virus● Escherichia coli● HIV● Marburgo virus● Mycobacterium leprae● Mycobacterium tuberculosis● SalmonellaThe posters were placed in the main stairs of the entrance at our school, by doing so, we assured that the most of the people that entered to the building would saw them.Design You can see all of the posters here: Created with flickr slideshow. Impact Since the first day that the posters were placed, the team observed that they caught the people's attention. They would stop their walk, in order to take a look and read them. We consider this happened because some of the posters contained information that people are interested in for different reasons, like Ebola virus or HIV. Some students and teachers asked different members of the team about the posters, and became interested in the things we do at iGEM.The posters had a great response in the CIDEB community.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/safety_posteparts From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Parts Inserta aquí la tabla :DReturn to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_parfutureplans From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Future Plans The next step to follow in order to accomplish the E.CARU project, must be to put the four pieces in which the team has been working (nhaS, irrE, BSTM1 opt. and L2+AIDA) into a single E.coli. As it was proposed at the beginning E. coli would contain two plasmids: one with the circuit composed by IrrE and L2+AIDA, and the other by NhaS and BSMT1 opt.; both with different antibiotics in order to know that both are inside the cell.It is expected that someday in the future, the theoretical design that was exposed in the abstract of our project can be finished. It means that all the four modules that conform E.CARU can be put together, not only as four separated pieces (see figure 1), but all together: BSTM1 opt. in front of NhaS as a reporter, and irrE with L2+AIDA in another plasmid, in order to give to E. coli resistance and the ability for binding silica respectively. Then, it could be used as the bio-filter proposed, and in a future used in a water treatment plant for processing saline water into usable or even potable water.Figure 1. Diagram representing the proposed circuit in just one E. coli.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_futureplproject abstract From 2014hs.igem.org Description Capture Module Aroma Module Resistance Module Union Module Parts Abstract iGEM CIDEB 2014’s project consists on a biological filter in which sodium ions are taken out of saltwater. To achieve this objective, the project reunited 4 different genes, each one giving E. coli a certain ability to perform a specific task.This year’s project consists of two devices: the first and main one is in charge of the removal of sodium ions. This device uses an aroma to report its effectivity. The second one is responsible of giving resistance to the bacteria to outstand conditions that would normally kill it, and also giving E.coli the ability to bind to silica or glass surfaces.Even though the project was originally composed by this two devices, for experimental purposes it was divided into four different modules. These modules are named after their function, and the name of the project “E.CARU” is an acronym of each one of them.The Capture module uses a completely new gene in iGEM that encodes for a protein that introduces sodium ions into the bacteria. The Aroma module is in charge of producing a mint-like odor in order to report the functionality of the Capture module. The Resistance module allows E. coli to withstand the salinity of the environment in which it is required to work, and finally, the Union module causes the bacteria to join to silica or glass surfaces, giving it the ability to act as a biofilter. Problem Water has been always known as a source of life, but nowadays there is not enough usable fresh water available in the world. The lack of fresh water around the world is an issue that presents a dangerous problem to our future. Only a small portion of Earth’s water is actually usable. Ninety-seven percent of the world's water is too salty for consumption or agricultural use. Furthermore, much of the fresh water is held in ice caps or other unattainable sources. This leaves approximately one percent of the global water as liquid and fresh; ninety-eight percent of which is groundwater (Bouwer, 2002).Different methods have been developed to solve these problems. One of them is desalination; converting sea water (rich in salts) into usable water, but this method is very expensive due to the great use of electrical energy, and the extraction process produces dangerous wastes to the environment (Cotruvo, 2010). For this reason, the project is focused on developing a biological machine capable of performing desalination, reducing costs and avoiding dangerous wastes during the process. For making this possible, E. coli needed to capture Na+ ions in saline environments and to be removed from the water after performing its task. Overview Before E. coli could be able to remove Na+ ions from water, it needed to aquire a resistance to adverse conditions, in particular excess salt. This could be possible through a protein called “IrrE”, which makes E. coli resistant to saline environments as well as UV rays and temperature changes. The protein NhaS (a new part), was used to give E. coli the ability to bind and capture Na+ ions. Also, the optimized version of the reporter “BSMT1”, a protein able to react with salicylic acid and release a Wintergreen odor, was used to know if nhaS was expressed. The final task that E. coli should perform was to bind its membrane to silica pearls, in order to be able to be removed from the water after taking out Na+ ions. In order to do this, a fusion protein named L2+AIDA was used. L2 gives E. coli the ability to bind silica, and AIDA acts as a tag for making L2 a membrane protein. With this ability E. coli could be removed from water through a bio-filter, made up of silica. The complete circuit is shown in figure 1. BSMT1 opt acts as a reporter for nhaS which is regulated by UV (to have a control over the NhaS expression), and IrrE with L2+AIDA are continuously produced.  Figure 1. Diagram representing our proposed circuit But we realized E. coli could have a genetic overload because the circuit was too big (approximately 5000 bp). Also the time we had to finish it was not enough, as well as most of the proteins we wanted to produce were putative or untested. So for a better understanding and for determine if each piece works we divided the project into four modules: capture, union, aroma and resistance, but the project is the result of their correlation. In fact our E. coli was named E. CARU (each letter by each module). Escherichia coli Capture Aroma Resistance Union Project Zoom In Bibliography/References ● Bouwer, H. (2002). Integrated Water Management for the 21st Century: Problems and Solutions. Journal of irrigation and drainage engineering, 193-200. ● Joseph Cotruvo, N. V. (2010). Desalination Technology: Health and Environmental Impacts. U.S: Taylor and Francis Group. Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/project_abstracmethods From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Methods Construction Plan and Protocols Experiments Construction plan and Protocols - Return to the TopHow do we plan to get the construction of our modules? Here is shown all the construction plan that we followed with all the aspects that we considered. Protocols and Construction Plan PDFIt appears your Web browser is not configured to display PDF files. No worries, just click here to download the PDF file. Experiments - Return to the Top In this section, the experiments performed in order to test the effectivness of the predicted models for our project will be described. The tested parts are the following: Capture ModuleAroma ModuleUnion ModuleCapture Module - Return to the TopUV ExperimentationThis experiment was designed in order to test the UV promoter's efectivness.Procedure:1. 2 Petri dishes were inoculated according to the "streak method" with transformed bacteria from a cultured petri dish, containing the nhaS and the RFP genes in their plasmids. (These colonies looked red due to the RFP) 2. The first step was repeated, this time with the transformed bacteria cultures containing the nhaS gene only, without the RFP gene. (These colonies looked white due to the absence of the RFP) 3. The bacteria were grown for one day in the incubator at 37°C4. The four petri dishes were exposed to UV radiation (302nm) for a 2 hour period 5. Pictures were taken of the bacteria cultures at 10 minute intervals during the 2 hour period Go to resultsViability test of the nhaS gene containing bacteria in salt - Return to the TopIn theory, the nhaS gene gives the bacteria a certain resistance to salt, but the exact percentage of increase in the resistance is unknown. Four experiments were designed in order to test: The viability of the nhaS transformed bacteria to survive in a salty environmentThe maximum ammount of salt in the medium which can be withstanded by the transformed bacteriaWhich of the two kinds of bacteria (red and white) gotten in the ligation, has the nhaS gene. In the ligation process of the nhaS gene, two different kinds of plasmids were obtained due to a possible mutation. The ones containing the RFP producing gene, and the ones that did not. Because it was uncertain the presence of the nhaS gene in the non-RFP producing bacteria these experiments were designed and performed in order to determine which one of the groups contain the nhaS gene.   Experiment #1Materials: ● NaCl concentration of 1%● NaCl concentration of 2.5%● NaCl concentration of 5%● NaCl concentration of 10%● NaCl concentration of 15%● 18 erlenmeyer flasks● A 1000 µl micropipette ● Micropipette peaks ● Bacteria with nhaS gene and reporter expression (red bacteria or RB)● Bacteria with nhaS gene and no reporter expression (with bacteria) ● Bacteria with no nhaS gene and no reporter presented (with bacteria without the gene)● 33 petri dishes Procedure: 1. Five concentrations of NaCl in Q water (1.0%, 2.5%, 5.0%, 10.0%, 15.0%) were prepared in separated flasks. 2. The three groups of bacteria (RFP+NhaS, NhaS and the control) were separated in different test tubes. 3. In a petri dish with LB agar and Chloramphenicol, 1 milliliter of the 1.0% concentration of NaCl was introduced; this process was repeated in the three petri dishes and in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.4. In both both petri dishes, 200 microliters of nhaS transformed red bacteria were introduced, and distributed the content with a sterile glass inoculation spreader in a sterile environment. 5. The steps 3 and 4 were repeated four times, but with the other four concentrations (2.5%, 5.0%, 10.0%, 15.0%.). 6. The steps 3 through 5 were repeated two more times, with a the remaining groups of bacteria.7. Three petri dishes with LB agar were inoculated with the control bacteria, introducing 200 microliters of it and spreading it with a sterile glass inoculation spreader. 8. The 33 total petri dishes were cultivated at 37º C for 24 hours. Go to resultsExperiment #2 - Return to the TopMaterials:●15 erlenmeyer flasks of 100 ml.●Cultive of nhaS red transformed bacteria.●Cultive of nhaS white transformed bacteria.●20 ml of each one of the next concentrations of NaCl: 1.0%, 2.5%, 5.0%, 10.0%, 15.0%Procedure: 1. In flasks of 100mL, different concentrations of NaCl in mQ water were preparated(1.0%, 2.5%, 5.0%, 10.0%, 15.0%), having a finale volume of 20mL, and were divided into 3 flasks of each concentration2. 200 µL of nhaS transformed white bacteria solution was placed in the flasks with each of the five different concentrations in a sterile environment, no more than 30 cm away from a Bunsen burner, to avoid sample contamination. 3. The previous step was repeated with the other two groups of bacteria. At the end there will be 15 flasks. of bacteria (five of each type). 4. The 15 flasks were incubated at 37 ºC during a day. Go to resultsExperiment #3 - Return to the TopMaterials: ● 25ml NaCl 1% + 25 ml CLB + 50 µl Cm ● 25ml NaCl 2.5% + 25 ml CLB + 50 µl Cm ● 25ml NaCl 5% + 25 ml CLB + 50 µl Cm ● 25ml NaCl 10% + 25 ml CLB + 50 µl Cm ● 25ml NaCl 15% + 25 ml CLB + 50 µl Cm ● 18 Erlenmeyer flasks● A 1000 µl micropipette● Micropipette peaks ● Bacteria with nhaS gene and reporter expression (red bacteria or RB)● Bacteria with nhaS gene and no reporter expression (with batceria) ● Bacteria with no nhaS gene and no reporter presented (with bacteria without the gene)● 90 petri dishes● 45 test tubes● Peptoned waterProcedure: First part1. Three samples of each salt concentration were produced in order expose our three types of bacteria to each different salt concentration independently. These concentrations were made into Erlenmeyer flasks; this represents a total of 15 Erlenmeyer flasks used in the beginning of the process.2. Three Erlenmeyer flasks were previously settled with the different bacteria. Each of these three Erlenmeyer flask contained a specific group of bacteria (red bacteria, white bacteria and controlled bacteria)3. With the micropipette and it’s respective peaks, 200µL of red bacteria solution were added to all salt concentrations (Just in one flask of the three ones from Nacl1% mix to NaCl 15% mix); this process was repeated in a sterile environment 30 cm near the bunsen burner in order to avoid contamination. 4. The micropipette peak was changed and 200µL of white bacteria solution were added to all salt concentrations (Just in one flask of the three ones from Nacl1% mix to NaCl 15% mix); this process was repeated in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.5. Last, but not least, with the micropipette and it’s respective peaks, 200µL of red bacteria solution were added to all salt concentrations (Just in one flask of the three ones from Nacl1% mix to NaCl 15% mix) ; this process was repeated in a sterile environment 30 cm near the bunsen burner in order to avoid contamination.6. The Erlenmeyer were incubated for 12 hours.See results of first partSecond part1. Poured, into 45 test tubes, 9 mL of peptoned water.2. The 45 test tubes are separated into 3 groups in order to specify 15 test tubes with peptoned water for each bacteria type.3. 1000 µl (1ml) of a certain colony of bacteria (25ml NaCl #% (1, 2.5, 5, 10, 15) + 25 ml CLB + 50 µl Cm with 200µl bacteria (red bacteria, with bacteria and controlled bacteria)) are poured into one test tube with peptoned water making a solution 1:10.4. 2 ml of 1:10 solution are collocated into 2 petri dishes. One ml for each one.5. Another ml of 1:10 solution is poured into another test tube with peptone water creating a new 1:100 solution.6. 2 ml of 1:100 solution are collocated into 2 petri dishes. One ml for each one.7. Another ml of 1:100 solution is putted into another test tube with peptone water creating a new 1:1000 solution.8. 2 ml of 1:1000 solution are collocated into 2 petri dishes. One ml for each one.9. Repeat from step 9 to step 12 using all concentration of all kind of bacteria.Go to resultsAroma module - Return to the TopQualitative experimentsIn the project, one of the modules (the aroma module) consisted in the production of a scented ester in (Winter Green). In order for the gene to work how it was supposed to work, the bacteria must be in an environment with a temperature higher than 32º C. This is because the gene has a constitutive promoter, but an RNA thermoswitch. which permit the synthesis of the protein. The thing is that even if the gene is expressed, it did not had any smell. The reason is because Winter Green odor is produced when the BSTM1 protein comes in contact with salicylic acid. The first experiment was designed only to prove that the protein is being produced and that the Winter Green odor can be smelled.Test tubes aroma experimentMaterials:●10 ml of BSMT1 opt. transformed cultive.●8 test tubes with 3 ml of LB medium and 3 µl of chloramphenicol.●A test tube inoculated with control bacteria (untransformed).●Salicylic acid at different concentrations (1mM, 2 mM, 3 mM and 4 mM).Procedure:First part1. 8 test tubes were inoculated with the BSMT1 opt. transformed bacteria.2. 3 ml of salicylic acid (ratio 1:1) were added to 4 test tubes, each one with different concentrations: 1 mM of salicylic acid2 mM of salicylic acid3 mM of salicylic acid4 mM of salicylic acid3. Incubate all test tubes at 37º C during a daySecond part1. In a rack were put: a test tube inoculated with the control bacteria, a test tube inoculated with BSTM1 opt. bacteria and salicylic acid, and a test tube inoculated with BSTM1 opt. bacteria but without salicylic acid.2. Random people was chosen and asked to smell the three test tubes and describe what the smelled.Petri dishes aroma experiment - Return to the TopThen in order to know if the RNA thermoswitch is working and at which concentration of salicylic acid smells the strongest, the following experiment was performed.Procedure:First part:1. 12 Petri dishes with an LB medium and the Chloramphenicol antibiotic were prepared.2.3 mL of a solution containing salicylic acid and mQ water was added to four Petri dishes, with the concentration of salicylic acid being of 10 mM. *The step noº 2, was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM. 3. 6 Petri dishes with LB medium were prepared. 4. 3 mL of a solution containing salicylic acid and mQ water was added into 2 Petri dishes, with the concentration of salicylic acid being of 10 mM. *The step noº 4 was repeated two more times changing the concentration of 10 mM of salicylic acid to 20 mM and 30 mM. 5. 200 µL of bacteria were added into all of the petri dishes. 6. 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 29 ºC for one day. 7. 2 Petri dishes containing Chloramphenicol from each of the three concentrations and 1 Petri dish without Chloramphenicol from each concentration were incubated at 35 ºC for one day. Second part: 1. Random people were chosen to smell the bacteria and a video was taken of their experience: - A group of bacteria with the aroma module at a temperature below 32 ºC. - A group of bacteria with the aroma module at a temperature above 32 ºC. - A controlled group of bacteria without the gene at a temperature below 32 ºC. - A controlled group of bacteria without the gene at a temperature above 32 ºC. 2. The people were asked to describe what they were smelling Go to resultsQuantitative aroma experiment - Return to the TopMaterials:● 25ml salicylic acid 2Mm + 25 ml CLB + 50 µl Cm ● 25ml salicylic acid 10Mm + 25 ml CLB + 50 µl Cm ● 25ml salicylic acid 30Mm + 25 ml CLB + 50 µl Cm ● 4 Erlenmeyer flasks● A 1000 µl micropipette ● Micropipette peaks ● Bacteria with BSMT1 opt. gene ● 36 Petri dishes ● 9 test tubes● Peptoned waterProcedure:1. One sample of each salicylic acid concentration is produced in order expose bacteria to all three concentrations independently. These concentrations are made into Erlenmeyer flasks; this represents a total of 3 Erlenmeyer flasks used in the beginning of the process. 2.One Erlenmeyer flasks was previously settled with the bacteria (which contained BsMT1 opt). 3. With the micropipette and its respective peaks, 200 µl of bacteria solution were added to all salicylic acid concentrations in a sterile environment 30 cm near the Bunsen burner in order to avoid contamination.4. The Erlenmeyer flask were incubated for approximately 12 hours.5. Poured, into 9 test tubes, 10 mL of peptoned water in all of them.6. The 9 test tubes are separated into 3 groups in order to specify 3 test tubes with peptoned water for each concentration with bacteria.7. 1000 µL (1ml) of a certain colony of bacteria (25ml salicylic acid #Mm (2, 10, 20) + 25 ml CLB + 50 µl Cm with 200 µl bacteria (which contained BsMT1 opt.) are poured into one test tube with peptoned water making a solution 1:10.8.4 ml of 1:10 dillution are collocated into 4 Petri dishes. One ml for each one.9. Another ml of 1:10 dillution is putted into another test tube with peptone water creating a new 1:100 solution.10. 4 ml of 1:100 dillution are collocated into 4 Petri dishes. One ml for each one.11. Another ml of 1:100 dillution is putted into another test tube with peptone water creating a new 1:1000 solution.12. 4 ml of 1:1000 dillution are collocated into 4 Petri dishes. One ml for each one.13. Repeat from step 9 to step 12 using all Mm dillutions.14. The total amount of Petri dishes are divided into two samples in order to have 2 Petri dishes of each 1:10, 1:100 and 1:1000 solution of the bacteria. This creates two groups of 18 Petri dishes.15. The first group is saved into an incubator with a temperature above 32o C. Approximately 35o C.16. The second group is saved into an incubator with a temperature below 32o C. Approximately 29o C.Go to resultsUnion module - Return to the TopEven if the construction of the union module was not finished, there was a planned experiment in order to characterize the module. As the function of the union module is to give E. coli the ability of binding to silica pearls the experiment was designed with the expectative of get results of how many bacteria bind to the silica in certain time, and the relation between how many silica pearls are and how many bacteria binds to it.Materials:● 1000mL (1L) of bacteria with L2+AIDA (silica module) in pSB1C3 plasmid cultive.● 500ml of control bacteria (untransformed).● 75 corning tubes of 50 ml.Procedure:1. Get the weight of each one of the 75 corning tubes2. In 50 corning tubes introduce 20 ml of bacteria transformed with L2+AIDA and in other 25 tubes, 20 ml of untransformed bacteria (control).3. Divide the corning tubes in groups (1, 2, 3, 4 and 5) of 5 control and 10 with L2+AIDA bacteria4. Centrifugate all groups at 5,000 rpm during 10 minutes. Throw the supernatan.5. Weight all the groups and subtract it to the previous weight (Step 1).6. Resuspend bacteria in 20 ml of LB medium.7. In each group, separate the corning tubes and add them the next grams of silica pearls:0.5 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.1 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.2 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.3 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.4 g of silica pearls -> 2 test tubes inoculated with BSMT1 opt. bacteria and 1 test tube with control bacteria.8. Wait during the next time:- Group 1 - 1 hr- Group 2 - 3 hr- Group 3 - 5 hr- Group 4 - 12 hr- Group 5 - 24 hr9. At their respective time separate the silica pearls of bacteria by decantation10. Centrifugate at 5,000 rpm during 10 minutes all the corning tubes. Throw the supernatant.11. Weight each of the corning tubes and subtract it to the initial weight (step 1).12. Compare both weights (Step 5 and step 11). - Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_methonotebook From 2014hs.igem.org Methods Notebook Results Interpretations Conclusions Future Plans Notebook In this section you can find our Notebook where we wrote everything that was done in the Wet-lab: First weeks in the lab!Week 2 of work in MayWeek 3Week 4Week 5Week 6Week 7Last WeekIf you want to download our complete Notebook you can click here --> DOWNLOAD NOTEBOOKMore experiments will be performed the weekend of June 21th and 22th. The results will be exposed in the Jamboree presentation.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/labwork_noteboosafety riskanalysis From 2014hs.igem.org Safety Questions Posters Lab Safety Cards Memory Game Risk Analysis Risk Analysis You can see and download the PDF version of this document, Right Here!OverviewIGEM CIDEB 2014 considers biosafety as important as every other points of the iGEM competition. Because of this reason, the team decided to perform a Safety Risk Assessment focused on the project and in the lab practices needed to accomplish it. In this assessment, a description of our host organism is made, along with the genetic modifications that were applied to it, including preventive measures to avoid its dissemination and appropriate identification and containment measures, in the case it was released into the environment. Also the overall potential risks of the project were included, taking in consideration all of the possible risks of working in our laboratory, along with preventive measures to reduce risk to a minimum.Organism's DescriptionEscherichia coli (E. coli) is a large and diverse genus of bacteria belonging to the Enterobacteriaceae. Although most strains of E. coli are relatively harmless, some can potentially affect humans and animals. Pathogenic kinds of E. coli can cause diarrhea, along with urinary tract infections, respiratory illness and pneumonia, among other symptoms. E. coli can be commonly found in the digestive tract of humans and many animals. It plays an important role in the decomposition and absorption of certain nutrients in the intestine that the body cannot break down by itself and to also prevent the digestive track to be colonized by other harmful bacteria.E. coli are capable of both aerobic and anaerobic cellular respiration, which is a characteristic that allows them to live in both oxygen rich and oxygen poor environments, which has allowed them to thrive in a wide variety of ecosystems.As a prokaryote, E. coli bacterium has no organelles, and its genetic information is not enclosed in a nucleus. E. coli protective layer consists on a cell wall and a capsule that protects it from the outside, potentially harmful environment. E. coli goes through binary fusion on a regular basis if given the right conditions, usually at 37° Celsius, and it is able to thrive and reproduce at a very fast rate.As previously mentioned, E. coli is one of the most diverse genera of bacteria, probably due to its adaptive abilities. Although there is a wide variety of different E. coli strains to choose from, not all of them have the same characteristics; some of them are pathogenic and are not safe to work with, which is the main reason why during the practices at the team’s laboratory, the E. coli’s strain that was used is the K12 DH5-α strain, which is one of the safest strains to work with, and one of the most used in biotechnology research. The K12 DH5-α strain is characterized by its poor abilities to colonize plant and animal tissue, and a low resistance to outside-lab environment, temperature fluctuation and different media composition causing the organism to die.E. coli’s K12 DH5-α inability to colonize intestinal tissue was experimented in 1978 in a work made by R. Curtiss “Biological containment and cloning vector transmissibility” showing that the K12 DH5-α strain is not likely to behave as a pathogen in mammal tissue. Due to these previous mentioned characteristics, it is classified as a Class 1 Containment under the European Federation of Biotechnology guidelines, and according to the United States Environmental Control Agency (EPA) E. coli K12 DH5-α strain opposes a very low risk for other organisms and under contained conditions of fermentation and are safe to work with.Genetic ModificationsIn order to accomplish the iGEM CIDEB 2014 project’s objective, E. coli went through some genetic modifications. The E. CARU project is divided into four different modules, each one of them adding a different characteristic to the bacterium. The four modules are: 1. Resistance 2. Capture 3. Aroma 4. Union1. Resistance moduleUnmodified E. coli K-12 is able to tolerate added salt of up to 10% concentration (M. Don, 2008), however, E. CARU was tested with higher amounts than those mentioned (For further information, check the Capture module in this wiki).In order to work with abnormal higher saline concentrations without killing the bacteria, IrrE, a gene that provides resistance to some adverse conditions for it, was introduced to E. coli.The gene IrrE up regulates the production of several stress responsive proteins, protein kinases, metabolic proteins, and detoxification proteins. It also down-regulates glycerol degradation. With this global regulatory effect, E. coli becomes more salt tolerant (UCL, 2012). The module’s sequence is as follows:The sequence begins with a constitutive promoter (BBa_J23119), followed by an RBS (BBa_B0034), the gene IrrE (BBa_K729001) and a terminator (BBa_B1002).2. Capture moduleOne of the most important genetic modifications in the project is the capture of sodium ions in order to desalinize water. This was made possible by taking advantage of NhaS, a putative gene which is characterized after its expression, “by its corresponding protein ability to bind and sequestering sodium ions.” (Ivey, Krulwich, 1994).The project’s circuit sequence is:Since NhaS is putative, iGEM CIDEB 2014 decided to test the module with a red fluorescence protein, which is simpler than the original reporter idea for the module, and this allowed us to test one gene at a time in each module.The sequence used for the NhaS experimentation is:An UV Promoter (BBa_I765001) was chosen to begin the circuit in order to control the NhaS gene’s expression in E. CARU. The promoter is followed by (BBa_B0034), the NhaS gene (BBa_K1255000), then the same RBS, an RFP reporter (BBa_E1010) and a terminator (BBa_B1002).Basically the same, just changing the RFP reporter for BSMT1 Opt (BBa_K1255001), which is the CDS that is able to produce a Wintergreen aroma. For further information look at the Aroma module in this document.3. Aroma moduleThe use of reporters differing from the usual fluorescence proteins is one of the objectives iGEM CIDEB 2014 team is pursuing by using aromatic reporters, like banana or, in this case, wintergreen odor.The Aroma module is used in order to prove the effectiveness of BSMT1 Opt CDS, for further use as an odor reporter for other teams and modules for this project. BSMT1 (Salicylic Acid Carboxyl Methyltransferase I) is formed as part of a different circuit, composed by a constitutive promoter (BBa_J23119), a riboswitch (RNA thermometer, BBa_K115017), a CDS that, when it is induced by salicylic acid, it releases an enzymatic product (methyl salicylate), responsible of wintergreen odor (BBa_K1255001), and a terminator (BBa_B1002).This sequence will help to test its effectiveness and future usage as an odor reporter, since other teams (MIT 2006) have just analyzed it theoretically. IGEM CIDEB 2013 uses a riboswitch to induce the gene expression at high temperatures.This piece (BSMT1 Opt) can replace RFP on capture module, or be added on union module; as wintergreen odor to demonstrate the presence of bacteria in silica beads or the capture of sodium ions on salty environments.This module will be tested on a culture medium, and induced by salicylic acid to produce WG (WinterGreen) odor.4. Union moduleThe main objective for iGEM CIDEB 2014 team is the construction of a biological circuit capable to capture sodium ions, and to remove them by using a silica-beads based bio-filter. In this module, the outer membrane of the bacteria is modified so it can bind silica or glass surfaces.This device is composed by a constitutive promoter (BBa_J23119), a common RBS (BBa_B0034), a fusion protein that of a set which includes the CDS L2 with its peptide signal and AIDA, in order to make the protein for binding silica; a membrane protein L2 (BBa_K888000); AIDA (BBa_K888001), and a terminator (BBa_B1002).This module was available and proportionated by UANL iGEM 2013 team, however, iGEM CIDEB 2014 made some modifications to it, ligating the peptide signal and L2.A silica bio-filter will be used to remove E. coli from the water, but, in order to have also qualitative evidence of E. coli’s attachment to silica beads, the aroma module’s function as reporter will indicate its presence in the silica.Potential RisksThe project uses E. coli K-12 as the host bacteria, causing it to be resistant to saline water, capture sodium ions, getting attached to silica surfaces and releasing the winter-green aroma as reporter.Potential Risks with the bacteria are minimum because, as previously mentioned in the organism’s description, the strain used is a non-pathogenic type. E. coli K12 has no known survival mechanism in living tissues, nor any of the health affecting symptoms that some varieties have.E. coli is absolutely safe, as P Kunhert states: “They are [E. coli K 12] classified as biologically safe vehicles for the propagation of many efficient gene cloning and expression vectors in all major national and international guidelines on biological safety for work with recombinant DNA technology” (1995).K-12 strain is defective in its cell wall components relevant to the ability to reorganize and adhere to the mucosal surface of colonic cells (Curtiss 1978). It does not have the type of glycocalyx required for attachment to the mucosal surface of the human colon.Performing genetic modifications in the project was not dangerous because the materials were handled carefully, in accordance with the “Laboratory Category One” guidelines published by the World Health Organization (WHO).The modified E. coli parts are safe for the environment and for human use. Variables are controlled by the team, as in the case of Capture module (NhaS) which is controlled by a UV promoter.Similarly, the Aroma module is controlled by a Riboswitch regulated by temperature in order to decide when and how is going to be activated, besides adding salicylic acid, which is only harmful in high doses for humans, and in the project it is used in very small amounts (2mm) to activate it.The Resistance module is also safe, since it gives the bacteria the ability to resist adverse conditions that would normally kill it, like high temperatures, high saline levels and high UV radiation, and nevertheless the bacteria would die in a normal environment.The Union module gives E. CARU the ability to bind glass and silica surfaces, which we will use in beads form with the presentation that is used commercially. No direct risks are related to the module itself, but Silica gel beads may contain toxic and potentially carcinogenic cobalt (II) chloride, which is added as a moisture indicator in commercially available product. This point is analyzed further in the “Union as a safety measurement” section.Lab MethodsIGEM CIDEB 2014 had to take in consideration some aspects about biosafety when working in the project. At all times an instructor was present as a supervisor during the practices.The team followed what the WHO’s category 1 laboratory guidelines indicated. The use of lab coat, long jeans, closed shoes and, if needed, tied hair was performed at all times while working on the lab. When managing dangerous substances gloves and/or special glasses were used depending on the situation.When working with the trans-illuminator, the team took care of not looking directly the UV rays, instead a plastic UV protective shield and glasses were used while working with UV.The laboratory was kept perfectly clean and dry, especially the place in which the instruments were located nearby electrical outputs. Glassware were reviewed and checked for cracks, before each usage; especially before applying under vacuum or pressure on them.The generated waste was always placed in a properly labeled waste container, placed handily to avoid spills.Hazardous SubstancesWhile working in the E.CARU project, specifically in the laboratory, the team was exposed to different substances that, if managed incorrectly, could cause harmful effects.The main substances used in the project were the following (click for further information):● Ethidium bromide● NaOH ● Acetic acid ● Methyl salicylate ● UV radiation ● Tris HCl ● EDTA (Ethylenediamineetetraacetic acid)● DSS (Dodecyl-sodium sulfate) ● Potassium acetate ● Agarose ● Calcium Chloride (CaCl)● Ethanol (Ethyl Alcohol) ● “Lysogenic Broth” (LB) broth and agar ● Buffers ● RNase ● Restriction enzymes ● Saline water ● Milli-Q waterUnion as a safety measurementThe Union module in the project has a genetic device that gives the bacteria the ability to anchor itself to silica surfaces. The team used this ability to use the bacteria as a biological filter. This biological filter would clean the water free of Escherichia coli to meet a solution for salty water. Why silica pearls? Because silica pearls are very common to find, and even though that they are known for being harmful for humans if ingested, it’s not the silica gel that is toxic, what is toxic are the substances that the silica pearl can absorb (silica gel is known for its characteristic of having a great ability for absorbing humidity). Silica is, for example, used as a safe food additive, this means no permit is needed when added.If people are not comfortable with using silica gel pearls for the biological filter, glass pearls can also be used, as the genetic device permits anchorage to glass surfaces, and glass has the advantage of not releasing any kind of substance if introduced in water, even for long periods of time, for the bacterium to attach. Also, for more efficiency in the de-contamination, glass recipients could be used, that way it would be easier for the anchorage of the bacterium.The functioning of the biological filter is very simple: due to the reason that after the first three modules of the project, salt water would still have the genetically modified bacteria, which will have sodium ions within its cellular membrane, a filtration process will be carried out by silica pearls, which will result in desalinized water without the before mentioned bacteria.For the filtration process to happen, no modification was used besides the genetic information that gives Escherichia coli the ability to adhere to the silica gel or to glass surfaces. Thus, when bacteria have already captured the desired amount of sodium ions, by adding silica or glass beads, the bacteria will attach in these and this will help remove the GMO (Genetically Modified Organism) in the water by an easy filtration of the water to be free of silica or glass beads containing the bacteria.The risks considered in the use of silica beads are minimal since "The silica gel is inert and considered a non-toxic product," according to CITUC (UC Toxicological Information Centre for its acronym in Spanish). Actually, if the pearls are not correctly removed, there is only one risk: choking hazard, which is prevented by the safety guidelines that are followed in the laboratory and warns that under no circumstance water should be ingested with or without the silica beads before being tested and approved.The biological filter, besides being the main purpose of our project, is one of the team’s safety measurements, because it prevents the bacterium from contaminating and reproducing in water by being removed.Contention MethodWhen treating with GMOs there’s no such thing as “overprotection”. iGEM CIDEB 2014 takes safety seriously and decided that the project should include at least one method to shut off E. coli in case of an unexpected emergency situation, at least in a theoretical way.The method proposed is simple, increasing the intensity of the UV light already used in the Project to turn off the organism.First of all, Ultraviolet or UV light is light with wavelengths from 100 to 400 nm. According to Meulemans in 1986, its spectrum is divided into 4 different categories, as the following table shows: Type of UV   Wavelenght  Vacuum UV  100 to 200nm  UV-C  200 to 280nm  UV-B  280 to 315nm  UV-A  315 to 400nm EPA (United States Environmental Protection Agency) states that disinfection by UV primarily occurs due to the germicidal action of UV-B and UV-C light on microorganisms. And that the germicidal action of UV-A light is small relative to UV-B and UV-C light. So, in order for UV-A light to cause a “shut off” effect in the organism, very long exposure times are necessary.What UV light does to microorganisms is that, once absorbed, it modifies and damages irreparably the DNA of the organism on the matters of question, in this case E. coli K12, by causing a photochemical damage. Therefore, RNA and DNA, molecules in charge of storing and carrying genetic information, can’t function properly, causing it to lose the ability to reproduce.As Wolfe in 1990 states, and with the information from EPA, 254 nm is the most potent wavelength that can cause damage in DNA. 254 nm belongs to the UV-C light category and the team is planning to use it that way to deactivate E. CARU in extreme situations.The UV light already used in the project is a promoter of the Capture module. This promoter is activated by 360nm, so it belongs in the UV-A category, and as it was mentioned before, this does not cause damage to the bacteria in short periods of time.According to our project, UV is one of the easiest methods to shut off our GMO. ConclusionsAfter doing an analysis of the possible risk related to our project and the safety measures that should be taken into consideration, it can be concluded that the project itself do not represents a considerable risk for human health, due to the poor resistance of the used strain of bacteria, even with the IrrE resistance module, the bacteria is still too weak to thrive in a non-controlled environment. Even though the bacteria cannot live outside the lab, all of the safety related issues to bio-contention of the organisms were all taken into consideration to avoid any possible contamination outside the laboratory. As previously mentioned, the bacteria does not oppose a significant threat for safety, but some substances used in the lab can be a major health concern if not managed properly. Because of this, the laboratory rules were made according to the WHO level 1 laboratory guidelines, and supervision was always present to avoid threats for the team. Bibliography/References● ACETIC ACID (80%, 99.5%, Glacial). Hazard Communication Sheet. Retrieved May 12, 2014, from: http://www.riskassessmentservices.co.uk/HazCom/Acetic%20Acid.pdf.● Agaroses Manual. Condalab. Retrieved May 12, 2014, from: http://www.condalab.com/pdf/agarose_manual_09.pdf.● “A toxic death for ethidium bromide”. (n.d.). Laboratory News. Retrieved May 13, 2014, from: http://www.labnews.co.uk/features/a-toxic-death-for-ethidium-bromide/.● Boric Acid. R.E.D. Facts. Retrieved May 11, 2014, from: http://www.epa.gov/oppsrrd1/REDs/factsheets/0024fact.pdf.● Calcium Chloride - MSDS. (n.d.). Calcium Chloride - MSDS. Retrieved May 13, 2014, from: http://www.calciumchloride.co.uk/calcium_chloride_msds.html.● CHEMWATCH. Sodium Dodecyl Sulfate. Santa Cruz Biotechnology, Inc. Retrieved May 12, 2014, from: http://www.calciumchloride.co.uk/calcium_chloride_msds.html.● Curtiss, R, 1978, Biological containment and cloning vector transmissibility, J. Infectious Dis. 137:668-675.● Don Sarah (2008). Optimal Conditions for the Growth of E Coli. Retrieved May 11, 2014, from: http://es.scribd.com/doc/11337868/Optimal-Conditions-for-the-Growth-of-E-Coli.● EDETIC ACID (EDTA). Summary Risk Assessment Report. Retrieved May 12, 2014, from: http://echa.europa.eu/documents/10162/5ed7db13-e932-4999-8514-378ce88ca51f.● Environmental Health and Safety (2007-09-10). "Silica Gel". Retrieved May 29, 2014, from: http://hazard.com/msds/mf/baker/baker/files/s1610.htm.● Environmental Protection Agency. Sodium Hydroxide. R.E.ED. Facts. Retrieved May 1, 2014, from: http://www.epa.gov/oppsrrd1/REDs/factsheets/4065fact.pdf.● Escherichia coli K-12 Derivatives Final Risk Assessment. EPA. Retrieved May 6, 2014, from: http://epa.gov/biotech_rule/pubs/fra/fra004.htm.● ETHANOL. Hazard Communication Sheet. Retrieved May 12, 2014, from: http://www.riskassessmentservices.co.uk/HazCom/Ethanol.pdf.● Ethylenediaminetetraacetic acid. Summary of Initial Risk Assessment Report. Retrieved May 12, 2014, from: http://www.safe.nite.go.jp/english/risk/pdf/03_summary/047sum.pdf.● Fotadar U, Zaveloff P, Terracio L (2005). "Growth of Escherichia coli at elevated temperatures". J. Basic Microbiology. 45 (5): 403–4.doi:10.1002/jobm.200410542. PMID 16187264.● EPA (2006). Ultraviolet disinfection guidance manual for the final long term 2 enhanced surface water treatment rule. Retrieved May 29, 2014, from: http://www.epa.gov/ogwdw/disinfection/lt2/pdfs/guide_lt2_uvguidance.pdf.● EPA Guidance Manual (1999). Alternative Disinfectants and Oxidants. Chapter 8. Retrieved May 29, 2014. ● General Information Escherichia coli (E. coli) . (2012, August 3). Centers for Disease Control and Prevention. Retrieved May 6, 2014, from: http://www.cdc.gov/ecoli/general/index.html.● UANL Mexico (2012). Recovery module. Retrieved on March 28, from: http://2012.igem.org/Team:UANL_Mty-Mexico/Project/recovery.● Helmestine Anne Marie, 2014. What happens if you eat silica gel beads? Retrieved May 16, 2014, from: http://chemistry.about.com/od/medicalhealth/f/What-Happens-If-You-Eat-Silica-Gel-Beads.htm.● IGEM biosafety 2013. Retrieved May 16, 2014 from: http://www.biofaction.com/wp-content/uploads/2012/04/igem-biosafety-2013.pdf.● IVEY Mark, KRULWICH Terry. (1994) Sodium ion binding proteins. Retrieved April 1, 2014 from: http://www.google.com.mx/patents/US5346815.● Jin Huh (2008). Part BBa_k112808. Retrieved May 16, 2014, from: http://2012.igem.org/Team:METU/KillSwitchOverview.● Jones, T. F. (2007). Investigation of Foodborne and Waterborne Disease Outbreaks. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. L. Landry & M. A. Pfaller (Eds.), Manual of Clinical Microbiology (9th ed., pp. 152-169). Washignton, DC: ASM press. ● Kunhert, P, J Nicolet, and J Frey. "Applied and Environmental Microbiology." Rapid and accurate identification of Escherichia Coli k-12 strands. Ed. P Kunhert. N.P., 31 Aug. 1995. Web. Retrieved May 13, 2014, from: http://aem.asm.org/content/61/11/4135.full.pdf.● McWilliams, M. Luria Broth and Luria Agar Media and Their Uses Protocol. American Society for Microbiology: Microbe Library. Retrieved May 12, 2014, from: http://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocol.● Material Safety Data Sheet. United Biochemicals. Retrieved May 12, 2014, from: http://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocol.● Methyl salicylate overdose Medline plus Medical Encyclopedia. (n.d.). U.S National Library of Medicine. Retrieved May 13, 2014, from: http://www.microbelibrary.org/component/resource/laboratory-test/3031-luria-broth-lb-and-luria-agar-la-media-and-their-uses-protocol.● MIT IGEM Team. (2006). MIT 2006. Retrieved on March 31, 2014, from; http://2006.igem.org/wiki/index.php/MIT_2006.● Normas de Seguridad Laboratorio Retrieved May 16, 2014, from: http://www.uv.es/gammmm/Subsitio%20Operaciones/7%20normas%20de%20seguridad_archivos/NORMAS%20SEGURIDAD%20LABORATO RIO.pdf.● Pérez, E. (n.d.). Boric acid poisoning: Medline Plus Medical Encyclopedia. U.S National Library of Medicine. Retrieved May 12, 2014, from: http://www.nlm.nih.gov/medlineplus/ency/article/002485.html.● Ribonuclease A. (n.d.). - Worthington Enzyme Manual. Retrieved May 12, 2014, from: .● Ribonuclease A. (n.d.). RCSB PDB-101. Retrieved May 12, 2014, from: http://www.rcsb.org/pdb/101/motm.do?momID=105.● "Silica Gel." Centro De Información Toxicológica. CITUC. Red Salud UC Facultad De Medicina. Web. Retrieved May 30, 2014, from: http://www.cituc.cl/temas/ficha1.php?id=22.● The known health effects of UV. (n.d.). WHO. Retrieved May 13, 2014, from: http://www.who.int/uv/faq/uvhealtfac/en/.● Type 1 Water, Milli-Q and Ultrapure Water System. (n.d.). Retrieved May 12, 2014, from: http://www.millipore.com/lab_water/clw4/type1.● UCL iGEM Team. (2012). irrE module. Retrieved March 31, 2014, from: http://2012.igem.org/Team:University_College_London/Module_5.● Zubieta, Chole et al. (2003). Structural Basis for Substrate Recognition in the Salicylic Acid Carboxyl Methyltransferase Family. Manuscript submitted for publication. Retrieved from www.plantcell.org; American Society of Plant Biologists.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/safety_riskanalyssafety labsafetycards From 2014hs.igem.org Safety Questions Posters Lab Safety Cards Memory Game Risk Analysis Lab Safety Cards Objective and DescriptionObjective: Safety Team did this activity in order to accomplish two targets: (1) to make people understand the importance of safety in a laboratory and (2) to help students keep in mind the correct ways to work on laboratory practicesDescription: Safety involves all the activities performed in the laboratory, and also giving safe methods for working, to avoid accidents or injuries; not only for the iGEM contestants, but also for general students. For this reason, the Safety Team from iGEM CIDEB 2014 designed and distributed some cards that included general information about how people must work on the laboratory in order to perform successfully their practices. The structure for each card was of 12cm x 7cm, including eight recommendations that discussed about how students should follow and perform their practices, including images.These cards were delivered by Safety team before an Experimental Science Laboratory practice to students of second semester. The team told them the importance of safety while performing lab work; they also encouraged them to work safer and proceed to give them the cards.The information contained in the cards was the following:1.     Remember to always use a lab coat,long pants, closed shoes and, in case of the girls, tied hair. It’s veryimportant to avoid accidents with every type of reactive you work with. 2.     Whenever dangerous substances are being worked with it’s necessary to use gloves and protective lenses. Skin andeyes are very sensitive. 3.     While doing a practice you shouldbe conscious of what you’re doing. Follow instructions and if you have anydoubt, ask before acting.4.     If you observe that somethingunusual is happening, don’t hesitate to communicate it to the teacher or to alab assistant.5.     Remember that some materials areheat conductors. If you heat something in the Bunsen burner, wait until itcools down and take it carefully in order to avoid skin burns.6.     Hazardous materials are neverthrown away in the drainage.7.     Never… Never add water to an acid.When preparing a solution like that, the water goes first, followed by theacid. 8.     Whenever you finish a practiceremember to leave your workplace and the material clean and in order.DesignThe cards had the following design:ImpactThe students showed interest in the cards and it was observed that all of them read the information before the laboratory practice began, that was a good sign because they were able to keep in mind the information given while working. Since they kept the cards with the information, they became aware that safety was not just for that moment, but for all the future lab practices in their lives.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/safety_labsafetycarteam advisors From 2014hs.igem.org Students Advisors Instructors Assistants Fun Advisors   Miss. Nashiely Moreira Information   Miss. Elsa Flores Lab Work   Mr. Félix Varela Software   Miss. Aracelia Alcorta Math Model   Miguel Loera Math Model   Lily Estrada Dry Lab Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/team_advisohp dnaweek From 2014hs.igem.org CINVESTAV DNA Week Explosion Collaborations Race for Science "Enlace Cientifico" Videogame DNA Week Objective and DescriptionEven though iGEM has been in our high school since 2012, most students still did not know what it is about. We have as a goal that future generations inside CIDEB show more interest and curiosity towards biology, and as a consequence, towards iGEM. We, as a team, consider important that if we want everybody to know what iGEM is and that our high school is involved in it, the starting point must be inside our own institution. With the purpose of spreading iGEM and our project, and taking advantage of the international DNA Day (April 25th), we organized the DNA Week.Image 1. Students listening and participating in the presentations.Our DNA Week consisted of a series of presentations and activities, which were given to all groups of the generation that have a chance to be part of the next iGEM CIDEB team. We asked permission to 3 different biology teachers, and we organized the dates and hours in a 4-day schedule, in May 12th, 13th, 14th, and 16th. We did a PowerPoint presentation with information about iGEM and our project. We also performed two activities referring to synthetic biology and to our project between the information given, with the purpose of keeping the audience interested in what we were talking about. THE PRESENTATION CONSISTED ON: What is iGEM? A general explanation of the iGEM competition. What is synthetic biology? The creation of biological machines in order to give new features to organisms. Talking about DNA A brief explanation of DNA and genes, which make every organism different from each other.   GAME TIME The first game represented the insertion of modified plasmids to the bacteria. They used toy guns to shoot the different plasmids into three different bacteria, taking into account the difference between red, green, and yellow fluorescence proteins in each bacteria.   IGEM in our high school CIDEB_UANL has participated in the iGEM competition for the last 2 years, obtaining the 3rd place in each one. CIDEB 2014’s project E.CARU consists in 4 modules: - The capture of Na+ ions - The productions of an aroma (Wintergreen) - Resistance to different environmental conditions - Binding to silica pearls. LET’S PLAY The last game consisted of a bacteria which two students had to transform into E.CARU, placing in it the characteristics that make it unique: muscles of resistance, a drawn silica pearl representing the binding of the bacteria, leaves of aroma and sodium ions (Na+), which represented the capture module.   Impact We achieved our objective. During the explanation, the students appearedsurprised and interested about the IGEM competition and our project.The response given by the rest of the students, of curiosity and participation, isa very important approach because we awoke their scientific interest and theirenthusiasm to do something related to it. By listening to us and playing the games, students came closer to theteam members asking more about how we were doing our project, what shouldthey do if they want to participate, what do we do in each one of our sub-teams(math model, human practices, safety, and other), and they were even asking us aboutthe posters we placed around the school. They showed a lot of interest.Even teachers approached the team to know about our work.Whilethe last year’s project was only known by a few students, this year is acompletely different situation. Now, thanks to activities like this, most ofthe students at CIDEB know about iGEM. This is a goodthing because some of them will form the new iGEMCIDEB generation next year, and the possibilities of gaining members who arereally interested has increased. Image 2. Student learning about our projectReturn to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/hp_dnaweemath aroma From 2014hs.igem.org Overview Capture Aroma Resistance Union Aroma Module The aroma module is based in the production of SAM/salicylic acid methyltransferase (BSMT1opt) in order to generate methyl salicylate, leaving a wintergreen odor. Since this module is not affected by external factors during its transcription, the established formula of mRNA was used.\begin{equation}\large \frac{d\left [ mRNA \right ]}{dt}=\alpha_{1}-d_{1}\left [ mRNA \right ]\end{equation}In the case of translation, a RNA thermometer affects the production of the protein BSMT1opt. When the temperature reaches 32°C, the RBS allows the translation of BSMT1 gene, but below 32° C does not.\begin{equation}\large RBS\left\{\begin{matrix}T\begin{equation}\large \frac{d[P]}{dt} = \alpha_{2} \cdot[mRNA] - d_{2}[P] - f_{post}\left\{\begin{matrix}TThe parameters for translation and transcription rates from Singapore 2008 iGEM team were used, as well as the transcription and traslation speeds carried out by E. coli, assuming a transcription speed of 70nt/s and a translation speed of 40aa/s. They were used in the equations below with the Wintergreen gene length (1198nt) and protein length (358aa) respectively:\begin{equation}\large \alpha_{1} = \frac{transcription speed}{gene length (nt)}\end{equation}\begin{equation}\large \alpha_{2} = \frac{translation speed}{protein length (aa)}\end{equation}\begin{equation}\large \alpha_{1} = \frac{(70)(60)}{1198} = 3.5\end{equation}\begin{equation}\large \alpha_{2} = \frac{(40)(60)}{358} = 6.7 \end{equation}After, it was needed to use the parameters for degradation rates of proteins and mRNAs obtained from Beijing PKU 2009 iGEM team:\begin{equation}\large d_{1} = \frac{1}{half-life(min)} + \frac{1}{30min} \end{equation}\begin{equation}\large d_{2} = \frac{1}{half-life(min)} + \frac{1}{30min} \end{equation}Since the half-life of BSMT1 has not been determined, a research was made and according to Zubieta (2003), the average half-life for salicylic acid methyltransferases are about 40min.Later, was used the information from Selinger’s team (2003) to determine the mRNA desgradation. They performed several experiments for finding the average mRNA half-life in E. coli . They used mRNAs of about 1100nt concluding they have an average half-life of 5min. Using the previous relation was found mRNA's half-life from BSMT1opt which was about 5.44min.\begin{equation}\large HL = \frac{1100(nt)}{5 min}\end{equation}With the previous data the degradation rates for both, the transcription and the translation of BSMT1opt were found\begin{equation}\large d_{1} = \frac{1}{5.44} + \frac{1}{30} = 0.21\end{equation}\begin{equation}\large d_{2} = \frac{1}{40} + \frac{1}{30} = 0.058\end{equation}For the simulation, the team used Simbiology® by plugging in the previously calculated data from the equations to find the amount of proteins E. coli would produce at certain time. The following graph was the result of the simulation: But for the translation, there was another factor taken in consideration, the “fpost”, which were the post-translational variables affecting the production of the functional protein.\begin{equation}\large \frac{d[P]}{dt} = \alpha_{2} \cdot[mRNA] - d_{2}[P] - f_{post}\end{equation}After researching it was found that BSMT1 is a special type of enzyme called Michaelis-Menten enzyme. BSMT1 reacts with salicylic acid producing methyl salicylate responsible for a Wintergreen odor. As BSMT1 will perform an enzymatic reaction, it was needed to know at which rate it will take place to produce methyl salicylate (Zubieta 2003). For that reason, the post-translational function considers the rate of methyl salicylate production as the variable that directly affects the final protein. The formula is called Michaelis-Menten Equation:\begin{equation}\large f_{post}=\frac{k_{cat}\left [ S \right ]\left [ E \right ]}{K_{m} + \left [ S \right ]}\end{equation}Where: “[S]” means the substrate concentration; “[E]”, the enzyme concentration (obtained by the rest of the translational formula); “Kcat”; is the turnover number; and “Km”, the substrate concentration needed to achieve a half-maximum enzyme velocity.“Km” and “Kcat” were established values for SAM (Zublileta, 2003) of 23 and 0.092, respectively. Later, was needed to know the optimal salicylic acid concentration to produce methyl salicylate. Based in the experimental data obtained by our team it was assumed 10mM in 1ml. “[E]” stands for enzyme concentration, or the protein produced. Substituting in the Michaelis-Menten Equation stays as below:\begin{equation}\large f_{post}=\frac{k_{cat}\left [ S \right ]\left [ E \right ]}{K_{m} + \left [ S \right ]}=\frac{0.092\left ( 10mM \right )\left [ P \right ]}{23 + 10mM}=0.027\end{equation}The enzymatic rate was used in Simbiology® to model the amount of BSMT1 which can releases the Wintergreen odor. The results are shown next:When both graphs (Graph 1 and Graph 2) were compared, it was concluded that the enzymatic reaction was too slow compared with the BSMT1 production, leaving only about ½ of releasing odor BSMT1 (about 600) compared with the not releasing odor BSMT1 (about 1300). The BSMT1 unable to release a Wintergreen odor is later degraded leaving a small amount of the functional BSMT1. It was assumed that the functional BSMT1 is the final product, which releases the Wintergreen odor, not too concentrated. It was assumed E. coli would release a slight Wintergreen odor based in the amount of proteins produced.Bibliography/References● DIBDEN, David J. G. (2005). In vivo cycling of the Escherichia coli transcription factor FNR between active and inactive states. Microbiology, 4063-4070.● SELINGER, Douglas R. M. (2003). Global RNA Half-Life Analysis in Escherichia coli Reveals Positional Patterns of Transcript Degradation. Genome Research, 216-223.● ZUBIETA, C. (2003). Structural Basis for Substrate Recognition in the Salicylic Acid Carboxyl Methyltransferase Family. Plant Cell, 1704-1716.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/math_aromproject resistance From 2014hs.igem.org Description Capture Module Aroma Module Resistance Module Union Module Parts Resistance Module Because our project is a biofilter which allows to remove salt from water, the bacteria needs to survive to extreme environmental conditions which normally it can’t do. We need E. coli to survive a high salinity environment to allow it to capture sodium ions and to remove the salt concentration of the water.In order to accomplish our project goal, we have to change E. coli metabolism and make it stronger, more resistant and more efficient than in normal E. coli bacteria.Last discoveries show irrE as a protein capable of change the E. coli metabolism, and giving it the ability to survive to bigger temperatures, bigger UV rays radiation and bigger salt concentration (UCL, 2012). Description The protein irrE originates from Deinococcus radiodurans, and initially, this gene provides resistance to radiation. But when transformed in E. coli, it protects it against salt, oxidative and thermal shock. (UCL, 2012) Also, different experiments from different iGEM teams, support the idea of the bigger salt resistance in E. coli with this biobrick. Image 1. This graph done by UCL iGEM 2012, shows how irrE biobrick increased the salt concentration resistance in E. coli compared with the results from Tu Delf iGEM 2010.irrE has been demonstrated to up regulate transcription of recA and pprA - genes which encode Recombines A and Radiation Inducible Protein. With respect to salt tolerance, irrE up regulates the production of several stress responsive proteins, protein kinases, metabolic proteins, and detoxification proteins. It also down regulates glycerol degradation. With this global regulatory effect, E. coli becomes more salt tolerant (UCL, 2012).Image 2. This diagram shows the effect of irrE protein on E. coli metabolism. DeviceInitially, IrrE and L2+AIDA, protein for binding silica (Union module), were joined together in only one circuit, but we needed to separate them because L2+AIDA has not been proved yet and it could affect the correct production of IrrE (see image 3).Image 3. Circuit for our project and for testing resistance and union modules. Parts of the Module IMAGE CODE DESCRIPTION   BBa_J23119   In the specific case of our bacteria, it helps to continuously transcribing the irrE gene in order to make bacteria resist high concentration of salt. This promoter has a length of 35 pb(Anderson, 2006).   BBa_B0034   This specific is RBS based on Elowitz repressilator. It is very common to see in many iGEM projects. It has a length of 12 pb (Mahajan, Marinescu, Chow, Wissner-Gross, & Carr, 2003). BBa_K729001 Gene that produces irrE, a substance that changes the bacteria’s metabolism and allows bacteria to survive to extreme conditions, some examples could be high UV rays exposition, or high salt concentration levels in an aquatic environment, oxidative or thermal shock. It has a length of 933pb (Sohrabi, 2012). BBa_B1002   Part made of 6pb responsible for transcription stop (Huang, 206). JustificationsThe resistance module is very important in our project because it allows the correct function of the bio-filter. Because the objective is to remove the sodium ions from the water, E. CARU, at the beggining of the process, will be surrounded by salt molecules which normally E. coli could not support.IrrE gives resistance to the high salt concentration of the water, allowing the Nhas gene to function and capture the sodium ions.Another important aspect in the usage of the IrrE gene, is because the promoter in the Capture module is activated with UV rays at 360 wv, intensity that can cause mutations to the bacterium. IrrE protects E. coli from UV rays and allows the bacteria to their work. Other teams that used IRREUCL 2012: They propose to confer salt tolerance on E. coli by linking the salt tolerance gene encoding the protein irrE (BBa_K729001) to a constitutive promoter (BBa_J23119). Image 3. Part designed by UCL 2012 for the irrE protein.Project Zoom InBibliography/References● Antiquity 2013. (2013, January 31). Part:BBa_B0034. Retrieved from http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034.● Huang, H. (2006, August 30). Part:BBa_B1002. Retrieved August 30, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_B1002.● iGEM2006_Berkeley. (2006, August 24). Part:BBa_J23119. Retrieved April 30, 2014, from http://parts.igem.org/wiki/index.php?title=Part:BBa_J23119.● Sohrabi, B. (2012, June 27). Part:BBa_K729001. Retrieved from http://parts.igem.org/wiki/index.php?title=Part:BBa_K729001.● UCLiGEM Team. (2012). IRRE module. Retrieved March 31, 2014. from http://2012.igem.org/Team:University_College_London/Module_5.Return to the Top Av. Lázaro Cárdenas to the East n/a Col. Mederos, Monterrey, Nuevo León, México. Pedro de Alba n/a Ciudad Universitaria, San Nicolás de Los Garza, Nuevo León, México. igem.cidebuanl@gmail.com iGEM CIDEB UANL 2014. CENTRO DE INVESTIGACIÓN Y DESARROLLO DE EDUCACIÓN BILINGÜE. UANLRetrieved from "http://2014hs.igem.org/Team:CIDEB-UANL_Mexico/project_resistanc1283Beijing HDFLS HighBeijing Haidian Foreign Language SchoolHigh SchoolThe UV-responsing tandem generators for a wintergreen odor and protective melittin.We design a bacteria that produces a wintergreen odor and protective melittin under extensive UV radiation. The biological system includes two generators: the UV-responsing wintergreen odor generator (BBa_K994000) and UV-responsing melittin generator. The melittin generator is composed of two transcriptional devices: RecA (SOS) promoter (BBa_J22106) and synthesized melittin gene with enterokinase digested sequence. The protective generator takes as input extensive UV radiation and produces as output fusion melittin. Melittin can improve immunity and scavenging free radicals to alleviate the harm from electromagnetic radiation, such as UV and blue light.1712BBa_K1283999http://2014HS.igem.org/Team:Beijing_HDFLS_HighBBa_K1283000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164620140http://2014hs.igem.org/files/presentation/Beijing_HDFLS_High.pdfhttp://2014hs.igem.org/files/poster/Beijing_HDFLS_High.pdfHigh SchoolLog in   Team:Beijing HDFLS High From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun   Overview Step1 Step2 We design a kind of bee necklace that contains protective melittin eyedropper, which can help you alleviate eyestrain after using computer for a long time. This is a basic overview of our design this year! This is the compositive generators of this year and last year. The biological system has dual functions. For details, you can refer to our project. Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Notebook From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2Team FoundedOur iGem team set up on October 14th,2013. We are voluntary to join our iGem team.Elected leadersWe had a meeting to elected a leader,a boy won in his speech and experience.BrainstormIn the begaining,we knew more information about igem from Dr Du and the last contestants,then each of us had a discuss and came up with their own idea of product. In the end, we chose to use a girl’s idea that is aim for the Fusion Melittin. Experiment DesignWe found information by surfing the Internet and reading up magazines. After sort out the references materials, then,we designed our project . Learn processWe studied the Melittin to knew what it is and where it comes from, for specific operation,we went to Research institute and university to conduct experiments,such as extraction of plasmid.Perfect websiteEveryone had job to do, we made Our wiki, it is a colourfui website because each memuber have mission to finish in our website.Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Noteboo/Project From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2Contents1 TITLE2 ABSTRACT3 BACKGROUND3.1 Blue light from computer induces eye retina producing free radical3.2 Melittin has funcition of scavenging free radical4 PARTS5 I: Parts of Protective Melittin Generators.5.1 Part A: Rec A (SOS) promoter5.2 Part B: Fusion melittin6 II: Parts of Tandem Generators.6.1 Part A: Wintergreen Odor Generator6.2 Part B: Protective Melittin Genertor7 METHODS7.1 METHOD I: The Protective Melittin Generator7.2 METHOD II: The Tandem Generators8 RESULTS8.1 Result I:The Protective Melittin Generator8.1.1 Plates8.1.2 Electrophoresis Map after Restriction Digest8.1.3 DNA Sequencing and Submitting Part8.2 Result II:Tandem Generators8.2.1 Plates8.2.2 Electrophoresis Map after Restriction Digest8.2.3 DNA Sequencing and Submitting Part9 STEPS9.1 Step1 Transformation and Miniprep9.2 Step2 Digestion9.3 Step3 Ligation9.4 Step4 Tansformation, Miniprep and Digestion again9.5 Step5 Sequencing TITLEThe protective melittin generator; The UV-responsing tandem generators for a wintergreen odor and protective melittin. ABSTRACTThe protective melittin generator is composed of two transcriptional devices: RecA (SOS) promoter (BBa_J22106) and synthesized melittin gene with enterokinase digested sequence. The protective generator takes as input extensive UV radiation and produces as output fusion melittin. Melittin can improve immunity and scavenging free radicals to alleviate the harm from electromagnetic radiation, such as blue light and UV.We further construct a biological system that produces a wintergreen odor and protective melittin under extensive UV radiation. The biological system includes two generators: the UV-responsing wintergreen odor generator (BBa_K994000) and the protective melittin generator. It not only can warn people of intense UV irradiation by a wintergreen odor, but also produce protective melittin. BACKGROUND Blue light from computer induces eye retina producing free radicalThe electromagnetic spectrum, in order of increasing frequency and decreasing wavelength, can be divided, for practical engineering purposes, into radio waves, microwaves, infrared radiation, visible light, ultraviolet radiation, X-rays and gamma rays. The eyes of various organisms sense a relatively small range of frequencies of EMR called the visible spectrum or light. Higher frequencies (shorter wavelengths) correspond to proportionately more energy carried by each photon. Blue light is the most energetic portion of the visible light spectrum, it's less energetic than UV radiation but it also has the ability to penetrate into tissue and cause cellular damage.Blue light is the hardest for the eye to focus on. We actually see a halo around bright blue light. Blue light scatters more inside the eyeball than other colors, producing more glare, more eyestrain and more fatigue. Intense blue light can cause damage to the retina, because blue is the hardest color for the retina to handle. The blue light induce that retinal produce free radicals. These radicals can lead to retinal pigment epithelial cell death. Then some light-sensitive cells lacks of nutrient, which results in visual impairment. Melittin has funcition of scavenging free radicalThe old paint and song implies that the early ancient civilization was conscious of the possible virtues from bee stings.Melittin is the main component (50~55%) of bee venom, which has multiple functions. PARTS I: Parts of Protective Melittin Generators. Part A: Rec A (SOS) promoterExtensive UV irradiation impings on heavily DNA damage and results in a single-strand DNA occuring, which activates RecA protein.Then,RecA protein leads to the expression of about 15 different repair proteins to rescue DNA damage.RecA protein is the central regulator in SOS repair system.Rec A (SOS) Promoter is available in registry part plate. Part B: Fusion melittin We synthesized fusion melittin gene with enterokinase digestion site. Melittin has the function of scavenging free radicals. II: Parts of Tandem Generators. Part A: Wintergreen Odor GeneratorThe generator was constructed last year, and can produce wintergreen odor under extensive UV radiation Part B: Protective Melittin GenertorThe generator is constructed this year, and can produce protective melittin under extensive UV radiation METHODS METHOD I: The Protective Melittin Generator METHOD II: The Tandem GeneratorsWe construct tandem Generators, using similar method with protective melittin generator RESULTS Result I:The Protective Melittin Generator Plates Electrophoresis Map after Restriction Digest DNA Sequencing and Submitting Parthttp://parts.igem.org/Part:BBa_K1283000 Result II:Tandem Generators Plates Electrophoresis Map after Restriction Digest DNA Sequencing and Submitting Parthttp://parts.igem.org/Part:BBa_K1283002 STEPS Step1 Transformation and MiniprepWe get Part A plasmid from registry, and construct Part B plasmid. Step2 Digestion Step3 Ligation Step4 Tansformation, Miniprep and Digestion again Step5 SequencingRetrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Projec/Human From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝That day was a big day for our iGEM team. We visited the Chinese Academy of Sciences Key Laboratory of Plant Molecular Physiology. At first, we met professor Qi Xiaoquan. He analyzed our project with us and give us some helpful suggestions. Then, a Postdoctoral called John Hugh Snyder who is working at here brought us to laboratories and introduced experimental instruments to us. We have learned how to use experimental instruments.It have a lot of good effects on doing experiments later. It broaden our horizons and experience something I'd never experience before. ＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝＝ After some works in our school, we took on the laboratory curiosity, excitement and a little nervousness to go to China Agriculture University to do the experiment . We were divided into four groups, and each group did experiment with one graduate student every time. We completed the experiments of our project in this university. Meanwhile we communicated with doctors, saw advanced instruments. They are interested in our project, and learned some projects about college iGEM competition through us. Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Huma/Team From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2Contents1 Team2 Instructor2.1 Sophia3 Students3.1 Ken3.2 Sukie3.3 Zhao Ziding3.4 Lu Yutong3.5 Chen Songhui3.6 Shi Yutailong3.7 Wang Haochuan3.8 Peter3.9 Liu Xiaoxian3.10 Lisa Hou Letao3.11 Ada Qian Yating3.12 Wu YuLun3.13 KeYu Zeng3.14 Niu Wanxing TeamHello, everyone, this is the team from Beijing, Haidian Foreign Language School. Our school deeply supports us and provides us with a lot of resource. We have came up with a great number of special, individual ideas, and chosen the best one as our project. The 16 students in our team have diffrence in dream, character and goal, but we all love one thing—iGEM. Our dear teathers are getting on well with us.We harvest joy and achievements in the whole process. No matter whether we can win and we will always have the passion to enjoy this competition and do something that is meaningful for all over the world. Instructor SophiaHello! I majored in molecular biology and recieved a Doctorate degree from The Institute of Botany, the Chinese Academy of Sciences, and published a paper about UV radiation-responsive proteins in rice Leaves by proteomic analysis in the academic journal: "Plant Cell and Physiology". The iGEM competition is interesting for me and my students. The process of preparing for the competition was hard, but fun, and exciting for us. It improved the ability of our students and promoted friendship between them. Team ideaDuring the early development of the project, nearly everyone on our team came up with multiple ideas about synthetic biology. Many of them were devoted to the improvement or protection of the environment. Others focused on detection and induction. I am very happy that our memembers are very smart and hardworking! Students KenHello everyone! I am Ken Hu QinKaiMy name is QinKai Hu, and my English name Ken. I am a 15 years old boy who comes from Taiwan. I am a member in our IGEM team. I love Biology. Though my grades in my class isn't very high, I still love studying. I am a outgoing and good at getting along with other. I like making friends, I love traveling and playing basketball. I can play saxophone. In the future, I want to be a biologist. My idea: Absorbing Automobile Exhaust FiltersThese filters can absorb Automobile exhaust. There are many dangerous chemical compositions, including mercury and heavy metals, it can lead people get cancer. These filters can absorb Automobile exhaust to make people healthier. SukieHi, I am Sukie SunYan, a 16-year-old student.I like playing the piano, reading books, drawing chinese painting and so on. In my daily life, I always believe that life is like a mirror, if you smile to it, it will smile to you, so never say never. I am glad to join this competition with my dear teacher and schoolmates. I believe, we can do it and we are the winner. After our hard work, we will achieve our goals.My Idea: Improve the quality of the pigment of our food.Because of food safety has become a hotspot of attention of everyone in the world, safety of food additives has also become an issue of great concern. However, whether in China or other countries, security concerns of food coloring.If, through the bio-genetic engineering of manufacturing a safe, inexpensive, commercially valuable pigment, can greatly alleviate this thorny issue. Zhao Ziding Hello everyone, my name is Zhao Ziding.Nicknames: Xiaobai Age: 17Date of Birth: 1997.02.03Hobby: Music, Guqin, games, sleeping Dream: Although it is not realistic, but I would like to cure cancer Education: High School Self-evaluation: I think I have a cheerful disposition. I never worry about anything. Maybe I am not the best one, but I will be the best. I am easy to get along with and I have good teamwork and organizational skills. Lu YutongHi everybody , My name is Lu Yutong,nickname is lol.I am a 17-year-old girl who is a Class Two Senior Two student . I like dancing,performing, managing, and singing! The hobbies are much more. I have written a good character, obtains teacher's performance frequently. From the first grade, I has been being various branches group leader. I had obtained many certificates,and have published many articles. Hoped that my impression can make you to satisfy! Hoped that my name can keep in your heart! Chen SonghuiMy name is Songhui Chen.I am 16 years old boy who come from Haidian Foreign Language School. My hobby is reading book, surfing on the internet ,listening to the music. My motto is "where there is a will, there is a way ". Shi YutailongHi!My name is Shi Yutailong.I was born in 1997.I am crazy dancing playing lol and learning biology.I have some Weakness.For example:a little lazy.So i want get better and learn morn about biology in this group.My ider:Silicon based human Wang HaochuanMy name is Wang Haochuan.I'm 17 now.I'm a playstation player,my favorite games species is raceing games.I'm learning biology too,so I want to get better and learn more about biology too~And I really want to take part in the game!My idea:The body's damaged cells repair quicker in this condition due to increased metabolism making recovery from illnesses easier and rapid. PeterI am glad to be here, my name is Peter Li Botao who is a 16 years old boy. My hobby is playing ice-hocky and basketball ect. Lots of my friends think i am hard-working, and i always keeping a happy mind. I want to be the pride of my school.My idea: Aviod human snoringStrong snoring sometimes can induce person to sudden death. When a person snoring at very high decibel, the bacteria on his nose will produce a special odor to wake him up. Liu Xiaoxian Everyone, I am a 15 years old girl whose English name is Wendy. My Idea: PM2.5-proof sprayAccording to the phenomena in our life that haze brings lots of trouble such as diseases and traffic accidents. So I suppose that I can use a kind of phagocytic cell and adaptation its express make it gobble up oxides of sculpture and nitrogen even some heavy metal. In severe cases, we can release this cell from the air by helicopter to improve our air. You can install this cell in cars or factories’ exhaust too. When the volcano erupted it can also absorb the small particles, make everyone live under a bright blue sky. Lisa Hou LetaoI’m Lisa. I am 16 and born in Beijing in April 30, 1998. My personality is a little bit complex, sometimes outgoing but sometimes won’t speak any word. My hobbies are singing, playing the piano, skiing, swimming and reading scientific books and my favorite kind of science book is Astronomy. To participate this kind of competition is for not only for board my horizon but also learn more knowledge of science. I’m not the best one in the team but I will do my best in our project. My idea: Plants Growing PromoterDue to the population growth, people had to use artificial ways to produce food, such as fertilizer, etc. Fertilizer can promote the growth of plants, crops, but at the same time have an impact on their health, the food people eat may also be affected，such as chloride ions, they accumulate in crops, when they go into human fat cells, it is difficult to discharge. They can cause respiratory tract Burns, edema, subsequently cause lung and heart crock up. So I wanted to study a kind of cells which can promote plant growth in a physical way rather than a chemical used as fertilizer. Through the synthesis of a cell and implanting the cells of plants, so that these cells can help absorb light, absorbs carbon dioxide, and role. So we can use a physical way to promote plant growth. Ada Qian YatingWelcome to watch myself-introduction.I am a good student and my name is qianyating.I am 15years old and born in Zhejiang province.I don’t like English and IGM because it's difficult for me,but l will stick to learning it well.I was a cheerful girl,so my hobbies is extensive.In my spare time ,l like sleepings,sleeping and sleeping.The endMajesty Leaking and very goodMy idea:How to reduce the harm of secondhand smoke on peopleWe want to design a kind of bacteria that can absorb the harmful component of secondhand smoke. Wu YuLunMy name is Wu Yulun a fifteen-year-old boy. My English name is Eric. And I'd like to listen to the music during my free time. I have broad interests. So I like reading books. For it can enrich my knowledge. I am an open and optimistic person who is easy to get along with. Also I'm a person with strong will and an independent mind. My favorite subject is biology. So I am great attracted in IGEM. This program interest me to try my best to learn it.My ideaAnti-skiddingWhen water contact with smooth glazed bricks, they will become rough. This bacteria avoid accidental harm from smooth glazed bricks. KeYu ZengA fifteen years old Senior 1 student, I am. My name is Zeng Keyu. I’m good at not only taking photos but also painting. In my opinion, they are the most important things for me. At the same time, I also enjoy studying chemical and biological.My idea:How to reduce the harm of secondhand smoke on peopleWe want to reduce second-hand smoke is a hazard to human project. Secondhand smoke has nicotine, tar and other harmful substances. We intend to carry out the project for nicotine. Find a way to neutralize or absorb nicotine substances.By reducing nicotine to reduce the harm of secondhand smoke on people. Can be implanted into a bacterial vector wrist or some belongings in. Niu WanxingI‘m Niu Wanxing, a sixteen-year-old girl in Beijing. In this competition, I am the responsible person of our product. My English name is Annie which means a cute little girl. I’m an Aries. Some people said that Arians are supposed to be courageous leaders but troublesome followers, but I don’t think so. I believe that I can be a good leader and I also can be an eligible member too. Although I’m not the best one, I will try my best to upgrade myself.my idea:Anti-radiation generatorThese days, more and more people work together with computer, i pad and many different kinds of electronic tools. But inside your useful tools are various distinct kind radiations. When the radiations go into your eyes, you would feel uncomfortable because of the blue light. So I want to come up with an idea that can protect our eyes from radiation.Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Tea/Safety From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2 SafetyActually we had safety training before we began the experiment. There are something we have to pay attention for it may affect our product. Firstly we have to wash our hands carefully before we touch the chemical reagent. Secondly, we must clean our tools after we finish our experiments. Thirdly, we have to wear the Latex gloves, the white coat, and the masks. Finally, we should read the experiments carefully, and do the steps in order.Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Safet/Fun From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2We are a very unit team, we like ice-cream and lollipop. Where the crazy is, where we are! Come and support us, it is our pleasure to answer you if anyone ask any question to us.Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Fu/Attribute From 2014hs.igem.org HomeTeamProjectNotebookSafetyAttributeHuman PracticesFun OverviewStep1Step2Contents1 Our Team2 Our Leaders2.1 Zhao ZiDing2.2 Sukie SunYan2.3 Ken HuQinKai2.4 Qian YaTing Our TeamAs a team, we believe that the most significant ability for us is the collaboration capabilities, so we did the experiment together. When we were doing experiment, we divided our team members into several groups, and all of us had the chance to experience the professional process with the help of the doctoral students, because of that, we could do the experiment in a proper way. On the other hand, each individual has its own advantages, so we also divide daily work into different parts, such as photographer, bookkeeper, and so on. Our Leaders Our leaders Zhao ZiDingA year eleven student, who is the most experienced team member, is our main leader. He always gives us the big direction and share some experience with us. Sukie SunYanSukie SunYan is a year ten student, one of the leaders in our team, she is major in daily transaction. Ken HuQinKaiKen HuQinKai is a year ten student, he is our leader too and he is major in dealing with daily things in our team. Qian YaTing She deals with the money in our team and controls the income and outcome of our team.Retrieved from "http://2014hs.igem.org/Team:Beijing_HDFLS_High/Attribut1263BioScience Dragons AZBioScience High School Phoenix, Arizona, United States http://phoenixunion.schoolwires.net/bioscienceHigh School501BBa_K1263999http://2014HS.igem.org/Team:BioScience_Dragons_AZBBa_K1263000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162320140High SchoolLog in   Team:BioScience Dragons AZ From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:BioScience_Dragons_AZ/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:BioScience Dragons AZ logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:BioScience Dragons AZ team.png Your team picture Team BioScience_Dragons_AZ Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project Our project is to engineer E. coli to harness solar energy and produce ethanol from glucose or other alternative sugars. The future application of this manipulation is in the creation of a biopanel. A biopanel will be like a solar panel powered by the E. coli that will convert solar energy into ethanol with a media of glucose provided from another renewable source. The goal is to create a simple and efficient method of renewable energy using daily resources such as sunlight to help in the process. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:BioScience_Dragons_AZ1272Penn Mishawaka INPenn High School, Mishawaka, INHigh School701BBa_K1272999http://2014HS.igem.org/Team:Penn_Mishawaka_INBBa_K1272000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=163420140High SchoolLog in   Team:Penn Mishawaka IN From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:Penn_Mishawaka_IN/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:Penn Mishawaka IN logo.png Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:Penn Mishawaka IN team.png Your team picture Team Penn_Mishawaka_IN Official Team Profile Contents 1 Team2 Project3 Headline text 3.1 Notebook3.2 Results/Conclusions3.3 Safety3.4 Attributions3.5 Human Practices3.6 Fun! Team Tell us about your team, your school! Project == The presence of arsenic in drinking water provides a complicated obstacle for developing countries. Some 100 million people are affected by arsenic poisoning worldwide, and the problem is exasperated by the unpredictable patterns of contamination. This necessitates frequent and comprehensive testing of well water; Bangladesh alone has 10 million water wells that must be checked twice a year. Effective arsenic tests have been created in the past, yet they often require expensive reagents, advanced technology and specialized technicians – not feasible for large-scale operations in the developing world. Our team endeavors to create a simple, biologically-based test for arsenic. Using existing BioBricks and building off the ideas of previous iGEM projects, we have designed two systems that work within E.coli to detect the presence of arsenic in a water supply and generate a strong, noticeable response. The first links an arsR promoter (BBa_J33201) turned on in the presence of arsenic to a firefly luciferase reporter gene (BBa_K325909) that emits a bioluminescent glow. Additionally in the system is a second luciferase reporter gene, (BBa_K325209) attached to a constitutive gene that will constantly emit bioluminescence of a different color to serve as an indicator of normal cell function. Ideally, the end result would be cells that will glow in a dark room, change colors (yellow to green) in the presence of arsenic, and stop glowing entirely if they are killed by arsenic or any other toxin. Additionally, we are in the process of designing a cascade sequence to detect and react to the presence of arsenic. A cascade sequence provides the benefit of customizability: we can control the reaction at many levels, both by ensuring when the sequence is turned on or off and by controlling the amount of output created by the system at different levels of arsenic. == Headline text Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:Penn_Mishawaka_IN1261Acton-BoxboroughRHSActon-Boxborough Regional High School Acton, MA 01720 http://ab.mec.edu/abrhs/High SchoolProject E. coli LuwakKopi Luwak coffee is the rarest and the most expensive type of coffee in the world, and is processed from feces of the Asian Palm Civet (Paradoxurus hermaphroditus). As the coffee berries are digested, bitter components of the coffee bean are broken down. Later, the beans are cleaned, roasted, and brewed to make the Kopi Luwak coffee. The problem with the procedure today is that farming industries keeps tens of thousands of civets living in battery cages, and force-feeds them only coffee berries. As a result of the treatment, the Asian Palm Civet population is declining. We propose to make this process humane, efficient, and sanitary by instead using bacteria to produce the coffee. We will accomplish this by genetically engineering E. coli with genes for various digestive enzymes, thus simulating the digestive process of the civets with coffee berries so we can finally save the Asian Palm Civets.1701BBa_K1261999http://2014HS.igem.org/Team:Acton-BoxboroughRHSBBa_K1261000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162120140http://2014hs.igem.org/files/presentation/Acton-BoxboroughRHS.pdfhttp://2014hs.igem.org/files/poster/Acton-BoxboroughRHS.pdfHigh SchoolLog in   Team:Acton-BoxboroughRHS/Team From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Our Team Our team of eleven students is from the Acton-Boxborough Regional High School and includes: Chris Kuffner Title: Founder, transformation lab master Chris is now a rising senior at ABRHS who is intrigued with the potential of synthetic biology and has a passion for STEM. He thinks that he is chilled out, but he is attracted to volatility. When he isn't trying to get himself killed on skis or wheels, he is working under a heavy course load so he can enter this controversial industry. Chris initiated this team with help from Mr. Mathieu, has done a large chunk of the research for this project and specializes in the transformation lab. Akash KapoorAkash is one of the team's first freshmen. He does find his way into the world of genetics, climbing the ladder of the double helix despite all the pressure from deadlines, video conferences, and registration dates. His coming to the Jamboree will reassure the team that he's still in the game. Epithet: The wanderer Madhuri Jois Madhuri Jois is a rising junior at Acton-Boxborough Regional High School. She is passionate about different fields of scientific research, including synthetic biology. Her interest for hands-on research is nourished through the iGem club. In addition to the sciences, Madhuri also volunteers at Emerson Hospital, and enjoys reading in her free time. Epithet: Poster Guru Charles TaussigRachel TaoKatherine LiuBarry HuangBen SternMy background interests are primarily biochemistry. I am mostly interested in neurobiology, but I also like synthetic biology. I also really like coffee, which is why I was so interested in our project. For our project, I primarily did research on synthetic biology and the processes we would use, as well as research of coffee. Epithet: Head Researcher William Huang I'm in 10th Grade and I contributed to the project by doing the lab grunt work. Victoria Chen Additional help was provided by: Wynne YaoJustin HongTara JawaharBenjamin PervierAnusha Purakayastha With sincere thanks to our coach and mentor, Aaron Mathieu. Aaron Mathieu is a biology teacher at Acton-Boxborough Regional High School in Acton Massachusetts, where he has been teaching since 2000. During his 18-year teaching career he has taught biology (including Advanced Placement Biology), bioethics, chemistry, physics, astronomy, physical science, forensics, and environmental science. Mr. Mathieu has been working with the BioBuilder Foundation for 3 years and is a BioBuilder Master teacher. Team attributions Our team has divided our project into several task forces: the wiki group, the project group, and the initial lab group. The wiki group is for the integration of information from all three groups, regarding lab data, and research, and also works to make sure all guidelines are met. The project group is the primary researching force, which collects all the data about what enzymes and system we'll need. The lab group carries out the laboratory steps in order to synthesize the bacteria which out project is based on. Also, we frequently have periods of time where team members do work outside of their main sub-group, which makes out progress run faster. Hangouts Not only did we 'hangout' in real life after meetings for recreation, we also had Google Hangouts. These series of Google Hangouts helped out team meet and make a lot of progress during the summer, since we couldn't necessarily have a place to meet in person. End of page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/TeamProject From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Project Description Kopi Luwak coffee is the rarest and the most expensive type of coffee in the world. This beverage is made from the feces of the Asian Palm Civet (Paradoxurus hermaphroditus) or Luwak, a cat-like omnivore that fills the niche of a racoon in Asia. The Asian Palm Civet eats raw coffee berries. As the berries are digested, enzymes in the animal's digestive tract break down components of the coffee bean that are responsible for its bitter taste. However, the beans themselves are not digested. The civet only digests the fleshy outer layer, so when it defecates, it leaves clumps of coffee beans that have been processed by its enzymes. The beans are then cleaned, roasted, and brewed to make the Kopi Luwak coffee. Due to the complexity of this process, Kopi Luwak is a very expensive item at $600 per pound. The outrageous price has made Kopi Luwak a novelty for the rich. Its allegedly excellent flavor is sadly something that most people are unable to afford. Another drawback of Kopi Luwak is that demand has driven businesses to animal cruelty in order to keep up production. A small civet farming industry has tens of thousands of civets living in battery cages being force fed coffee berries. Civets, being omnivores, are no more capable of surviving on coffee than humans. As a result their population is diminishing. We propose to make this process more humane, efficient, and sanitary by using bacteria instead of civets to process coffee berries into Kopi Luwak beans. We will accomplish this by inserting genes that code for proteins found in the civet's digestive tract into a hardy bacteria that can withstand the pH levels required for the proteins to operate. We intend to add genes for salivary amylase, pepsin, pancreatic amylase, trypsin, and chymotrypsin, sequenced from palm civets or closely-related species. Once the bacteria have been transformed, we will attempt to simulate the digestive process of the civet on coffee berries and analyze our results. Although this project seems unconventional, putting animal enzymes in bacteria has been done before to great effect. Take rennet cheese for an example. In order to obtain the enzymes required to produce this cheese, a calf must be slaughtered; the material (rennet) is taken from the dead animal's digestive tract. Now, due to growing demand for this cheese, bacteria containing the rennet enzymes are used instead. Today, the sale of these cheeses is a popular and profitable industry. By putting animal enzymes in bacteria, we create a digestive platform that is capable of processing more than just coffee berries. End of page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/ProjecSimulations From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Simulations Your browser does not support the HTML5 canvas tag. RBS Promoter ORF Terminator Backbone End of Page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/SimulationSafety From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Safety Safety is an important process while doing laboratory work in order to stop cross-contamination, and keep the safety of our team members and others. Also, we kept the safety of the delicate environment in mind when disposing and handling certain substances. In the 3A Assembly Before each session of the 3A assembly lab, we had to clear our workspace of any equipment that may cause injury, obstruction of a normal lab workspace, discomfort of the lab runners, and generally, other laboratory labs that we did not want to disturb. With a cleared workspace checked, we washed down the work space with seventy-percent ethanol alcohol. This would ensure the existence of any microorganisms pre-existing in our workspace will not contaminate our apparatus and samples directly, or indirectly by means of secondary chemicals. Also, specific measure were taken so that the E. coli bacterium and other laboratory substances such as enzymes, buffers, or distilled water do not improperly escape into the environment, regardless of how it was used; if it was part of the lab or in the laboratory workspace, then it must be disposed of properly. This process includes properly disposing and ensuring proper post-treatment of pipette tips after every use of a unique substance to prevent cross-contamination of any substances and waste products. Members of our laboratory were required to wear surgical gloves when transferring materials to apparatus such as agar plates, glass test-tubes, Erlenmeyer flasks, etc. Gloves were also worn when handling such substances directly such as sterile glass beads, in order to reduce in-lab contamination and possible external leakage. In the Project lab At the time of the project amylase lab during the week of June 9, we will have specific safety protocols. This not only includes regulations described above in the 3A assembly lab, but also additional precautions since it is a slightly more complex and involved lab. Lab occupancy will be limited to having a maximum of 5 people in the small lab space at a given time. This includes intermediate times between major steps of the lab to take notes and clarify the following instructions for the next set of steps. A specific period at the end of each set of steps will be dedicated to record information so that the data may be in a format appropriate to other applications (i.e. the Lab page of this website), and appropriate disposal and hygiene protocols to each step, including discarding pipette tips, cleansing the workspace with ethyl alcohol, removing physical apparatus not needed for the next few steps, and washing skin that may have come into direct or indirect contact with lab equipment, such as hands. iGEM Safety Questions Researcher, Public and Environmental Safety The organisms we are working with is non-pathogenic k12 strain E. coli from Carolina Labs and a highly competent E. coli from New England BioLabs, which both present low individual and community risk. They are under Risk Group 1 of WHO's (World Health Organization) Classification of Infectious Microorganisms by Risk Group. Our Lab space would be listed as Risk Group 1 under WHO's Relation of Risk Groups to Biosafety Levels, Practices and Equipment. Sterilizing chemicals such as ethanol and bleach pose some danger to individuals, for which gloves are worn. Such sterilizing chemicals are used to prevent the E. coli from escaping into the environment, but no serious danger will come if the transformed E. coli escapes. Also, the parts used for the transformation of the E. coli do not form threat to the environment. BioBrick Part Safety All our parts are listed under our Research Page by code from the iGEM registry of biological parts. None of these parts pose safety issues. NSTA BioSafety Comittee Following NSTA Regulations Along with following safety regulations of our own, we followed the NSTA (National Science Teacher's Association) guidelines for safe laboratory practices handling microbes. This is a recognized protocol that our school's science department uses for safety in a laboratory experiment. Only handling microorganisms with known sources The only sources for such microorganisms should be from biologicla supply houses or university laboratories. The genus and species name should be known. Microorganisms should never be cultured off one's own body as they potenially are pathogenic. Treating all microorganisms as potential pathogens Any microorgansim may potenially present pathogenic properties. Students with comprised immune systems should not participate in such laboratories. Petri dishes should also not be completely open to the air. Personal protective equipment and hygiene Students should wear safety goggles, aprons, and gloves. Open-toed footware should not be worn, and any lose clothing or hair should be pulled back. Washing hands Hands should be washed before and after laboratory procedures to disinfect skin surfaces. Disinfecting work areas before and after use Either 70% alchohol or 10% bleach should be used to disinfect work areas before and after laboratory steps. Ethanol and any paper towels used to clean up the ethanol should be kept away from hot objects such as burners, so that any fumes do not ignite. Sterilizing equipment An autoclaving procedure should be used to disinfect equipment used before the labs. 70% ethanol and 10% bleach can also be used to sterilize. Never pipetting by mouth Liquid cultures should be transferred by a pipette bulb or a pipetting device. Labeling everything clearly Cultures, media, chemicals, and disinfectants should be clearly marked by name. Any hazardous substances should be clearly marked as well. End of page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/SafetResearch From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Research Pre-Research E. Coli vs. Yeast E.coli can live in a higher range of pH and should be easier to engineer, but may be less productive.Yeast will most likely be more be more productive in protein production in the long run but is otherwise inferior to the use of E. coli, so our decision is to use the latter. Also, we used a k12 strain of E. coli from Carolina Labs and a highly-competent strain of E. coli from New England BioLabs. DNA Sequences We need an open reading frame (orf) a ribosome binding site (rbs), a promoter, at least 3-5 enzymes:(pepsin, salivary amylase, trypsin, chymotripsin, and pancreatic amylase), and a terminator, to create a fully functional plasmid to digest the coffee beans.We planned on using the amylase orf that already existed in our ordered tray, in part to treat the coffee beans for breaking down complex carbohydrates to simple sugars. This will make the resulting coffee less bitter and more sweet. The part BBa_K523006 codes for amylase and also includes it's own secretion mechanism, which enables the displaying of amylase on the cell’s membrane. This image shows the template for a typical plasmid requirement Parts The Promoter A promoter is a DNA sequence that tends to recruit transcriptional machinery and lead to transcription of the downstream DNA sequence. The part we anticipated to use as a promoter was BBa_J23100; however, our Amylase ORF already comes with its own LacZ promoter, and ribosome binding site. The Ribosome Binding Site A ribosome binding site (RBS) is an RNA sequence found in mRNA to which ribosomes can bind and initiate translation in bacteria. The part we anticipated was BBa_B0030. This part is considered a strong RBS in terms of initiating transcription. It has many 'relatives', such as BBa_B0031, BBa_B0032, BBa_B0033 and BBa_B0034. The Open Reading Frames Protein domains are portions of proteins cloned in frame with other proteins domains to make up a protein coding sequence. Some protein domains might change the protein's location, alter its degradation rate, target the protein for cleavage, or enable it to be readily purified. The open reading frame we actually ended up using in the main lab and the high efficiency lab was almost solely amylase, which as described, has its own transcription and secretion mechanisms. The Terminator The Terminator is the 'last' region of the plasmid, where RNA polymerase stops transcription. There are two main types of terminators in prokaryotes, rho-dependent terminators, and rho-independent terminators. Rho-dependent termination bases its function on the Rho protein, which causes the RNA polymerase to fall disassociate form the plasmid. Rho-independent termination does not use Rho to disassociate the RNA polymerase, but instead stops by the DNA sequence, here's how it works. The DNA sequence contains a two regions that are rich in cytosine and guanine. When this is transcribed, those regions on the RNA form hydrogen bonds to itself: effectively forming a 'loop' which disassociates the DNA polymerase from the DNA. Within the category of rho-independent terminators, are terminators that stop transcription on the forward strand in forward direction (forward terminators), on both strands and directions (bi-directional terminators), and on the reverse strand and direction only (reverse terminators). One specific part that seems promising is the rho-independent, forward terminator BBa_B1006, which has a 99 per cent success rate of terminating the transcription, the highest efficiency of all the terminators. The Backbone A plasmid is a circular, double-stranded DNA molecules typically containing a few thousand base pairs that replicate within the cell independently of the chromosomal DNA. A plasmid backbone is defined as the plasmid sequence beginning with the BioBrick suffix, including the replication origin and antibiotic resistance marker, and ending with the BioBrick prefix. Anticipated Parts Pepsin This gene will help digest protein in the coffee bean. We needed to find a pepsin gene more similar to the Paradoxurus hermaphroditus (Asian Palm Civet), because there are many different variations. Any secretion most likely means lysis with acid: we do not have the acid proof backbone in our plate.Although we planned on getting this pepsin part synthesized using the template from the existing planned pepsinogen part BBa_M143, analysis of the sequence revealed multiple errors. The part claims to be optimized for production in E. coli; however, it includes the human signal peptide which would not work if synthesized in E. coli. By the time we realized this, it was too late to plan and synthesize a new part. So we decided to just plan the part, submit it, and get it synthesized in the future. Resources Jumhawan, Udi et al, Selection of Discriminant Markers for Authentication of Asian Palm Civet Coffee(Kopi Luwak): A Metabolomics Approach NCBI Taxonomy Browser Martinez, Federico Luis, Quality Enhancement of Coffee with Acid and Enzyme Treatment Quality Enhancement of Coffee with Acid and Enzyme Treatment: Patent Application Marcone, Massimo F, Composition and properties of Indonesian palm civet coffee(Kopi Luwak) and Ethiopian Civet Coffee End of Page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/ResearcLabs From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Laboratory Practices High Efficiency Lab (experimental) Materials competent cells microcentrifuge tubes pipettes plasmid ice bucket thermocycler SOC media Chlor plates and agar plates Procedure Thaw frozen cells and pipet 50 ul into a tube Add 3ul of plasmid DNA, flick tube to mix Put on ice for 30 minutes Heat shock in the thermocycler for 30 sec at 42 degrees (C) Return to ice for 5 minutes After the 5 minutes, pipet 950 ul of SOC into the tube Incubate at 37 degrees for 2 hours, shake Warm plates to 37 degrees in incubator Spread 500 ul of medium onto the chlor and LB plates, incubate overnight. Main Project Lab Materials Reagents: E. coli ( K12 and competent E. coli)50 mM cold sterile calcium chlorideAmylase plasmidLB broth2 LB plates2 LB/chlor plates Equpment and Supplies: Inoculating loopsMicropipettorsmicrocentrifuge tubesBeaker of iceThermocycler Procedure (colony lift method) Mark one sterile microcentrifuge tube K (K12) and the other C (competent) Add 250 ul of cold sterile 50mM calcium chloride to each tube, using sterile tip and aseptic technique. Place both tubes on ice. Pipet 200 ul of E. coli, K12 into K and competent into the C tube. Mix cells in the tube by tapping, shaking or vortexting. Do this promptly – cells don’t resuspend well if left untouched in the cold solution. Return tubes to ice. Add 2 ul of plasmid solution directly into the cell suspension. Tap with finger to mix. Don’t splash or make bubbles. Return tubes to ice. Incubate both tubes on ice for 15 minutes if time is available. Label an LB plate and LB/chlor plates. Following 15 minute incubation, heat shock the cells in both tubes. It is critical that cells receive a sharp and distinct shock by doing the following: set thermocycler for 42 degrees (C) for 90 seconds Immediately return both tubes to ice for at least one minute, optimally for 5-30 minutes. Gentle shaking aids recovery. Use micropipettor to transfer 250 L of LB broth to each tube. Gently tap tubes to mix. Spread cells on plate as follows: 100ul into each according half Spread bacteria using a sterilized loop. Use a fresh loop for each plate. Let plates sit for several minutes to let cell suspension soak into agar plate. Tape set of four plates together. Incubate upside down in 37 degree C. incubator for 12-24 hours. Disinfect your lab bench, properly dispose of contaminated materials, and wash your hands before leaving the lab. Method: The methods are derived from the Genomic Biology workshop of David Micklos, Dolan DNA Learning Center of Cold Spring Harbor Laboratory: www.dnalc.org. 3A Assembly Kit (igem) The 3A Assembly Kit and following protocols will take you through the process of 3A Assembly, and by the end you will have assembled your own composite part in the lab. The kit includes two parts: Part A (BBa_J04500) and Part B (BBa_J04650), which when assembled together will form a RFP (red fluorescent protein) generator. Your cells will turn red! Step 1: Growing the E. coli Streaking the Agar Stabs (About 15 active minutes and 16-24 hours of incubation are needed.) 70% ethanolPaper towelsLab marker/SharpieAgar Stab: Part A - BBa_J04500 (kit)Agar Stab: Part B - BBa_J04650 (kit)Inoculating loops (kit)LB agar plates - Amp/Kan (kit) Clean the lab bench by wiping down with 70% ethanol and paper towels.Part A (BBa_J04500) and Part B (BBa_J04650) are both maintained on pSB1AK3 plasmid backbones, which means they are ampicillin- and kanamycin-resistant. Label the agar plates with the names of Part A and Part B.Notice how each zig-zag overlaps with the previous one just a little, and only at the end.Use an inoculating loop to transfer some cells from the Part A agar stab to the appropriately labeled Amp/Kan agar plate. There is a hole in each agar stab from where it was inoculated. Dip an inoculating loop into the stab at the same location, and streak the bacteria onto the agar plate in a zig-zag pattern. Using a fresh inoculating loop, streak onto the agar plate again creating a new zig-zag pattern that overlaps the first. This will help ensure that you will have single colonies to pick from. Streak gently, and try not to puncture the agar.Repeat step 4 for Part B..This prevents other bacteria from settling, and growing, on your agar plate.Place the agar plates into the incubator with the agar side facing up, lid facing down (see insert). Incubate the agar plates at 37° C for 14-16 hours. Alternately, incubate at room temperature for 24-30 hours.Once your agar plates have grown up you can store them in your fridge (4° C) until you're ready to grow up your cell culture.Plates can be stored at 4° C for up to 3 weeks. Growing up Cell Cultures (About 30 active minutes and 16 hours of incubation are needed.) 70% ethanolpaper towelsLab marker/Sharpie14ml culture tubes (kit)10ml of LB broth - Amp/Kan (kit)Inoculating loops (kit)Agar plate: Part A - BBa_J04500 (see previous step)Agar plate: Part B - BBa_J04650 (see previous step)Rotator/Shaker Clean the lab bench by wiping down with 70% ethanol and paper towels.Remove the agar plates for Part A and Part B from the incubator or 4° C fridge.Label one 14ml culture tube for each Part. Add 5ml of LB broth (with ampicillin and kanamycin) to each culture tube.Use an inoculating loop to pick a single colony from each agar plate and inoculate the LB broth, in the appropriately labeled culture tube. Do not use the same inoculating loop more than once! Press lightly on the snap caps of the 14ml tubes, the caps should be a bit loose to allow for air flow.Incubate for 16 hours at 37° C, in a rotator or shaker. Rotation helps the cells grow faster, and prevents them from settling at the bottom.After incubation, the cell culture should be cloudy. You can now firmly press down on the snap caps to seal the tubes and store the cell culture at 4° C until you're ready to move onto the next step. Step 2: Miniature Preparations (About 1 active hour is needed.) 70% ethanolPaper towelsMarker/SharpieCentrifuge/microcentrifuge2 Beakers/waste collection containersBleachBuffers: P1, RNAse A, P2, N3, PB, PE (kit)1.7ml Microcentrifuge tubes (kit)Qiagen spin columns (kit)Distilled water (kit)Cell culture: Part A - BBa_J04500 (see previous step)Cell culture: Part B - BBa_J04650 (see previous step)Nanodrop machine (optional) Note: If you haven't already, add the RNAse A to both Buffer P1 aliquots. Once you add RNAse A, you must store Buffer P1 at 4° C until use.Clean the lab bench by wiping down with 70% ethanol and paper towels.Make sure the cap of the culture tubes creates a firm seal. Spin down the two culture tubes in a centrifuge. Spin for 3 minutes at 8000 rpm.You should see a pellet of collected cells at the bottom of the tube. Slowly pour the supernatant (media at the top of the tube) into the beaker. Be sure not to do it too quickly or the cell pellet might dislodge. Pour some bleach into the beaker to sterilize the solution.Pipet 250ul of Buffer P1 to each 14ml culture tube. Pipet up and down gently to re-suspend the cell pellets.Label one 1.7ml microcentrifuge tube each with the part name. Transfer the resuspended cells to the appropriate microcentrifuge tube.Pipet 250ul of Buffer P2 to each microcentrifuge tube. Close the tubes, and flip them upside down gently 5x.Carefully open the tubes, and pipet 350ul of Buffer N3 into each one. Close the tubes, and flip them upside down gently 5x to mix. The solution will turn clear and slightly “chunky”.Spin down the samples in a microcentrifuge for 10 minutes at 13,000 rpm. This spin will separate cellular debris onto the side of the tubes.Label one Qiagen spin column for each part. Carefully pipet the supernatant (the clear liquid) to the appropriate spin column. Try not to transfer the white debris pelleted onto the side of the tubes.Spin down the spin column tubes. In a microcentrifuge, spin for 1 minute at 13,000 rpm.Remove the filter tube (top) from the collection tube (bottom). Be sure not to confuse the two samples! Pour the flow-through in the collection tubes into the other beaker. Place the filter tube back into its original collection tube.Pipet 500ul Buffer PB to each spin column. Spin down the samples. In a microcentrifuge, spin for 1 minute at 13,000 rpm.Remove the filter tube from the collection tube. Be sure not to confuse the two samples! Pour the flow-through into the other beaker. Place the filter tube back into its original collection tube.Pipet 750ul Buffer PE to each spin column. Spin down the samples. In a microcentrifuge, spin for 1 minute at 13,000 rpm.Remove the filter tube from the collection tube. Be sure not to confuse the two samples! Pour the flow-through into the other beaker. Place the filter tube back into its original collection tube.The spin columns should appear empty, but spin down the samples for 1 minute at 13,000 rpm again. This will remove any remaining buffer from the filter tubes.Label one clean 1.7ml microcentrifuge tube each with a part name. Transfer the appropriate filter tube to the clean 1.7ml tube.Pipet 50ul of distilled water to the center of each filter tube. Let the samples sit for 1 minute, then spin down the samples. In a microcentrifuge, spin for 1 minute at 13,000 rpm.If you can, calculate the concentration of the DNA, and mark this on the microcentrifuge tubes. They can be stored at -20° C for long-term storage, or at 4° C for short-term storage.To determine the concentration of DNA using a Nanodrop, open the Nanodrop program on the attached computer.Select the "Nucleic Acid" option. Pipet 2ul of distilled water onto the bottom sensor. Gently close the top arm, and click "OK" to activate the machine. After the machine has finished activating, click on "Blank".Lift the arm. Wet a Kimwipe with distilled water, and use this to gently wipe the top and bottom sensor clean.Pipet 2ul of your miniprepped Part A DNA sample onto the bottom sensor, then click on "Measure". Write down the concentration on the side of the tube.Repeat steps 3-4 for your miniprepped Part B DNA sample.Lift the arm. Wipe the top and bottom sensor with the damp Kimwipe. Step 3: Restriction Digest (About 30 active minutes and 50 minutes of incubation are needed.) 70% ethanolPaper towelsIceContainer for iceLab marker/SharpiepSB1C3 linearized plasmid backbone (25ng/ul) (kit)Part A (25ng/ul) (kit)Part B (25ng/ul) (kit)RFP Control (20ng/ul) (kit)0.6ml tubes (kit)NEB buffer 2BSANEB enzymes: EcoRI, SpeI, XbaI, PstIThermocycler, or waterbath and thermometer Clean the lab bench by wiping down with 70% ethanol and paper towels.Keep all enzymes and buffers used in this section on ice.Thaw NEB Buffer 2 and BSA in room temperature water. Re-homogenize both by inverting the tubes, and flick/spin them to collect the liquid at the bottom of the tube.Label four 0.6 tubes: Part A, Part B, pSB1C3 (linearized plasmid backbone), and RFP ControlAdd 500ng of DNA to the appropriate tube. Add distilled water to the tubes for a total volume of 42.5ul in each tube.Pipet 5ul of Buffer 2 to each tube.Pipet 0.5ul of BSA to each tube.In the Part A tube: Add 1ul of EcoRI enzyme, and 1ul of SpeI enzyme.In the Part B tube: Add 1ul of XbaI enzyme, and 1ul of PstI enzyme.In the pSB1C3 tube: Add 1ul of EcoRI enzyme, and 1ul of PstI enzyme.In the RFP Control tube: Add 1ul of EcoRI enzyme, and 1ul of PstI enzyme.The total volume in each tube should be approximately 50ul. Mix well by pipetting slowly up and down 5x. Be gentle, and do not vortex. Spin the samples for 5 seconds in a microcentrifuge, or flick them to collect all of the mixture to the bottom of the tube.Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermocycler, but a waterbath and an accurate thermometer works well also!The digested DNA can be stored at 4°C for a few days. For longer storage, keep at -20°C. Step 4: Ligation (About 15 active minutes and 50 minutes of incubation are needed.) 70% ethanolPaper towelsLab marker/Sharpie0.6 tubes (kit)Distilled water (kit)IceContainer for iceT4 DNA Ligase Reaction BufferT4 DNA LigaseMicrocentrifugeThermocycler, or waterbath and thermometerRestriction Digest: pSB1C3 linearized plasmid backbone (see previous step)Restriction Digest: Part A (see previous step)Restriction Digest: Part B (see previous step)Restriction Digest: RFP Control (see previous step) Clean the lab bench by wiping down with 70% ethanol and paper towels.Thaw T4 DNA Ligase Reaction Buffer at room temperature. Keep the T4 DNA Ligase in the freezer until you're ready to use it.Label one 0.6ml tube as New Part.Add 2ul from the pSB1C3 linearized plasmid backbone digest.Add 3.3ul from the Part A digest.Add 3.9ul from the Part B digest.Add 1ul of T4 DNA Ligase Reaction Buffer.Add 0.5ul of T4 DNA Ligase (keep this at -20°C until use!).Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.Label one 0.6 tube as Ligation Control.Add 2ul from the RFP Control digest.Add 6.5ul of distilled water.Add 1ul of T4 DNA Ligase Reaction Buffer.Add 0.5ul of T4 DNA Ligase.Mix by gently pipetting up and down 3x. Do not vortex; this inactivates the enzymes. Place tube in microcentrifuge for a quick 5 second spin or flick the tube to collect the mixture at the bottom.Incubate at 16°C for 30 minutes, then at 80°C for 20 minutes. We use a thermocycler, but a waterbath and thermometer combination works great too! The ligated products can be stored at -20°C. Step 5: Transformation (About 1 active hour and 12-24 hours of incubation are needed.) 70% ethanolPaper towelsLab marker/SharpieIceContainer for iceTimerNEB10 cells (see Growing step for preparation instructions)2.0ml microcentrifuge tubes (kit)Inoculating loops (kit)Ligation: New Part (see previous step)Ligation: Control (see previous step)DNA: RFP Control (20ng/ul) (kit)SOC media (kit)LB agar plates - Chloramphenicol (kit)Sterile glass beads (kit)Waterbath and thermometer Note: SOC media gets contaminated easily, so be careful when handling. if possible, wear gloves, and only open the container when you need to.Clean the lab bench by wiping down with 70% ethanol and paper towels.Keep all materials on ice unless otherwise specified! This will help make the cells more competent and easier to transform.Label a 2.0ml microcentrifuge tube as Transformation Control, another as Ligation: New Part, and one more as Ligation Control.Add 5ul of RFP Control DNA (20ng/ul) into the Transformation Control tube.Add 2ul of the New Part ligation product into the Ligation: New Part tube.Add 2ul of the RFP Control ligation product into the Ligation Control tube.Place the tubes on ice to pre-chill them.Thaw one competent cell aliquot tube on ice (this takes about 5-8 minutes).Gently flick the tube of competent cells, then pipet 50ul of competent cells into each 2.0ml microcentrifuge tube.Try to keep the cells as cold as possible by holding just the top of the tube, not the bottom where the cells are.Incubate the DNA and cell mixtures on ice for 30 minutes. During this incubation, pre-heat the waterbath to 42°C.Place the tubes into the waterbath for 60 seconds. Immediately place the tubes back on ice for 5 minutes.Add 200ul of SOC media to each tube. Gently tap the tubes with your finger to mix.Incubate the tubes at 37°C for 2 hours. During this time, prepare the agar plates by labeling them. Add 3 - 6 glass beads per plate.Pipet 200ul of the Transformation Control onto the appropriate plate. Spread evenly over the surface of the agar by gently shaking the plate back and forth. The beads will do the work for you!Repeat step 11 for the other two transformations.Place the agar plates into the incubator with the agar side facing up, lid facing down. Incubate the agar plates at 37°C for 12 - 14 hours. Alternately, incubate at room temperature for 24 hours.Check for red colonies the next day, and post your results online in the next section! Plates can be stored at 4° Celsius for up to two weeks. End of page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/LabFrom 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Home Goal The goal of ABRHS was to create a safe alternativeto theproduction ofdigested Kopi Luwak coffeebeans. For the iGEMcompetition, we geneticallyengineered the bacteria,E. coli, to produce digestiveenzymes that would do thesame work.(read more) The Team Our team consists of eleven students from the Acton-BoxboroughRegional High School. Together, we promoterespect for self,others, and learning.Meet the Team Asian Palm Civet, photographed by Praveenp under Wikimedia Commons The Kopi Luwak Acknowlegements MIT Lincoln Labs New England BioLabs BioBuilder End of page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHhttps://www.neb.com/~/media/NebUs/Navigation/logo-NEB.pngNotebook From 2014hs.igem.org HomeProjectNotebookThe TeamSimulationsSafetyResearchLabs Notebook First Few Weeks In the first few weeks, we had introduced iGEM to students, taken sneek-peeks on projects done by past iGEM teams, done a banana lab and small exercises. The banana lab was a test lab to see how different modified E. coli would produce a banana smell from an inserted banana enzyme gene. One example of a demonstration was modelling the process of producing a plasmid of recombinant DNA with restriction enzymes and ligase using pipe cleaners, candies, labels and scissors. Up to February 26 2014 Our team has done some moderate research on final topics. The final project will be picked soon. These are a list of the possible candidates: Fat/sugar/protein converterGenetically produced fuel/BioDeiselKopi Luwak CoffeeBacteria that absorbs CO2/fossil fuel by product/catalytic converterSkin regenerating BacteriaBacteria with memory/synthetic neuronsSynthetic/modified mitochondria or chloroplast February 27 2014 Narrowing it down. Genetically produced fuel/BioDeiselKopi Luwak Coffee (COLI Luwak Coffee)Bacteria with memory/synthetic neurons / Battery? March 6 2014 Final Decision by the Team: Kopi Luwak Coffee (project name is E. COLI Luwak) March 12 2014 Our team has officially designated team members to certain tasks for the project. These Include: The team wiki Research on Kopi Luwak project Working on the 3A assembly kit for genetic recombination After this meeting, we carried out the 3A assembly protocol twice (see protocols 1-5 in lab tab). We did not achieve growth on the chloramphenicol plates either time, possibly due to our failure to properly freeze the competent cells. April 17 2014 Our team is finishing up the 3A Assembly Lab for the second time. We have decided to plan certain agendas: We need to finish the 3A assembly lab. We need to make an official notebook for our actual Project progress. We need to investigate the past two 3A labs for errors, and mistakes to fix in the future. May 1 2014 Our team has received the DNA Distribution Plate Kits from iGEM which contain various DNA sequences (for enzymes, promoters, etc) on plasmids that we can use for our project. We have decided to initially use the following according to our project: Pepsin Amylase (salivary and pancreatic) Trypsin (trypsin and chymotripsin) We also have the Standard Registry of Biological Parts by iGEM to research more possible parts. May 8 2014 Our team has focused on several things this meeting: The iGEM Jamboree Fee Our Project-lab Notebook More on the parts we will use- very few of the original parts are in the catalog More frequent meeting dates! We plan to generate money to help pay for the Jamboree fee by having a bake sale. Also, we will find and research every single part we will need to use, including regulatory parts such as promoters, operators, etc and devise a specific plan for the project-lab, for which we keep an official physical notebook. May 15 2014 Today we assigned specific iGEM parts for research: RBS Promoters Terminator Pepsin and Amylase The DNA backbone We have put aside the idea of using trypsin as one of our enzymes since it has a complex process for forming its shape or conformation with multiple genes. Furthermore, we need to work on transferring our vast amounts of research from paper to the site, and the safety page. We have additionally working on our idea of a fundraiser bake-sale, and decided to meet on Tuesdays and Thursdays. May 22 2014 Today we integrated information about our research on the type of part, and specific parts. We will officially decide what parts to order by next Tuesday. May 29 2014 Today we clarified a few deadlines including the abstract, the ordering of the parts, the registration of the Jamboree, and the safety tab, as well as the detailed planning for the lab. June 5 2014 The following deadlines were clarified: The iGEM wiki freeze at June 20. The iGEM poster. The iGEM registration finalizations. We will have our lab during next week, for amylase in E. coli. Also, Safety, Simulations, Research, Lab and Team tag websites need to be updated. June 10 2014 Today, the High Efficiency lab (see lab tab) lab was started and finished using part BBa_K523006for alpha amylase. This lab failed, leading to the conclusion that our competent cells had not been properly maintained and were no longer effective. We also plan to have a Google Hangout on thursday, since many people are busy. The following topics were discussed and worked on. The iGEM wiki (Research Tab update, Home page, Team page, etc.) The iGEM poster. The iGEM possible T-shirts Testing the bacteria with beans (or cherries), needs to take place as well. June 16 2014 Due to the failure of the High Efficiency lab, we performed a new procedure: the Main Project Lab (see lab tab). This procedure does not rely on the competent cells, but instead uses CaCl2 to catalyze transformation.We did not see any growth the next day, but we had some leftover cells that we had not put on the plates. These leftovers were plated in the hope that they had transformed, but they had not. End of page Retrieved from "http://2014hs.igem.org/Team:Acton-BoxboroughRHS/Noteboo1288Shenzhen SZMS地址：深圳市人民北路深中街18号 18 shenzhong street Renmin North Road Luohuo district Shenzhen Guangdong Province ChinaHigh SchoolE. coli Plant-sitterOur project is to create an ?E. coli Plant-sitter? that achieves smart maintenance of healthy plant growth through the application of synthetic biology to temperature moderation. We hope that our toolkit will simplify the process of plant cultivation and make synthetic biology accessible to people?s daily life. Hothouse plants whose preferential temperature ranges from 25~30℃ are exposed to the threat of low productivity and unhealthy growth in overly cold environments. Therefore, we came up with a system that aids the healthy growth of these plants by protecting them from overly low temperatures through smart temperature control. Our system of temperature moderation, including temperature sensing and temperature regulating, is able to produce heat once the temperature drops below 27℃ and stops heating when the temperature reaches 27~28℃. In this way, the temperature is to be kept at 27℃, ensuring the healthy growth and high productivity of hothouse plants.1803BBa_K1288999http://2014HS.igem.org/Team:Shenzhen_SZMSBBa_K1288000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165120140http://2014hs.igem.org/files/presentation/Shenzhen_SZMS.pdfhttp://2014hs.igem.org/files/poster/Shenzhen_SZMS.pdfHigh SchoolLog in   Team:Shenzhen SZMS/Human Practice/BIT's 5th World DNA and Genome Day From 2014hs.igem.org Home Team Members Project Modelling Lab Safety Gallery Human Practice BIT's 5th World DNA and Genome Day was held in Dalian, China from April 25th to 28th, 2014. Two of our team members(Zhang Yang and Pan Sixuan) attended the meeting among the nine student delegates of Shenzhen Middle School. During the meeting, we introduced our project to and communicated with the attendees present, including Nobel Prize winners and research conductors in colleges and institutions all around the world. Contents 1 Plan 1.1 BIT's 5th World DNA and Genome Day1.2 Our goal 2 Preparation3 Course4 Significance5 Gallery6 External Links Plan BIT's 5th World DNA and Genome Day BIT's 5th World DNA and Genome Day is to be held from April 25th to 28th, 2014 in Dalian, Liaoning Province in northern China, during which professors and scientists (including 10 Nobel Prize winners in Physiology or Medicine, Chemistry and Physics) from all over the world will conduct inspiring lectures and sharing sessions with regard to the development and prospect of life science. Our goal Our project is to make an E. coli Plant-sitter. As there will not be enough time for us to comprehensively introduce our product at the conference, we focused on presenting our idea, introducing the iGEM competition, and demonstrating what we can come up with and achieve as high school students inspired by the beauty of science and technology. Preparation The poster in English The poster in Chinese Our Human Practice group came up with two hand-made posters using coloured paper to present our project. In order to provide more convenience, one poster is in English while the other one is in Chinese. Our Human Practice group spent two busy evenings making the posters, finishing them just in time. Our posters consist of colorful words which serve as introduction of our project and pictures of our team members working and discussing. Course During the 4-day conference in Dalian, we listened to specialists’ lectures and learned about research methodology on the most advanced genetic technology, which can be applied to our experiments as well. Moreover, but we also got a chance for face-to-face communication with the Nobel laureates such as Dr. Hartmut Michel, Dr. Thomas C. Südhof, Dr. Akira Suzuki and Dr. Ada Yonat and the professors such as Prof. Wang Min, Prof. Tan, etc. We exchanged name cards with these experts and hoped that we would get further communication with them via email. We presented our posters on the Poster Exhibition section to promote our project. Some professors and specialists were attracted to our project and they are surprised that high school students also began to do scientific research. We earnestly introduced our “E. coli plant sitter” project to those who were interested. Some experts even gave us some comments on our message board next to the posters. A biology researcher says that we should “pay attention to the deterioration of E. coli culture medium.” Dr. Roderick J. Ryan, a researcher from Eastern Health, Monash and Deakin University, Australia said that our project was “very impressive.” One of the staff told us that a professor studying plant genes stood in front of our posters for a long time and asked the committee staff for our contact information. On the last day of the conference, we were given an interview by a publisher from Nanjing. We discussed about the relation between middle school and high school’s education and our interest in biology. Significance For our trip to Dalian, we not only got access to the most advanced scientific research, but also learned about the researchers’ methods, inspiration and spirit. We believed that what we have learned in this trip will help our team work better in our following research! Gallery Find more photos here:Team:Shenzhen SZMS/Gallery#World DNA and Genome Day External Links the official website in English and in Chinese 睹大师风采，扬深中精神 on school website 发现深中精神 on school website Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/Human_Practice/BIT%27s_5th_World_DNA_and_Genome_DayProject From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice <<Back to wiki main pageFind more about our project below!Contents1 Background2 Plasmid and Working Details2.1 Temperature Sensing2.2 Temperature Regulating Background E. coli Plant-sitterOur project is to create an “E. coli Plant-sitter” that achieves smart maintenance of healthy plant growth through the application of synthetic biology to temperature moderation. We hope that our toolkit will aid the process of plant cultivation, enlarge the public understanding of synthetic biology, and make the methods and technology of synthetic biology accessible to people’s daily life.Daily life – yes, on earth our Plant-sitter can be well applied to the growth of plants – but what about…Wait for it –MARS!Mars, lying four hundred million kilometers from earth, has the most earth-like seasons due to Mars and Earth’s similar tilts of rotational axes. As a result, in order for men to make use of Martian resources and to develop its potential as an alternative human residence in the universe, a Mars Migration Program was issued SpaceX, indicating that founding a human community on Mars requires 1) releasing carbon dioxide to make the atmosphere denser and to create raw materials for photosynthesis; 2) cultivating plants beside human residence to generate oxygen.– which is where our E.coli Plant-sitter helps.Although there will be basic heat supply in the houses, the healthy growth of the plants is still under threat as Mars’ surface temperature varies from -143℃ to 35℃. Therefore, we came up with a system that protects the plants from overly cold environment through smart temperature control, keeping the temperature at around 27℃, aiding the plants who are burdened with the glorious purpose of generating oxygen to defend against the Jotunheim weather on Mars.Plants whose preferential temperature ranges from 25~30℃ are exposed to the threat of low productivity and unhealthy growth in overly cold environments. Therefore, we came up with a system that aids the healthy growth of these plants by protecting them from overly low temperatures through smart temperature control. Our system of temperature moderation includes two parts, temperature sensing and temperature regulating. Plasmid and Working Details Compile Function Introduction J23106 Promoter It promotes the transcription of the following parts. More R0051 CI regulated Promoter It promotes the transcription without CI. When CI is binding to it,it is repressed. More K115016 27℃ RBS It only promotes the translation when the temperature is over 27℃ More K115017 32℃ RBS It only promotes the translation when the temperature is over 32℃ More B0030 strong RBS It can promotes the translation strongly. More C0051 Coding sequence It is the coding sequence of CI Protein. More 1.7.7.2 (KEGG) Coding sequence We got this part from bacillus subtilis best7613 strain.It catalyzes the reaction which converts Nitrate to Nitrite. More K145015 Coding scquence It is the coding sequence of GFP. More B0015 Terminator It can stop the transcription. MoreThe process of temperature sensing involves a promoter, a 27℃ RNA thermometer, and cI. The process of temperature regulating involves a cI regulated promoter, an enzyme which catalyzes an exothermic chemical reaction, a 32℃ RNA thermometer, and a GFP LVA device which serves to indicate the occurrence of a change in temperature. The promoter in temperature sensing part is consistently on and thus so is the process of cI’s transcription from DNA to RNA. When the temperature is below 27℃, the 27℃RNA thermometer takes a curved, locked structure which does not enable translation from RNA to protein cI, which in turn allows the activation of the cI regulated promoter, the production of the enzyme, the occurrence of an exothermic reaction which heats up the environment, and the expression of GFP LVA tag which gives out green light to indicate that the temperature is below 27℃ and that our device of temperature control is doing its work. When the temperature rises and reaches 27 ℃ as a result of the exothermic reaction, the 27℃ RNA thermometer enables the translation of cI protein, which in turn represses the cI regulated promoter, ending the release of heat through the exothermic reaction by stopping the production of the enzyme which catalyzes that reaction. As a result of an inactive promoter (the cI regulated), the GFP also stops its expression and is decomposes, indicating that the temperature has risen from below 27 ℃ to 27 ℃and that our system of temperature control has done its work.If the 27℃ RNA thermometer fails to stop the heating process at around 27℃ and the temperature keeps rising up to 32℃, the 32℃ RNA thermometer is activated, leading to the presence of protein cI, which places a double-check on the inhibition of the heating process. Temperature Sensing This process involves BBa_J23106 (promoter), BBa_K115016 (27℃ RNA thermometer), and cI (Repressor protein cI).BBa_J23106 is consistently on and thus so is the process of cI’s transcription from DNA to RNA. When the temperature is below 27℃, the 27℃ sensitive RNA thermometer does not enable translation from RNA to protein cI, which in turn allows the activation of BBa_R0051, the production of the enzyme, the occurrence of an exothermic reaction which heats up the environment, and the expression of GFP LVA tag which gives out green light to indicate that the temperature is below 27℃ and that our device of temperature control is doing its work.When the temperature rises and reaches 27 ℃ as a result of the exothermic reaction, BBa_K115016 (27℃ RNA thermometer) enables the translation of cI protein, which in turn represses BBa_R0051, ending the release of heat through the exothermic reaction by stopping the production of the enzyme which catalyzes that reaction.As a result of an inactive promoter (BBa_R0051), the GFP also stops its expression and is decomposes,indicating that the temperature has risen from below 27 ℃ to 27 ℃and that our system of temperature control has done its work.If BBa_K115016 (27℃ RNA thermometer) fails to stop the heating process at around 27℃ and the temperature keeps rising up to 32℃, BBa_K115017 (32℃ RNA thermometer) is activated, leading to the presence of protein cI, which places a double-check on the inhibition of the heating process.The process by which our temperature-control system operates may be better illustrated in the following flow chart: Temperature Regulating The process of temperature regulating involves BBa_J45503 (cI regulated promoter), an enzyme which catalyzes an exothermic chemical reaction, BBa_K115017 (32℃ RNA thermometer) and a GFP LVA device which serves to indicate the occurrence of a change in temperature.The following is a flowchart explaining how our E. coli Plant-sitter works.Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/ProjecModelling From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice <<Back to wiki main pageIn order to assess the efficiency of our E.coli babysitter, we have made math models for the core system in it, EC 1.7.7.2, which functions as to heat up the environment to provide a more suitable temperature for the plant.EC 1.7.7.2 is basically a ferredoxin-nitrate reductase; with the required substrates nitrate and reduced ferredoxin, EC 1.7.7.2 is able to catalyze the reactions below:nitrite + H2O + oxidized ferredoxin ⇄ nitrate + reduced ferredoxin + 2H+HNO2 + NH3 = N2↑+ 2H2Oobviously as suggested above,the first reaction is a reversible one; the heat we need is actually from the second reaction since the first one is basically to convert nitrate provided by our special LB into nitrite.On the other hand, the warmth released by the first reaction is so little that it has nearly no effect on the result. Consequently,we make only the math models for the second reaction, whose substrate is nitrate. As calculated, the general warmth that would be released is 334.55 KJ/mol, which is a theoretical datum.However,considering the fact that we do not have adequate data to carry out our math models, we finally decide to use proportionality instead of accurate data. Both of the following charts are established by Excel.The first one is a model under the condition that purely only EC 1.7.7.2 is functioning, which means, the concentration of it remains unchanged: (grey line → temperature inside the systerm)The second one is a model under the condition that RNA Thermometer in the first part of our entire plasmid is functioning to start the heating-up system which is mostly EC 1.7.7.2 in certain extent of external temperature (<27℃). Since it is being manipulated by the other system, the concentration of EC 1.7.7.2 can no long remain unchanged, but changed over time: (black line → temperature inside the systerm)General information about EC 1.7.7.2 can be found in the following link: http://www.genome.jp/dbget-bin/www_bget?ec:1.7.7.2.Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/ModellinLaboratory Open Day From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman PracticeLaboratory Open Day of Team:Shenzhen SZMS was held on May 14, 2014, in which students from our school, Shenzhen Middle School, were invited to visit our laboratory at Room C201. They can see our equipment freely, and we offered a chance to some 40 students that they could try doing agarose gel electrophoresis.Contents1 Preparation2 Course3 Safety Protection4 Photos5 External Links Preparation poster #1 poster #2We had been preparing for the Day since May 7, when the first edition of the plan was made. According to this plan we would arrange 2 rounds of do-it-yourself courses in which the students are offered a chance to try some experiments which we often do. Other main activities include introducing synthetic biology and iGEM.We began propaganda work on May 10 Saturday by publishing the news on WeChat, a famous social network app in China. We also published our contact information, and the places of first round were almost filled in 6 hours. A lot of students spread the news by forwarding it. Three posters were also published with the news. On two of the posters write "Laboratory Open Day" and the time, while the third one tells how to apply to take part in the experiments. What's more, we printed the posters and posted them in school. Course At 4 PM on May 14, 2014, we opened the door of our lab and brought the visitors in. First of all, Yufei Jing, Yang Zhang and Ziyun Ye instructed about the instruments, the usage of instruments, and lab safety rules. Then, the participants are divided into four groups to do the agar gel electrophoresis experiment under the tutoring of our team members. Other visitors are guided to a lab tour.The electrophoresis is finished at about 5:10 PM. Some groups got significant results while some groups failed in the experiment. All of the participants are excited about experiencing new experiments and learning things out of the textbooks.After the experiments, we gave all visitors post cards with our project comic pictures and the iGEM logo on. Moreover, we prepared two message boards for the visitors and they left messages there. Many students said that this experience helped them get a brand new view on biology and biological experiments. Safety Protection Matters needing attention for the Day.We put the tutoring of lab safety as one of the most important part of the open lab day. In order to ensure the safety of visitors and experiment participants, we arranged the team members to guide the visitors at all time and offered lab safety instruction at the very beginning of this activity. The lab safety guidelines include:Do not eat in the lab!Do not touch the solutions without wearing gloves! Do not take out solutions from the refrigerator!Do not use the instruments without the guiding of our team members!As a result of our arrangements, there are no reported ailments during and after this activity. However, since we did not emphasize about the correct usage of the pipette, one pipette was damaged due to false operation. Photos Find more photos here:Team:Shenzhen SZMS/Gallery#Laboratory Open Day External Links File:Laboratory Open Day of Team-Shenzhen SZMS plan.pdf:the plan (in Chinese).File:PPT used in Laboratory Open Day of SZMS.pdf:the PPT used (in Chinese).Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/Human_Practice/Laboratory_OpeGallery From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice <<Back to wiki main pageHere are some pictures of Team:Shenzhen SZMS.Contents1 Photos1.1 Lectures in Secondary Schools1.2 Laboratory Open Day1.3 World DNA and Genome Day2 Our cuties Photos Lectures in Secondary Schools Learn more:Team:Shenzhen SZMS/Human Practice/Lectures Laboratory Open Day Learn more:Team:Shenzhen SZMS/Human Practice/Laboratory Open Day World DNA and Genome Day Learn more:Team:Shenzhen SZMS/Human Practice/BIT's 5th World DNA and Genome Day Our cuties Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/GalleLab From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice <<Back to wiki main pageHere you can find out how we build our plasmid. The plasmid map and information of parts used is given in Team:Shenzhen SZMS/Project. ScheduleNo. Procedure Backbone Status of the Plasmid1[K115016] Spe1,Pst1 Double Digestion, Electrophoresis detection，DNA refinement;[C0051] X ba1,Pst1Double Digestion, Gel extraction;Combination1C3(K115016)[K115016+C0051]2[J23106] Spe1,Pst1 Double Digestion, Electrophoresis detection，DNA refinement;[K115016+C0051] X ba1,Pst1Double Digestion, Gel extraction;Combination1A2(J23106)J23106+[K115016+C0051]3[J2s3106+ K115016+C0051] EcoR1,Spe1Double Digestion, Electrophoresis detection，DNA refinement;[B0051] EcoR1,Xba1 Electrophoresis detection，DNA refinement;Combination1C3(B0015)[J23106+K115016+C0051]+B00154[B0030] Spe1,Pst1 Double Digestion, Electrophoresis detection, DNA refinement;[EC:1.7.7.2] Xba1, Pst1Double Digestion, Gel extraction;Combination1A2(B0030)B0030+EC:1.7.7.25[R0051] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[B0030+ EC:1.7.7.2] Xba1,Pst1 Double Digestion, Gel extraction;Combination1A2(R0051)R0051+[B0030+ EC:1.7.7.2]6[B0030] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[K145015] Xba1,Pst1Double Digestion, Gel extraction; Combination1A2(B0030)B0030+K1450157[B0030+ K145015] EcoR1, Spe1Double Digestion, Electrophoresis detection,DNA refinement;[B0015] EcoR1, Xba1Double Digestion,Electrophoresis detection,DNA refinement;Combination1C3(B0015)[B0030+ K145015]+B00158[R0051+B0030+ EC:1.7.7.2] Spe1, Pst1Double Digestion,Gel extraction;[B0030+ K145015+B0015]Xba1, Pst1 Gel extraction;Combination1C3(B0030+K145015+B0015)[R0051+B0030+ EC:1.7.7.2]+[B0030+K145015+B0015]9[K115017] Spe1, Pst1Double Digestion, Electrophoresis detection,DNA refinement;[C0051] Xba1, Pst1Double Digestion,Gel extraction; Combination1C3(K115016)[K115017+C0051]10[J23106] Spe1,Pst1Double Digestion, Electrophoresis detection,DNA refinement;[K115017+C0051] Xba1, Pst1Double Digestion, Gel extraction;Combination1A2(J23106)J23106+[K115017+C0051]11[J23106+ K115017+C0051] EcoR1, Spe1Double Digestion,Gel extraction;[B0015]EcoR, Xba1Double Digestion, Electrophoresis detection,DNA refinement,Combination1C3(B0015)[J23106+ K115017+C0051]+B001512[The first part] Spe1,Pst1 Double Digestion;[The second part] Xba1,PstL Double Digestion;Gel extraction; Combination1C3(The first part)1st+2nd13[The first and second parts] Spe1、Pst1Double Digestion; [The third part] Xba1、Pst1Double Digestion;Gel extraction; Combination1C3(The third part)1st +2nd+3rdDone Log Book Date Event Results Problem summaryApr.3B0015(chl) B0030(chl) Plasmid TransformationDone (yet this B0030 is single error)Apr.4Single clone selected from the work on Apr.3 (In order to be pithy, this process is omitted from the table in all the following daily work)DoneApr.5Plasmid preped and culture restored from the bacterial suspension on Apr.4 (omitted later for pithiness and convenience as well)DoneApr.6K115016(CHL) K115017(CHL) C0051(CHL) transformationDoneMay.2J23106(AMP) K082003(CHL，as GFP+LVA) transformation (help offered by Mentor Wang)DoneMay.5Digestion: K115016(S/P) C0051(X/P) C0051(E/S) B0015(E/X)ElectrophoresisGel extraction16℃ overnightDoneMay.6Product from 5.5（CHL）transformedFailedMight be the problem during the plasmid prepMay.7Plasmid mentioned above reprepedDoneMay.13Digestion: C0051(E/S) B0015(S/P) K082003(E/S)ElectrophoresisGel extraction16℃ overnightDone, C0051-B0015K082003-B0015 gainedMay.14Products from 5.13（CHL）all transformedDone, C0051-B0015K082003-B0015 gainedMay.15Gel confirmation (products from 5.14), comparison with gel of B0015Done, product longer than the original plasmidMay.27Digestion: C0051-B0015(X/P) K115016(S/P) K115017(S/P)ElectrophoresisGel extractionCombinationTransformation（CHL）Done,K115016-C0051-B0015K115017-C0051-B0015gained.May.28B0030(AMP) transformationDone; informed that R0051 requires PCR in order to get the primer of EC 1.7.7.2May.29Digestion: J23106(S/P)K115016-C0051-B0015(X/P)K115017-C0051-B0015(X/P)ElectrophoresisGel extractionCombinationTransformation（AMP）Done,J23106-K115016-C0051-B0015(27℃CI-protein)J23106-K115017-C0051-B0015（32℃CI-protein）gained.May.30The result of sequencing of C0051-B0015K082003-B0015 receivedCorrectly combinedJun.2Digestion: B0030(S/P) K082003-B0015(X/P)ElectrophoresisGel extractionCombinationTransformation（AMP）DoneJun.8The results of sequencing of J23106-K115016-C0051-B0015(27℃CI) and J23106-K115017-C0051-B0015（32℃ CI）received.Correctly combinedJun.9Digestion:B0030-K082003-B0015(E/S)J23106-K115017-C0051-B0015(E/X)ElectrophoresisGel extractionCombinationTransformation（AMP）Done with transformation;Yet failed in the electrophoretic analysis.DisconnectedJun.10ciF1 ciF2 ciF3: primers to synthesize EC 1.7.7.2 and PEP (additional PEP R0051-B0030-) gained.Jun.11With the original blue 2X Master Mix PCR round 1,2,3 implementedFailed; only primer founded in the electrophoretic analysisJun.12Round 1 from 6.11 repeated in the morning;Round 3 PCR with 2X hi-fi Master Mix at noonFailed in the morning but done in the afternoon;R0051-B0030-1.7.7.2 recycled product gained.might be the problem of MIXJun.13Digestion: 27℃ CI（S/P）R0051-B0030-1.7.7.2（X/P）32℃ CI（E/X） B0030-K082003-B0015(E/S)ElectrophoresisGelextractionCombinationTransformation.Failed, no culture founded on the dish.Unknown error; might be the problem of extracted gel.Jun.15Digestion: 27℃ CI（S/P）R0051-B0030-1.7.7.2（X/P）32℃ CI（E/X） B0030-K082003-B0015(E/S)ElectrophoresisGelextractionCombinationTransformation.According to the result of electrophoresis, the length of the digested and combined part is the same as that of the primitive plasmid; yet we cannot exclude the problem of plasmid prep; might have failed.A tube was missed during the digestion yet was made up later; do not know about its possible effect yet.Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/LaE.coli Plant-sitter heals the world From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman PracticeE.coli Plant-sitter heals the world is a song based on Michael Jackson's Heal the World written by members of Team:Shenzhen SZMS. Lyric There's a place in your heartAnd I know that it is loveAnd this place could be muchBrighter than tomorrowAnd if you really tryYou'll find there's no need to cryIn this place you'll feelThere's no hurt or sorrowThere are ways to get thereIf you care enough for the livingMake a little space, make a better placeThere’s a chill Each plant diesAnd the farmers watch and sighAs they lie in the coldWithering from tomorrowAnd if one really triesAs we’ve done in days and nightsThere’s a way to be soughtAmid the cold and iceThe plants are safe from freezingIf we guard them from the cold wutheringMake a warmer space, make a better placeHeal the worldMake it a better placeFor you and for me and the entire human raceThere are people dyingIf you care enough for the livingMake a better placeFor you and for meE. coliMake it a better placeFor wheat and for beans and the Greenhouse-grown-raceThere’ll no longer be crops dyingWith our plant-sitter operatingMake a better placeFor all those in needIf you want to know whyThere's a love that cannot lieLove is strongIt only cares for joyful givingIf we try we shall seeIn this bliss we cannot feelFear or dreadWe stop existing and start livingThen it feels that alwaysLove's enough for us growingMake a better world, make a better worldIf you want to know whyThere’s an exothermic reaction’s enzymeHeat is strongWhich comes out below twenty-seven degreeIf we try we shall seeIn this coldness that needs heatThe switch is onPlants stop shaking and get heatedThen it feels that alwaysWarmth’s enough for them growingMake a warmer world, make a better worldHeal the worldMake it a better placeFor you and for me and the entire human raceThere are people dyingIf you care enough for the livingMake a better placeFor you and for meAnd the dream we would conceived inWill reveal a joyful faceAnd the world we once believed inWill shine again in graceThen why do we keep strangling lifeWound this earth, crucify it's soulThough it's plain to see, this world is heavenlyBe God's glowWe could fly so highLet our spirits never dieIn my heart I feelYou all are my brothersCreate a world with no fearTogether we'll cry happy tearsSee the nations turnTheir swords into plowsharesWe could really get thereIf you cared enough for the livingMake a little space to make a better placeHeal the worldMake it a better placeFor you and for me and the entire human raceThere are people dyingIf you care enough for the livingMake a better placeFor you and for meHeal the worldMake it a better placeFor you and for me and the entire human raceThere are people dyingIf you care enough for the livingMake a better placeFor you and for meHeal the worldMake it a better placeFor you and for me and the entire human raceThere are people dyingIf you care enough for the livingMake a better placeFor you and for meThere are people dyingIf you care enough for the livingMake a better placeFor you and for meThere are people dyingIf you care enough for the livingMake a better placeFor you and for meYou and for me / Make a better placeYou and for me / Make a better placeYou and for me / Make a better placeYou and for me / Heal the world we live inYou and for me / Save it for our childrenYou and for me / Heal the world we live inYou and for me / Save it for our childrenYou and for me / Heal the world we live inYou and for me / Save it for our childrenYou and for me / Heal the world we live inYou and for me / Save it for our childrenRetrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/E.coli_Plant-sitter_heals_the_worlTeam Members From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice <<Back to wiki main pageThere are 17 students and 3 instructors in our team. Their personal information is given here: Name Photo Self-intro Work Qian Yuhan钱雨涵 Yuhan Qian, a senior 2 student, is a member of the Experiment Group who has learnt about Molecular Biology and Bioinformatics herself. According to what she said, although staying in the laboratory with the equipment was delightful, learning the theories was also essential for speculating the possible results, analyzing the phenomena and finding the factors that can lead to failures. team leader, experiment group member Wang You王悠 You Wang,now a senior 2 student, has learned the expansive possibility of synthetic biology to faciliate human life since senior 1.Amazed and enchanted by the magic of it, she attends iGem with great passion, exploring more surprise and potential in this field.In SZMS iGem Team, she exercises herself by contacting head quaters, searching for parts, designing plasmid and doing experiments.To build communication with other iGem Teams, she also attend the iGem meeting in Hongkong. "iGem is not only a competition", smilingly comments she,"It's a impressively memorable lesson." experiment, human practice, theory Lin Jingfei林静菲 Lin Jingfei, a senior one student in Shenzhen Middle School, finds her great interest in synthetic biology. After participating the Human Practice Group, she helped make the advertisement pamphlet and posters. She also helped the group leader, Jing, instruct the visitors on the Open Day of the Laboratory. Believing that the art and science can be combined together, she is trying to contribute more and more for the 2014 SZMS Team. human practice Pan Sixuan潘思璇 Sixuan is a sophomore student and a member of the Theory group for SZMS iGEM Team. Motivated by an interest of chemistry and molecular biology, she helped plan and visualize the plasmid by using Snapgene. Almost drawn herself in published papers and DNA sequences, she fortunately did not get blind but managed to build the model that fits our objective. With good skills for communication, she went to Dalian for the BIT’s 5th Genome Day and promoted our project there by presenting our posters. “By gradually accessing to the field of genetic research, I perceived the possibility of making miracles through genome studies,” She said. Theory group member, responsible for researching the theoretical construction of our project, plasmid planning and visualization, promoting our project on BIT’s 5th Genome Day and project presentation Cai Peiwen蔡佩汶 Peiwen Cai, a disguised senior 2, has delayed her high school life for one more year because of her exchange year in Germany, where she had totally set her mind free and lived wildly. Under such an circumstance, she discovered that her enthusiasm was never for advertising or political campaign but for biomed. This lifechanging discovery has motivated her to join SZMS iGem team and led her ever since then into the track of biomed. Challenge never tears her down but builds up her sense of humor.To crack down the human neuro-dilemmas with medicine is one of her two lifelong goals, the other one of which is to be a lobbyist. Former theory group member, mainly in math model group and wiki group.Tech girl working for the team. Zheng Bingmiao郑冰苗 Bingmiao Zheng is a senior one student from Shenzhen Middle School. Bingmiao is interested in biology and design, so she chooses to be a member of Human Practice of our project, Plant-Sitter, for the 2014th iGEM competition. She helped Jing make the hand-made poster for BIT's Genome Day and do some publicity, like the Open Day of the laboratory. Zhang Yang张杨 This man, who is a senior two student, attended the BIT’s 5th Genome Day to introduce our team to those people who attended this international meeting. Some professors from America and China show high interest in our project. Zhang Yang likes experiments a lot. It just like that he is always in the laboratory. He does learn a lot through this competition. He takes part in almost all parts of the team except the building of the model. Jing Yufei景羽飞 Yufei Jing is a senior two student from Experimental Curriculum, Shenzhen Middle School. She love biology because it is a subject with many unknowns, that makes her full of curiosity. She is the leader of the Human Practice Group, arrange the activities of this group and also draw many illustrations for our project. Zhou Yineng周益能 Zhou Yineng is a senior one student from Standard Curriculum, Shenzhen Middle School. As a biology lover, he spent a lot of time in the laboratory, which helps him gain a lot of experience of experiment, and as a result, he has become one of team members who have done a complete cycle including digestion, electrophoresis, gel extraction, combination and transformation, although he even didn't know what transcription means at first. human practice, experiment, wiki. Feng Yanming冯彦铭 Feng Yanming(Danny Feng) , a senior-two student from Shenzhen Hgih School of Science(HSS). He is the Vice-President of Students’ Union and the President of IT Club in the School. He is keen on computer science, web designing, programming and viedo making(Included Viedo clipping&making special effect by using AE.) “A lof of my classmates asked me why I’m good at computer, I just told them, the curiosity is the best teacher of learning.” He said, "It's a whole new chance&chanllenge for me to attend the iGem competition. I love biology, but I don't know much about the experiment of molecular biology before. " When Danny came to the lab for the first time, he even didn't know how to use the pipettor. But he worked hard, and made a big progress."It's really a valuable experience for me. I've studied a lot during the experiment, and I really enjoyed the time I stayed with my team members, they are very friendly to me." Web designing,video making,human practice Yan Ming闫铭 Ming Yan, one of the senior one students in SMS IGEM team, finds her interest in biology after going through the whole process of the IGEM competition. She has met many excellent people (including her teachers, teammates, and many students who are also interested in synthetic biology) because of participating the competition. She enjoys the competition every much. experiment Yang Hongkang杨泓康 Yang Hongkang is a senior one students and a member of SZMS iGEM Team’s theory group. Having an interest in biochemistry and mathematics, and a career expectation in mathematical and theoretical biology, he participated in iGEM competition as to take a step toward his goal. With the passion for his work, he designed the key reactions of the exothermic system, established the early program of the enzyme engineering, and participated in the math-modelling of the project. Theory group member, responsible for the constructing of the chemical reactions and mathematical model. Chen Qingyi陈清怡 Qingyi Chen, a senior 3 student in Shenzhen middle school, has strong propensity toward biology and statistics which are the fundamental consists of the society. She is very ambitious and motivate to the tasks. Having virtue of tenacity and inquisitive, She is always curious about the truth and dedicates in solving problem. For her, biology is very amazing world which construct creatures. The complex structure of protein and the beauty of plants always draw her passion. Hoping to discovery further about biology, she always stays hungry to learn more about biology. Theoretical member. She helps the team to find out innovative ideas and research the feasibility of specific ideas. Wang Zhaobo王照博 Wang Zhaobo is a senior 1 student in Shenzhen Middle School. He is an important member of Human Practice who is interested in Biology. Also he is a member of Shenzhen Photographs Alliance and he becomes an awesome cameraman in our iGEM. “Delighting you always” he always photograph Yang Yuwei杨雨薇 Yuwei is a senior two student who is dedicated into bio-medical engineering and psychology from the International Curriculum from Shenzhen Middle School. As a member of human practice group for the 2014th SZMS iGem Team, she believes that the creativity and imagination which are mostly demonstrated in art work can also be applied into the fantastic micro cellular world. For improving human's living condition and deepening the research in diseases or creatures, she steadfastly intends to go far in bio-med and psychology fields. " People are DNA's way of making more DNA", she believes that one day the secrets of nature will be exposed and hopefully she will become one of those "precursors". Human practice group member, responsible for propagandizing the project and publicity such as opening lectures with teammates in other schools. She also takes charge of organizing the team trip plan during the competition days and contacting with the committee for the invitation letters, etc. Xiao Linfan肖琳凡 Linfan XIAO has been nurturing her interest in life science since kindergarten age (actually it was derived from music and wildlife conservation, but that's another story). She has placed her focus on biomed, chemistry, zoology, and is now on her hitchhiking journey to the amazing galaxy of synthetic biology. In both her argumentative essays and her life, she firmly holds that the art and science of this vigorous field will prosper and thrive, contributing to the future advancement of humanity. Theory group leader, responsible for organizing the project's theoretical construction, plasmid design, and experiment plan. Ye Ziyun叶子运 Ziyun Ye, a senior two student from Shenzhen Middle School, is interested in biology especially the application of biological technology. In the “Plant Sitter” project for 2014 IGEM, he takes part in plasmid construction. Although he had no experience of experiment of molecular biologybefore 2014, he tried a lot and soon got skills of simple experiment. “Just try more,” he said. experiment Liu Chenxi刘宸希 Chenxi Liu, a senior two student in Shenzhen Middle School, has participated in biological study for years. Being enthusiastic on biology in younger age, she actively practices biological engineering with her teammates. In the theory group and experiment group, she works hard cooperates with others well. She believes that biological engineering is an interesting subject worth life time of hard work and research. Theory group, experiment groupRetrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/Team_MemberiGem Safety Q&amp;A From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice <<Back to wiki main pageHere are our answers for iGEM Safety Questions.Contents1 Q&A1.1 Question 11.2 Question 21.3 Question 31.4 Question 42 Reference Q&A Question 11.Would any of your project ideas raise safety issues in terms of:researcher safety,public safety, orenvironmental safetyOrganisms:E.coliRisk Group 1 (no or low individual and community risk) - WHO Laboratory Biosafety ManualBSL 1 - The list of human to human transmission of pathogenic microorganismsScindapsus aureusRisk Group 1 (no or low individual and community risk) - WHO Laboratory Biosafety ManualRisks:Potential pathogenicity to team members since e.coli is infectant and would cause disease when accumulating.Potential risk of the release of modified gene out of the lab and possible pollution of other organisms.Addressing issues:Suicide systemLab Safety Rules: Good microbiological techniques(GMT)General requirements for laboratory biosafety GB-19489 2008Safety training:WHO Laboratory Biosafety ManualGeneral requirements for laboratory biosafety GB-19489 2008Biosafety in the Microbiological and Biomedical Laboratories Manual,3th edition CDC/NIH Accident out of plan:Release of modified gene and possible genetic pollution if the containment procedures go wrong.Release of bacteria and possible health problem of team members.Addressing issues:Suicide system to prevent genetic pollution.Protection suits for researchers.Medical examination in hospital.Complete sterilization in the lab. Question 2 2.Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes,Did you document these issues in the Registry?How did you manage to handle the safety issue?How could other teams learn from your experience?All the biosafety levels of the parts in our project are BSL 1.Suicide system Question 3 3.Is there a local biosafety group, committee, or review board at your institution?If yes, what does your local biosafety group think about your project?If no, which specific biosafety rules or guidelines do you have to consider in your country?No.WHO Laboratory Biosafety ManualGeneral requirements for laboratory biosafety GB-19489 2008Biosafety in the Microbiological and Biomedical Laboratories Manual,3th edition CDC/NIH Question 4 4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?Safety level examination of parts and devices in lab.Clear biosafety hierarchy system of BioBrick parts(or devices) and publish the classification on the website of iGem for participants to consult.Suicide system to prevent genetic pollution when release of organism happens. ReferenceThe list of human to human transmission of pathogenic microorganisms;General requirements for laboratory biosafety GB-19489 2008.Retrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/iGem_Safety_Q%26Lectures From 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman PracticeIn order to encourage junior high school students to love biology, we held two lectures in secondary schools near Shenzhen Middle School. The first one was held on June 3, 2014 at Lianhua Middle School(莲花中学), and the second one was held on June 6 at junior high part of Shenzhen Middle School(深圳中学初中部). Lianhua Middle School Lecture at Lianhua Middle School Lecture at Lianhua Middle SchoolThree members of our team Zhang, Jing and Wang went to LMS on June 3, 2014, where they did the first publicity to the junior highs.Approximately 50 students gathered to attended our lecture. They showed high interest in Biology especially Biochemistry and Synthetic Biology. We showed them some paintings we drew with E-coli expressing GFP, RFP and YFP. Some students complained ‘why this interesting competition does not allow junior high students to attend.’Some of them think our project is useful to farm that our project can help the farmers in cold areas grow their plants better. While some others thought our project could be used in some other fields like planet conquering. Shenzhen Middle School (Secondary School) Lecture at Shenzhen Middle School Lecture at Shenzhen Middle SchoolDuring the lecture given in Shenzhen Middle School(Secondary School) on June 6, an increasing amount of curious students came to our lecture. Despite the fact that they are currently only a group of 12- to 14-year-old students, they were excellent audiences and were strongly motivated by their interest in biology to ask us any questions, some of which even refer to the principle of this competition. What these intelligent students asked, is not only the functions of the current design, but also the possible improvements of it. What we have seen from them is exactly a spirit desperately needed for the future scientific workers.In the end of our lecture, we even received future challenge from some students, who wanted to take part in iGem next year and were confident to defeat us in the future.What our lecture has brought to these young enthusiastic sharp minds is not only the information about genetic biology and our project, but also an idea, a strongly motivated drive to get into this field further. External Links Find more photos here:Team:Shenzhen SZMS/Gallery#Lectures in Secondary SchoolsRetrieved from "http://2014hs.igem.org/Team:Shenzhen_SZMS/Human_Practice/LectureFrom 2014hs.igem.org HomeTeam MembersProjectModellingLabSafetyGalleryHuman Practice<<Back to wiki main pageWelcome to homepage of Team:Shenzhen SZMS! Our project is called E. coli Plant-sitter. Contents1 Team1.1 Attributions2 Project3 Lab4 Modelling5 Safety6 Human Practice7 Fun8 More Info TeamShenzhen Middle School (深圳中学 Shēn Zhèn Zhōng Xué) is an high school in Luohu District, Shenzhen, Guangdong ever since 1947. In December 2013, a group of 17 SZMS students who have enthusiasm in biology were gathered together in the little yet omnipotent lab on campus; ever since then, this team of 17, Shenzhen SZMS has been established. AttributionsHey here! Check out our 17 members!Team:Shenzhen SZMS/Team Members. ProjectOur project is to create an “E. coli Plant-sitter” that achieves smart maintenance of healthy plant growth through the application of synthetic biology to temperature moderation, which can be applied to either hothouse cultivation on Earth or the Human Migration Program on MARS.Read more:Team:Shenzhen SZMS/Project LabWanna find more about how we bulid our plasmid? Check here:Team:Shenzhen SZMS/Lab ModellingWe bulit a model to estimate how the enzyme works. Check here:Team:Shenzhen SZMS/Modelling SafetyCheck our answers to the Safety Questions here:Team:Shenzhen SZMS/iGem Safety Q&A Human PracticeCome and meet the friend of our E.coli babysitter, E.coli the Fluorescent!Sincere greeting from the iGem lab in Shenzhen Middle School!Since all of our E.coli the Fluorescent are brilliant in both languages, the fourth group of them are advertising for their friend, E.coli Plant-sitter: "The soulmate of plant, E.coli babysitter hug you warm and give you heat all the time."Our human practice plan includes:Take part in BIT's 5th World Annual World DNA and Genome Day in Dalian from April 25 to 28, 2014. Details:Team:Shenzhen SZMS/Human Practice/BIT's 5th World DNA and Genome DayOpen our laboratory and invite students in our school to come and experience what we usually do, such as agarose gel electrophoresis. Details:Team:Shenzhen SZMS/Human Practice/Laboratory Open DayHold lectures at junior high schools in Shenzhen to encourage junior high school students to love biology. Details:Team:Shenzhen SZMS/Human Practice/Lectures FunTheme song for our project called E.coli Plant-sitter heals the world! A song that always arises in our minds whenever we think of iGem :)Find more photos here:Team:Shenzhen SZMS/Gallery! More Infoschool offical website (in Chinese) school intro (in English)school info on WikipediaRetrieved from "http://2014hs.igem.org/Team:Shenzhen_SZM2014hs.igem.org/wiki/images/thumb/1/17/IGEM_basic_Logo_white_stylized.png/100px-IGEM_basic_Logo_white_stylized.png1302Anatolia GreeceThessaloniki High School Greece www.anatolia.edu.grHigh SchoolMethane Gas 'Sensor'Methane gas(CH4)is a non-toxic greenhouse gas;however it's flammable and can form explosive mixtures with air.It affects the radiation of Earth directly and indirectly by reacting with oxidizers,halogens and some halogen-containing compounds.It is an asphyxiate gas and may displace oxygen in an enclosed space environment.Asphyxia may occur if the oxygen concentration is reduced to below 19.5% by displacement.When structures are built on or near landfills,methane can diffuse into buildings?interiors and expose occupants to significant levels of methane.Being socially conscious of the situation,we participate in iGem 2014 with an experiment according to which,we will synthesize a bacterium that recognizes methane gas and emits aroma in response,using two genomes.Having this in mind we're aiming to develop methane detection methods,thus helping in taking precautionary actions for future exposure.It introduces a new way of activity that may result in improved indoor air-quality.This could be later developed in a kit by companies active in the field.1511BBa_K1302999http://2014HS.igem.org/Team:Anatolia_GreeceBBa_K1302000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166620140High SchoolLog in   Team:Anatolia Greece From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:Anatolia_Greece/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:Anatolia Greece logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:Anatolia Greece team.png Your team picture Team Anatolia_Greece Official Team Profile Contents 1 Team2 Project3 Notebook4 Results/Conclusions5 Safety6 Attributions7 Human Practices8 Fun! Team Tell us about your team, your school! Project Anatolia_Greece Methane gas (CH4) is a non-toxic greenhouse gas; however it is highly flammable and can form many explosive mixtures with air. It affects the radiation of Earth both directly and indirectly by reacting with oxidizers, halogens and some halogen-containing compounds. Methane is also an asphyxiate gas and may displace oxygen in an enclosed space environment. Asphyxia may occur if the oxygen concentration is reduced to below 19.5% by displacement. When structures are built on or near landfills, methane can diffuse into buildings’ interiors and expose occupants to significant levels of methane. Being socially conscious of the situation, the team of Anatolia College of Thessaloniki, we participate in iGem 2014 with an experiment according to which, we will synthesize a bacterium that recognizes methane gas and emits banana aroma in response, using the x genome and the y genome . Having this in mind our team is aiming to develop methane detection methods, thus helping in taking precautionary actions for future exposure. Such a bacterium would be useful not only for experimental reasons but also for the welfare of our society. It introduces a new way of activity that may result in improved indoor air-quality and consequently improve standards. This could be later developed in a kit by companies active in the field. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:Anatolia_Greece1298CoBRACochrane High School, Cochrane, AB http://cochrane.rockyview.ab.ca/High SchoolCoBRA, Cochrane Alberta - Stopped In Their Little Blue TracksWhile mountain pine beetle are a natural part of the southern Rocky Mountain ecosystem, recent beetle outbreaks are larger than those of the past. Decades of fire suppression have created large tracts of older pine forest that provide a highway for beetle expansion. The lack of fire, combined with a recent warming trend, means that the beetles are now occurring farther east, farther north and at higher elevations than ever before. This is cause for concern as the mountain national parks form the margin between the beetle outbreak in British Columbia and commercial forests in Alberta. Our project will focus on building a bacterial plasmid that will produce and secrete the chitinase enzyme which will in turn be able to break down the chitin rich membranes of the BSF while leaving the tree unaffected and thus able to use its own defences to deal with the mountain pine beetle.1401BBa_K1298999http://2014HS.igem.org/Team:CoBRABBa_K1298000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166120140http://2014hs.igem.org/files/presentation/CoBRA.pdfhttp://2014hs.igem.org/files/poster/CoBRA.pdfHigh SchoolLog in   Team:CoBRA/HumanPractices From 2014hs.igem.org    A healthy forest is diverse. It has trees and plants of different ages, species, heights and genetic make-up. Diversity provides good quality wildlife habitat and helps limit the size and number of wildfires and insect outbreaks. In the forests of the Canadian rocky mountains,specifically the national parks, the activity of mountain pine beetle plays a role in creating forest diversity. Healthy forests are in a constant state of change in part through natural disturbances like avalanches, landslides, floods, fire and the activity of insects and diseases. Forests have evolved with these processes for thousands of years. Without them, the natural balance is lost. While mountain pine beetle are a natural part of the southern Rocky Mountain ecosystem, recent beetle outbreaks are larger than those of the past. Decades of fire suppression have created large tracts of older pine forest that provide a highway for beetle expansion. The lack of fire, combined with a recent warming trend, means that the beetles are now occurring where they haven't been observed before: farther east, farther north and at higher elevations. This is cause for concern as the mountain national parks form the margin between the beetle outbreak in British Columbia and commercial forests in Alberta. It is important to note that the beetle itself doesn’t kill the trees and that the detrimental effects associated with the mountain beetle are produced by Grosmannia Clavigera, or blue stain fungus (BSF). This fungus causes irreversible damage to the trees internal systems by converting the trees natural defenses into a carbon based food source. This clogs the various nutrient channels within the tree and the fungus uses those channels as a means to spread and eventually kill the tree. Our project will focus on building a bacterial plasmid that will produce and secrete the chitinase enzyme that will in turn be able to break down the chitin rich membranes of the BSF while leaving the tree unaffected. These genes have been cloned from interior spruce, and lodgepole pine tree, which are native to this ecosystem. Thus, the trees will be able to use their own defenses to deal with the mountain pine beetle.        As outlined in our project introduction, the coniferous forests of the great Rocky Mountains are of unparalleled importance. Not only for the sake of our economy, but also far more importantly for maintaining our local ecology and the biodiversity of these forests. Throughout this diverse forest, trees have become infected with Mountain Pine Beetles. The beetle burrows through the tree and leaves Grosmannia clavigera or blue-stain fungus in its wake. The blue-stain fungus plugs the phloem of the trees, which carries nutrients and phytochemical defenses of the tree. The trees’ main defenses are chemicals called terpenes, which the blue-stain fungus preys on. Essentially, the blue-stain fungus and the mountain pine beetle have a mutualistic relationship in which the beetle provides the fungus with a method of transport from tree to tree, and the fungus prevents the tree’s terpenes from killing the beetle. We hope if we eliminate the fungus from this relationship, we can decrease the rate at which the beetle is infesting the forests to a healthy rate.    This graphic depicts the enormity of the infestation. Historically, the beetle was killed off by the harsh winters of the Canadian Rockies, but with a series of milder winters, the beetle population has exploded exponentially, killing off the trees that provide food and habitat for most of the other organisms that live in this ecosystem.    Despite the negative effects of the beetle has always been an important part of the Rocky Mountain pine forest ecosystem, as in the past it has killed off diseased or old trees, and the beetle is a source of food to many birds such as woodpeckers and other insects. Our team does not want to completely eradicate the beetle, as this may cause additional ecological problems, but we do want to slow the beetle to the point at which it is killing trees in a sustainable manner. The best way to do this is to attack the blue stain fungus, which is a key to the beetle surviving the trees’ defence systems. Hopefully, if we eliminate the fungus, the beetles rapid expansion will be slowed to a more sustainable and healthy rate. The wood that has been infected with this fungus is usable as a building material but the rate at which these trees are being killed is unsustainable. The forestry industry cannot process all the lumber affected by the blue stain fungus, as the sheer volume of raw lumber coming in is simply too much, and as a result useable wood will be left to rot.    According to the British Columbia Forestry Department 16.3 million hectares of forest have been affected and 40 million acres have been completely destroyed, which is approximately the amount of wood harvested in ten years. Due to this damage, the supply of softwood lumber that could be harvested from the affected areas could drop between 32 and 67 percent. To combat this devastating problem, the government of British Columbia alone has already invested $884 million to mitigate the impact of the beetle. Additionally, the government of Canada has spent $340 million and is committed to spending another $800 million. Despite the government’s efforts, the Canfor Sawmill in Quesnel BC, and the West Fraser Sawmill in Houston BC, have been forced to close, resulting in the loss of 434 jobs, as their supply of softwood lumber is no longer usable. Obviously, this loss of jobs has had a devastating affect on the local economies. Sources http://www.vancouversun.com/Interior+sawmills+closed+beetle+epidemic+erodes+supply/9079750/story.html http://www.pc.gc.ca/eng/docs/v-g/dpp-mpb/sec4.aspx http://www.pc.gc.ca/eng/docs/v-g/dpp-mpb/sec3.aspx http://www.nrcan.gc.ca/forests/insects-diseases/13381 http://www.for.gov.bc.ca/hfp/mountain_pine_beetle/ http://en.wikipedia.org/wiki/Blue_stain_fungus http://fgya.foothillsri.ca/resource/enhanced-mountain-pine-beetle-decision-support-tool Retrieved from "http://2014hs.igem.org/Team:CoBRA/HumanPracticesFrom 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:CoBRhttp://2014hs.igem.org/wiki/images/f/f8/CoBRA-IGenLogo2.pngLabNotebook From 2014hs.igem.org Entry #1: Below is a link to our project guidebook. You can jump to the specific section for your job.iGEM Notebook is here.Entry #2: This is an excellent diagram illustrating the interactions between the mountain pine beetle, the blue-stain fungus and the pine tree. Will this help us narrow our research?Entry #3: Correspondence Between Adam Sibbald and Dr. Matt Bryman (January 19th 2014):Hello Adam,Thank you for contacting me, and your interest in collaborating with the Tria team around your iGEM competition. I have forwarded you email and contact information along to the Network Director, Dr. Janice Cooke, as well as our research team for an information they can provide. Normally I'd be happy to provide you with information, however I will be away for the next two weeks without email contact and would be unable to help at the early, and critical stages of your project development.The interactions amongst major biological components of the MPB system (the beetle, pine tree hosts, fungal associates, and bacterial/other associates) are very complex, and you are correct that you want an in-depth perspective before deciding on the research direction you with to explore. I would encourage your team to read more about the life cycle of the beetle before settling solely on an E. coli derived solution.If I could recommend some resources for you, the Tria project website has a series of research papers on more specific attributes of the MPB epidemic which could be useful once you team has decided on its final direction. Also, I would recommend reviewing the MPB website operated by Alberta Environment and Sustainable Resource Development who are the hands on Government organization who conduct monitoring and control activities in Alberta. Another site to review would be that of the Canadian Forest Service which provides a more Canadian perspective on the issue. Likely you have come across these as you researched the topic, but I'm still providing links below:- Tria Project Website: http://www.thetriaproject.ca- AESRD MPB website: http://mpb.alberta.ca/- CFS website: http://www.nrcan.gc.ca/forests/insects-diseases/13381I will follow up with you once I return from absence and see if there additional information I can provide. I wish you and your team the best as you choose your research topic and move ahead in a fun competition.Cheers,MattMatt BrymanNetwork ManagerThe NSERC TRIA Network (TRIA-Net) Entry #4: Correspondance Between Ms Bennett and Dr Janice Cook, TRIA Project Lead, University of AlbertaHi Stephanie,Apologies for the delay in responding to your email. We sent out a request to our TRIA members to see if anyone would be interested in mentoring your iGEM team in this project. I was disappointed that no one was available to mentor your team on this. I am probably not the most appropriate person to guide your group, but I would be willing to take a stab at it. I have spent quite a lot of time thinking about synthetic biology products that TRIA would be interested in, and how we could provide guidance. My lab is not really in the business of biotechnology, so this is a stretch for me. After some thought, I think that there are two avenues that the Cochrane students could take. The first avenue would be more aligned with TRIA research: here, your students could try to develop a system that synthesizes different mountain pine beetle pheromones or tree chemicals that can cause changes in beetle behaviour that could help in controlling the outbreak. An example of a commercially available beetle pheromone is verbenone, which is emitted by beetles to signal that an attacked tree is fully occupied, and that incoming beetles should find another tree. Similarly, maybe there is the potential to synthetically create a blend of tree chemicals that could be used as a repellent (or as a trap). I have cc’d two TRIA researchers on this email who have expertise in this area; if you wanted to follow up on these ideas, I would hope to convince them to provide some mentorship on this project. The second avenue is quite far from TRIA research: here, your students could use mountain pine beetle killed timber (infected with the mountain pine beetle fungal associates) as a feedstock to create new products from this wood. Given that the fungus is degrading the wood, the wood composition will be different than conventional softwood, and potentially could be used to generate different extractives, such as for the biofuel industry. This would be fundamentally exciting, but is very far from what I do. I have a colleague on campus who may be able to help you on this project, if your team was interested in this avenue. Apologies again for the delayed response. Please let me know if you would like to proceed with one of these projects (or if you’ve found another mentor), and we can decide next steps. Best wishes, JaniceAssociate ProfessorDepartment of Biological SciencesUniversity of AlbertaEntry #5: Mountain Pine Beetlehttp://www.nrcan.gc.ca/forests/insects-diseases/13397This site has the scientific name of the mountain pine beetle (Dendroctonus ponderosae), as well as its' life history and how it attacks and damages the tree.Entry #6: Genome Of Blue-Stain Fungus Evolved To Bypass Tree Defence In Mountain Pine Beetle Epidemichttp://www.publicaffairs.ubc.ca/2011/01/24/genome-of-blue-stain-fungus-evolved-to-bypass-tree-defense-in-mountain-pine-beetle-epidemic-ubc-research/"Now, researchers from UBC and the BC Cancer Agency’s Genome Sciences Centre have conducted a detailed genome analysis and identified genes in Grosmannia clavigera that are responsible for the fungus’s ability to bypass the lodgepole pine’s natural fungicide – and use it as a carbon source for fungal growth."Entry #7: Pheromone Production In Bark Beetleshttp://www.ncbi.nlm.nih.gov/pubmed/20727970"The first aggregation pheromone components from bark beetles were identified in 1966 as a mixture of ipsdienol, ipsenol and verbenol. Since then, a number of additional components have been identified as both aggregation and anti-aggregation pheromones, with many of them being monoterpenoids or derived from monoterpenoids."Entry #8: Verbenone useshttp://www.fs.usda.gov/Internet/FSE_DOCUMENTS/stelprdb5373188.pdfThis site explains how verbenone can be put to use to combat the mountain pine beetle. This chemical is a temporary solution and only works in places where the beetle has not completely infested the area.Entry #9: Beetle factshttp://www.for.gov.bc.ca/hfp/mountain_pine_beetle/Updated-Beetle-Facts_April2013.pdfInteresting facts about the beetle dealing with winter temperatures at the bottom of this page.Entry #10: Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum Scientific name: Grosmannia clavigeraKingdom: FungiPhylum: Ascomycota Class: SordariomycetesOrder: OphiostomatalesFamily: Ophiostomataceae/p>Genus: GrosmanniaSpecies: G. clavigerahttp://www.pc.gc.ca/docs/v-g/dpp-mpb/sec3/dpp-mpb3a.aspx“Mountain pine beetle carry fungus spores near their mouthparts”“the chewing action empties the spores into the tissues of the tree”“After the beetle introduces the fungi to the tree, *mycelia grow rapidly into the sapwood, slowing the movement of fluids and disrupting the tree’s defences against the invading beetles”*Mycelia is the vegetative part of a fungus that grows in thread-like structuresMycelia grows in the phloem and blocks the flow of fluids, which would block the flow of sap. This sap would trap the beetles and kill them if the fungus was not presentGuy that researched blue stain fungus genome and identified genes that are able to convert natural tree fungicide into food source: Joerg Bohlmann (EMAILED)Michael Smith Laboratorieshttp://www.cfs.nrcan.gc.ca/bookstore_pdfs/28144.pdfThe fungus bypass the trees natural defences due to a genetic change that allows them to turn these defenses into a carbon based food source. Mountain Pine Beetles serve as a vector for three different types of blue stain fungus, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum Based on the fungi’s isolated location (Alberta, BC, Colorado, California, etc.) different fungal isolates immerge with varying temperature tolerances and optimal growing conditions. “The precise role of these fungi is not known, but they are thought to help the beetle overwhelm the defences of the host tree… and provide necessary nutrition throughout the lifecycle of the beetle”Grosmannia clavigera is the more aggressive of the three fungi on most host trees. http://www.pnas.org/content/108/6/2504.shortWe establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. *Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.*Terpenoid - Any of a large class of organic compounds including terpenes, diterpenes, and sesquiterpenes. They have unsaturated molecules composed of linked isoprene units, generally having the formula (C 5H 8)Entry #11: Random thoughts and ideasFemale pine beetles would release pheromones (aggregation pheromone) once inthe tree to attract male beetles for reproduction. They are also capable ofreleasing pheromones (anti-aggregation pheromones) that contradict thesepheromones that give the message to other beetles that the tree is alreadyoccupied. These pheromones only have asmall area around the beetle where they are actually effective. I was thinkingif there is any way to take the anti-aggregation pheromone and to neutralize itwith the aggregation pheromone. if we neutralize this pheromone will this stop the male beetle from finding thefemale beetle? Or will the time it will take for the two beetles to meet justbe slowed down and give time for the trees natural defenses to kick in and killthe female beetle? Obviously this will take some more research but thoughtI should just write my thoughts down before they slipped away. Entry #12: Chitinase Info.http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Chitinase.html Great website from Princeton about Chitinase. Definitely worth a read. Entry #13: Female pine beetle pheromonesFound an article relating to Entry 11. It states that the aggression pheromone is called Trans-Verbenolhttp://link.springer.com/chapter/10.1007/978-3-642-88448-1_1#page-1Entry #14 February 11, 2014 Chitinase InfoChitinases are enzymes that hydrolyze the N-acetylglucosamine polymer chitin, and they occur in diverse plant tissues over a broad range of crop and noncrop species. The enzymes may be expressed constitutively at low levels but are dramatically enhanced by numerous abiotic agents (ethylene, salicylic acid, salt solutions, ozone, UV light) and by biotic factors (fungi, bacteria, viruses, viroids, fungal cell wall components, and oligosaccharides). Different classes of plant chitinases are distinguishable by molecular, biochemical, and physicochemical criteria. Thus, plant chitinases may differ in substrate-binding characteristics, localization within the cell, and specific activities. Because chitin is a structural component of the cell wall of many phytopathogenic fungi, extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Plant chitinases have different degrees of antifungal activity to several fungi in vitro. In vivo, although rapid accumulation and high levels of chitinases (together with numerous other pathogenesis-related proteins) occur in resistant tissues expressing a hypersensitive reaction, high levels also can occur in susceptible tissues. Expression of cloned chitinase genes in transgenic plants has provided further evidence for their role in plant defense. The level of protection observed in these plants is variable and may be influenced by the specific activity of the enzyme, its localization and concentration within the cell, the characteristics of the fungal pathogen, and the nature of the host-pathogen interaction. The expression of chitinase in combination with one or several different antifungal proteins should have a greater effect on reducing disease development, given the complexities of fungal-plant cell interactions and resistance responses in plants. The effects of plant chitinases on nematode development in vitro and in vivo are worthy of investigation.Source: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2619419/Entry #15 February 12, 2014 BSF Genome infohttp://treephys.oxfordjournals.org/content/32/8/943.fullhttp://www.pnas.org/content/108/6/2504.fullEntry #16 Chitinase experimenthttp://www.biomedcentral.com/1472-6750/13/46In this experiment, a chitinase is expressed by an e coli bacteriaEntry #17 The Economic, Social and Environmental Costsof the Mountain Pine BeetleStage One Report_The BC Experience and Lessons for GAER (Final)_ January 2007.pdfEntry #18 New Direction to Consider - February 18, 2014From Dr Janice CookExpressing the chitinase in the bluestain fungus itself (Grosmannia clavigera would be an appropriate choice). In an ideal world, the chitinase would be secreted by the fungus into the cell wall space, and chew up the chitin. Then the fungus (particularly fungal growth) is compromised because of the degradation of the cell wall.You want to have the chitinase turned on when the fungus is introduced into the tree, so that its growth is compromised. So maybe there is a fungal gene that is strongly upregulated by something about the tree, like monoterpenes? If Joerg's group (or Colette's group) has identified one or more of these genes (which I'm pretty sure they have), then you can identify the promoter for this gene from the G. clavigera genome sequence. This can be used to create the construct that you would use to create the transgenic fungus, which then could be introduced into the tree (or at least, for proof of concept, onto culture plates that contain the monoterpenes) and see if you can turn on the gene. If the gene gets turned on, then you can see what happens to the fungal cell wall.From Magda Pop - in an earlier gmail - which could be what Dr Cooke is speaking toYou could use a (pinII) promoter which is induced by monoterpenes(stress tree chemicals) to control a chi gene => when the tree gives offmonoterpenes, the (pinII) promoter is activated to produce chitinase enzyme,which degrades the chitin from the cell wall of the fungus ***Note: the pinIIpromoter might be available in Vancouver (http://www.researchgate.net/publication/6167104_Testing_of_a_heterologous_wound-_and_insect-inducible_promoter_for_functional_genomics_studies_in_conifer_defense)Or instead of pinII, use a stress/terpenoid-induced promoter that dr Bohlmannrecommends? From Patrick Wu in response to Dr CookIf we engineer the fungus directly, however, I'm not too certain if the fungus will be willing to keep those genes as it reproduces. Suicidal organisms tend not to be considered evolutionary fit, so those genes might simply disappear in one or two generations...But we mentioned a different organism during our brainstorming session, Agrobacterium tumefaciens. This is an organism that usually infects plants and can introduce DNA into them (which is how scientists can transform algae--for cheap!). What can be done, if we can't express the chitinase in E. coli, is to instead grow Agrobacterium that can introduce the chitinase genes into the fungus. From there, the fungus can create the chitinase inside itself.There seems to be at least a few papers on Agrobacterium being able to introduce DNA into blue stain fungus. I've attached one here.Blue Stain Fungus Transformation.pdfThe only issue with this idea is accidental transformation of the trees along with the fungus, since the bacterium was originally a plant pathogen. That might be something to explore further... hm.Entry #19 Building From Latest Consideration - February 19, 2014There seems to be an overall concern regrading the sustainability of Dr Cooke's suggestion. How to keep that suicidal gene going in the BSF. So to build on that, here's Dr Bohlmann's suggestion and Magda's response to that.From Dr BohlmannWe have a small paper in press on spruce and lodgepole pine chitinases which I will send you as soon as have the pdf of the corrected page proofs. You can also look for the paper at: http://dx.doi.org/10.1016/j.phytochem.2014.02.006. (the link does not work) I should be online within the next couple of weeks. The paper does not address your questions about engineered promoters for controlled expression, but includes some basic information on simple IPTG-inducible expression in E. coli.From Magda in response to Dr BohlmannI must say I am not clear how dr. Cooke's idea would work in practical terms.Assuming that (1) the team can use Agrobacterium to introduce a chitinase gene into the BSF, and (2) the engineered BSF would keep the gene, my question is: How do you get the transgenic BSF onto the trees? Would the plan be to add the transgenic fungus to already infested trees, and hope that it would not only destroy itself but the wild type BSF too? Or would the plan be to replace the wild type fungus with the transgenic one altogether (would that even be doable at all)? Unless I'm totally off track here, I am not sure how feasible or safe any of this can be made.Dr. Bohlmann's message seems more interesting to me. I couldn't open the link he sent (gives me an error) but from what I understand his lab is about to publish data on expression of spruce and pine tree chitinases in E coli (under IPTG induced promoter). He doesn't say if it worked or not or if it did, how well, but it seems to me that is the avenue worth pursuing. Maybe you can pick his brain a little more re the paper he is about to publish? Maybe his lab managed to get some expression and you can ask him to send you the gene(s) so you can use a different promoter? I am going to go back to what I said in an earlier message: simple is better. My suggestion would be to start with a working (maybe even constitutive) promoter and a chitinase that worked in a previous (dr Bohlmann's?) E coli construct. You could also compare with different chitinases from the registry? Add a secretory tag (Lisa's earlier point)? I think there would be plenty of interesting stuff to try in the relatively harmless laboratory E coli before attempting to engineer the fungus that is known to harm the trees.Source: WikipediaIsopropyl β-D-1-thiogalactopyranoside (IPTG) is a molecular biology reagent. This compound is a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, (this is a standard promoter) and it is therefore used to induce protein expression where the gene is under the control of the lac operator (which chitinases are under).IPTG, unlike allolactose, is not hydrolyzable by β-galactosidase, its concentration therefore remains constant in an experiment. Like allolactose, IPTG binds to the lac repressor and releases the tetrameric repressor from the lac operator in an allosteric manner, thereby allowing the transcription of genes in the lac operon, such as the gene coding for beta-galactosidase, a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. But unlike allolactose, the sulfur (S) atom creates a chemical bond which is non-hydrolyzable by the cell, preventing the cell from metabolizing or degrading the inducer. The concentration of IPTG therefore remains constant and the expression of lac p/o-controlled genes would not be inhibited during the experiment.According to Dr Bohlmann, if we grow E. coli in a solution of IPTG, the bacteria will take it up and undergo transcription. If we engineered an E. coli construct with a spruce/pine tree chitinase, then that transgenic E. coli will produce the tree chitinase.Entry #20 Clarification of Terms From Latest Consideration - February 21, 2014From MagdaA constitutive promoter is normally ON, meaning that a coding region downstream from it will be always transcribed under normal/regular conditions. For example, if you placed the coding region for the chitinase downstream from a constitutive promoter, under normal conditions of growth the bacterial cells will make the chitinase enzyme continuously (ie all the time). From what I understand, you'll get quite a few constitutive promoters in your kit, either by themselves or with additional regulatory sequences for fine tuning of expression. For example, this is a promoter part by itself: http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100, while this is a composite part for high expression: http://parts.igem.org/wiki/index.php?title=Part:BBa_K314100. You will also get this promoter: http://parts.igem.org/wiki/index.php?title=Part:BBa_R0040, which behaves as a constitutive promoter unless you engineer the cells to also express its repressor (TetR). Therefore, even without trying to replicate dr Bohlmann's results, my feeling is that the team would have plenty of interesting things to try once you get your hands on a working chitinase construct.A secretory tag would be a protein fragment that would be attached to your protein of interest (the chitinase), and would serve as a signal for the cell to export/secrete that protein. Essentially, if you need your protein to be secreted, you fuse its coding region with the coding region for the secretory protein. For your purposes, it would be important for the chitinase enzyme to get outside the bacteria, which is where its substrate/target would be (ie the chitin in the cell wall of the fungus). The alternative would be to build in a suicide-switch along with the chitinase, so cells will produce the chitinase and then would lyse to release/expose the enzyme to allow it to access the chitin.I searched the registry but was unable to find a secretory tag - it's probably because of my limited knowledge of the registry and how it works. One part I found that looks of interest to me is this: http://parts.igem.org/wiki/index.php/Part:BBa_K103006; It's in your distribution kit but it's not for secretion. It is for display of the protein of interest on the outer cell surface. I don't think it's ideal (to have the enzyme displayed may constrain/impair its activity) but that's all I could find. I am sure that experienced iGEM-ers will have a lot more ideas on this.Entry #21 Meeting with David and Himika - February 25, 2014Entry #22 Origin of the chitinases - Wednesday March 8The chitinases were cloned from interior spruce (Picea glauca x engelmannii) and lodgepole pine (Pinus contorta) trees.This information was found in the second sentence of the abstract of Bohlmann's most recent paper. ( "cDNA for six chitinases were cloned from interior spruce (Picea glauca x engelmannii) and four from lodgepole pine (Pinus contorta)." ) The next sentence contains the names of the six chitinases from the interior spruce and the four from the lodgepole pine. Of the three chitinases that we are looking to get from Dr. Bohlmann, two were cloned from the interior spruce (PgeChia1-1 & PgeChia1-2) and the last was cloned from the lodgepole pine (PcChia1-1).Entry #23 Ken RaffaThis is a link http://labs.russell.wisc.edu/raffa/ leading to the website of Ken Raffa, a leading Entomologist who has an advanced understanding of the forest ecosystem. His expertise was recommended to us by Dr. Bohlmann Entry #24 Nucleotide/ Protein Sequences: Saturday March 8PgeChia1-1 http://www.ncbi.nlm.nih.gov/nuccore/HM219843PgeChia1-2 http://www.ncbi.nlm.nih.gov/nuccore/HM219844PcChia1-1 http://www.ncbi.nlm.nih.gov/nuccore/HM219849Also, we have 10ng of each Chitinase on the way from UBC. The nucleotide and protein sequences for all chitinase clones are available in NCBI GenBank. Accession numbers for all genes are listed in the "Experimental" section of the paper under subheading "Nucleotide sequence accession numbers". The expression vector that we used was pMal-c4x from NEB:https://www.neb.com/tools-and-resources/interactive-tools/dna-sequences-and-maps-tool . The genes were cloned into BamHI (right after) and before HindIII restriction sites.Entry #25 Schematic Drawing of Project: Monday March 10 Entry #26 DNA sequencing: Sunday March 231. Template from BioBasic.com sent to us by Lisa O.2. Open BioBasics.com, NCBI website where gene sequence is located, Google Help:Biobrick Prefix and Suffix from iGEM, NEBCutter V2.0Checking on Cutsites in your DNA:a. NEBcutter have openb. Open Help:BioBrick Prefix and Suffix in iGEM - copy and paste prefix (gaattcgcggccgcttctag - first tag) into NEB text box.c. Open NCBI - click on FASTA - copy and paste chitinase class gene into NEB text box after prefixd. Open Help:BioBrick Prefix and Suffix in iGEM - copy and paste suffix (tactagtagcggccgctgcag- only tag) into NEB text box after chitinase gene sequencee. Hit "submit" (right hand side) for cutsites in NEBcutterf. Go to bottom right - click "custom digest"g. Click on restriction enzymes: XbaI, SpeI, PstI, NotI, and EcoRIh. Click on "digest"You will now see only those cut sites and can see where the restriction enzymes cut into your gene. We will also get the exact number of nitrogen bases in our gene sequence. Before Custom digest After Custom digest with our 5 restriction enzymes. 3. Fill in Gene sequencing sheet as per Lisa's instructions. Used PgeChia1-2 as template.4. Fill in Gene synthesis optimization request as per Lisa's instructions. This is for the protein sequence. Use NCBI - choose protein id= click on its link ; choose FASTA; copy and paste into optimization sheet.Entry #27: A quick link to our wiki and project proposal - Thursday, March 27http://2014hs.igem.org/Team:CoBRAEntry #28: Paper by Dr. Raffa - Thursday, March 27http://digitalcommons.usu.edu/cgi/viewcontent.cgi?article=1058&context=barkbeetlesThis paper by Dr. Raffa explores the relationship between the fungi, beetles, and trees. The paper suggests that there are many factors that determine whether a tree is able to fight off the beetle / fungus combination such as "vigor of the tree, site and stand conditions, and the size of thebeetle population". So despite the fact that some conifer species have naturally occurring chitinases, these tress could still die in a high density beetle infestation by the fungi contributing to the death of the host tree through mycelial penetration of host tissue or the combined action of toxin release and its effect on conifer defenses. Therefore, our project may not be strong enough to aid the trees in areas of high beetle population.On the other hand, the paper seems to suggest that a healthy population of trees would be able to resist the fungi and beetles if the beetle population is low enough. The paper also confirms that our prediction that "beetle-caused death ofany particular tree is relatively low"http://www.agrihouse.com/press-release.php?id=17This experiment with a compound called chitosan was used to increase resin output from the tree. It shows that sheer resin output will overwhelm and force out any fungi carrying beetles. As a result, the pine beetle egg count in the tree was reduced, and blue stain fungus was not found the the trees that were treated with this chitosan. It is unclear whether this experiment was performed in a high density or low density beetle population. It is also unclear whether this experiment had any effect on the surrounding forest community, like woodpeckers.Entry #29: Workshop #2 - Saturday April 5 Still need to do the following:1. Shaking incubator - DIY2. Timers for Day 33. NEB Biobrick enzymes - E, X, S, P, buffer and ligase4. BioBasics - chitinase enzymesEntry #30: Gene synthesis of Chitinase genesThe Chitinase genes that we received from Dr. Bohlmann needed to go through some additional changes before they were usable. First of all, these pieces contained the PstI cut site in their coding region, which could wreak havoc on our restriction digest, by cutting the Chitinase at the wrong place with the enzymes. In addition, the biobrick prefix and suffix were not present, so the Chitinase parts could not be ligased to any of our other parts. Here are some screenshots of the original parts from the NEB cutter site, with all PstI, EcoRI, XbaI, SpeI and NotI cut sites displayed.PcChia 1-1 PgeChia 1-1PgeChia 1-2As you can see these parts have illegal cut sites in them. We sent the base pair sequence and protein sequence to Biobasics and asked them to invoke a silent mutation where the illegal cut sites are, and to add the biobrick prefix and suffix. Here are the resulting cut sites on the genes. PgeChia 1-1 and PcChia 1-1 (Identical pictures)PgeChia 1-2The illegal cut sites have been removed and the biobrick prefix and suffix have been added.Entry #31: Preparing Media and Growing Cultures & Why Won't My Cultures Grow?Preparing media and growing cultures :http://depts.noctrl.edu/biology/resource/handbook/media.pdfWhy won't my cultures grow :http://www.sciencebuddies.org/science-fair-projects/project_ideas/microbio_cultureproblems.pdfEntry #32 - Wet Lab Experiment Wednesday May 7 - Inoculation1. Inoculated 5 mL LB media (broth) with a single colony of DH5a cells from petri plate into a 15 mL Falcon tube.2. Incubated each falcon tube in shaking incubator at 37 deg celsius overnight.Entry #33 - Wet Lab Experiment Thursday May 8 - Making competent cells and bacterial transformation1. 8 am added 100 uL of DH5a mixture to 9.9 mL LB liquid media in to a new 15 mL falcon tube. Did this for two tubes.2. Placed new falcon tubes into shaking incubator and will leave for 6 hours at 37 deg celsius in shaking incubator.3. 4 pm made Competent cells (Chitinases - added 40 uL ddH2O to each vial diluted to 1:10 then used 2 uL; RFP - added 500 uL ddH2O to 50 pg vial - all added; J04500 - added 10 uL to spot on kit - all added)4. 5:30 pm began transformation - made a note that we were working with a very, very small pellets5. 7 pm incubated 5 plates overnightEntry #34 - Wet Lab Experiment Friday May 9 - Evidence and Analysis of Transformation of DH5-a Competent cellsEvidence for Test Trial #2All chitinase plates had these "growths" on them; translucent, round-smooth "colonies", few in numbers, all grown on LB/Amp Colonies should look like this All iGEM DNA (J04500 and J04450) colonies looked like this; strange peripheral edges; more transparent on edges not uniform solid growth; J04450 did not flouresce red as can see by negative colour under purple lightRFP colonies should be red within 18 hours at 37 deg CAnalysis1. All 5 plates did not transform properly. Confirmed by Lisa O, David L and Dr Bohlmann (emailed this evening)Conclusions 1. Grow up another vial of DH5-alpha cells for another transformation2. Check for negative growth on LB/Chlor media3. Double check concentrations - used 1:10 diluted Pge1-1, Pge1-2, and Pc1-1; used 100 ng/uL of RFP; used less than 100 ng/uL of J04500Entry #35 - Wet Lab Experiment Saturday May 10 - Evidence of Antibiotic Test; Transformation Redo1. 7 am added 100 uL of DH5a mixture to 9.9 mL LB liquid media of each to three falcon tubes. Incubated in shaking incubator.Evidence of Antibiotic Test Analysis1. Tubes on far left show negative growth in each. DH5a cells from LB plate were inoculated in LB/Chlor showing no growth as the antibiotic killed them. The second tube is LB/Chlor with no cells and showing no growth. The antibiotics are sterile.2. Tubes in middle picture show positive growth of our DH5a cells in LB media. 3. Tubes in far right together illustrating cloudiness we're looking for.Conclusions for antibiotic test1. Growth is not being compromised by our Chloramphenicol antibiotic2. Transformation failure could be because:a. concentrations aren't rightb. time in hot water bath not long enough - let's increase from 45 sec to 60 sec or let's decrease to 30 secc. need greater pellet size when making competent cells.Transformation Lab1. Performed a control (LB) and test (LB/Chlor) for both competent and transformation lab. 2. Tried two different concentrations for the Chi-DNA; 10 ng/uL and then diluted it 1000X to 1pg/uL3. Heat shocked for 60s and on ice for longer4. Changed up the competent test by putting on ice before adding last 100 uL of CaCl2Results from Saturday's Test Trial #3 Entry #36 - Wet Lab Experiment Sunday May 11 - Results from Sunday's Test Trial #4 Conclusions:1. DH5a cells have been compromised2. Antibiotic plates don't seem to be working correctlyAction:1. Make new sleeves of antibiotic plates, in particular Amp and Chlor2. Use newly arrived competent DH5a cells and transform them with our chitinase plasmids.Entry #37 - Wet Lab Experiment Monday May 12Results After Growing New Antibiotic Plates and Receiving New Competent DH5a cellsConclusions:1. All 6 control LB and experimental LB/Antibiotic plates saw superb colony growth2. All LB/antibiotic plates with no cells had no growth showing us our antibiotic plates are working3. Both LB plates with no cells had no growth showing us our LB media is not contaminated.Actions:1. Inoculated LB media with DH5a cells from the LB plates - this is to grow a stock of DH5a cells for competency2. Restreaked 6 new antibiotic AMP plates to get better colony growth in preparation for Miniprep tomorrowEntry #38 - Process for making Chitinhttp://ispub.com/IJMB/10/2/14186This is a process for making chitin. We could use this chitin to achieve proof of concept Entry #39 - Chitin for Sigma Aldrichhttp://www.sigmaaldrich.com/catalog/search?interface=All&term=chitin&N=0&mode=match%20partialmax&focus=product&lang=en&region=CAThis is a link to a site at which we could buy chitin from shrimp shellsEntry #40 - Sites with Lysis ideasThese sites have ideas for a way to lyse cell while leaving the protein intacthttp://www.embl.de/pepcore/pepcore_services/protein_purification/extraction_clarification/cell_lysates_ecoli/Future goalsIdea #1http://www.pnas.org/content/108/6/2504.shortWe establish that Gc is heterothallic, and report evidence for repeat-induced point mutation. We report insights, from genome and transcriptome analyses, into how Gc tolerates conifer-defense chemicals, including oleoresin terpenoids, as they colonize a host tree. RNA-seq data indicate that terpenoids induce a substantial antimicrobial stress in Gc, and suggest that the fungus may detoxify these chemicals by using them as a carbon source. *Terpenoid treatment strongly activated a ∼100-kb region of the Gc genome that contains a set of genes that may be important for detoxification of these host-defense chemicals. This work is a major step toward understanding the biological interactions between the tripartite MPB/fungus/forest system.*Terpenoid - Any of a large class of organic compounds including terpenes, diterpenes, and sesquiterpenes. They have unsaturated molecules composed of linked isoprene units, generally having the formula (C 5H 8)Idea #2 - Monday March 10 from Magda Surface display: Instead of sending things to the media outside the cell, you can also stick things onto the outside of the cell to be displayed. As Magda suggested, this normally works just fine, however sometimes it can impair the function of your protein. The only way to tell for sure is to try however- usually you fuse the protein (as described for the secretion tag) to the N terminus (start) of the protein for one construct, and the c terminus (end) of the protein for a second construct, and test both to see if one works better. The part that Magda suggested has been shown to work. http://parts.igem.org/wiki/index.php/Part:BBa_K103006. An alternative would be something like this http://2012.igem.org/Team:Penn/SurfaceDisplayBBa. It won best biobrick, and was also shown to work. Chitinase APPEARS to be a monomer (meaning that the enzyme is formed from a single protein chain, and not from more than one protein associating together (Protein subunits)).I think this article brings strong support in favour of targeting the blue stain fungus with a bacteria-displayed chitinase. And the fact that the surface display part (http://parts.igem.org/Part:BBa_K811005) is available in the registry, and even won the best biobrick award (!) adds to the promise of this project. Thanks for digging these out, LisaIdea #3 - Saturday March 29 from Richard LeeArming Trees Against Pine Beetle Invasions Retrieved from "http://2014hs.igem.org/Team:CoBRA/LabNotebooTeam From 2014hs.igem.org   The Cochrane Bio-Researchers in Action (CoBRA) is a team of smart and dedicated students passionate about science with the goal to not only make a difference in the scientific community but to solve problems that affect our own community. Our team, with the support of local companies, uses synthetic biology to come up with clever and effective solutions for problems that have had a huge impact on our community.       Katrina Berube(Wet Lab/Human Practice) Lauren Van Dyke    (Wet Lab) Autumn Bernard    (Wet Lab) Katie Goetjen  (Visual Arts) Rhett Devlin   (Wet Lab)     Kristian Smits(Wet Lab/Visual arts)     Joao Moraes(Wiki Page/Wet Lab) Richard Lee  (Wet Lab) Jacob Rovere  (Wet Lab) Cassandra Bourcher      (Visual Arts)     Adam Sibbald(Wet Lab /Human Practice             /Business) Ben Luft(Wet Lab) Marius Anastasiu(Human Practice/Business) Matthew Pipa    (Wet Lab) Kayla Sage   (Wet lab)     Edith Henneberg (Documents/Treasurer)     Jim Reilly & Stephanie Bennett      (Teachers) Retrieved from "http://2014hs.igem.org/Team:CoBRA/TeaProject From 2014hs.igem.org Restoring self-preservation in the lodgepole pine trees: Inhibiting blue stain fungal proliferation using class I chitinase activity in transformed E. coli DH5alpha cells    CoBRA iGEM Project Proposal Our research is to determine whether chitinase genes in transgenic E.coli will be produced and to what degree to reduce the BSF disease in pine trees. The CoBRA iGem team will attempt to engineer a new DNA biobrick containing a specific promoter gene, a gene of interest (one of three different Class I chitinases; PgeChia1-1, PgeChia1-2, and PcChia1-1,)3 and a terminator gene. This construct will then be placed in E.coli bacteria so that this bacteria, when subjected to pine tree resins, will secrete the chitinase which will kill the Gc.    3N. Kolosova, J. Bohlmann, and C. Breuil. "Cloning and characterization of chitinases from interior spruce and lodgepole pine."Elsevier (2014): 1-8. Print.    Background Information For our project, the CoBRA iGEM team has decided to attempt to manage the devastation that the mountain pine beetle (Dendroctonus ponderosae) has caused to the pine trees in forests along the Rocky Mountains in Alberta and British Columbia. This beetle burrows into the bark of the lodgepole pine of these Mountains and with the help of a fungus called the Blue Stain Fungus(Grosmannia clavigera), lays its eggs in the tree. The winter then kills the beetle off, but the eggs, which are protected from the cold by the tree, survive the winter and mature to be able to start the cycle all over again. The Gc spreads its mycelium into the phloem, and then feeds on this essential structure, thus choking off the supply of glucose to the tree. Research also shows the the BSF survives the pine trees defensive resin production, by using the monoterpene chemicals as its food source.    These processes put the tree under tremendous stress and often kills it. The Mountain Pine Beetle has always been a factor in this ecosystem, but historically, winters have been harsh and cold enough to keep the beetle population in check. With global warming beginning to produce noticeable increases in winter temperatures, more and more beetles have been able to survive, and the population has reached a level where it is capable of ravaging huge chunks of forest, not only destroying the ecosystem, but rendering the lumber unusable.    Looking at this problem from several angles, our team decided that the best way to combat this epidemic would be by killing off the Blue Stain Fungus or Gc. One reason is that the BSF is helpful, but not essential to the MPB survival. Secondly, by focussing on the BSF, we are attempting to minimize our disruption of MPB predator populations, such as woodpeckers and other birds. Thirdly, our objective is to focus specifically on the pathogenic BSF and not destroy the symbiotic relationships of a forest fungal ecosystem.    Chitin is a structural component of the cell wall of many pathogenic fungi including BSF. Chitinases are enzymes that hydrolyze the polymer chitin breaking it down. Extensive research has been conducted to determine whether plant chitinases have a role in defense against fungal diseases. Expression of cloned chitinase genes in transgenic plants has provided further evidence for their role in plant defense. The level of protection observed in these plants is variable and may be influenced by the specific activity of the enzyme, its localization and concentration within the cell, the characteristics of the fungal pathogen, and the nature of the host-pathogen interaction. The expression of chitinase in combination with one or several different antifungal proteins should have a greater effect on reducing disease development, given the complexities of fungal-plant cell interactions and resistance responses in plants.  Our project goal is to determine if a cloned chitinase cDNA can be successfully expressed in transgenic E.coli. Using recombinant DNA techniques our team will create an entirely new DNA biobrick, this biobrick will placed in the pSB1C3 vector and contain a specific inducible or constitutive promoter (LacI or TetR), a specific cDNA (one of three class 1 chitinases; PgeChia1-1, PgeChia1-2 and PcChia1-1), and a stop codon or terminator gene. This construct as previously mentioned will be placed into lab grade Top 10 and k12 lab strain E.coli bacteria thus allowing these new, genetically altered bacteria to successfully produce and secrete the chitinase enzyme thus showing proof of concept. It is important to note that our engineered bacteria will not be used outside of a controlled lab setting during our for the current project.Materials and Method:  As our ultimate goal is to express the chitinase gene in transgenic E.coli, we followed standard recombinant DNA techniques. To begin, our specific gene of interest, the chitinase, is not available in the iGEM registry. We optimized our DNA using BioBasics Inc. Pc 1-1Pge 1-1Pge 1-2Before optimizationAfter optimizationCompetent Cells and Transformation  After obtaining our optimized plasmids from BioBasics Inc , chemically competent cells were created using DH5α E. coli cells. Cells were grown in 5mL of LB broth at 37oC in a shaking incubator overnight. The following morning, 100μL of cells were grown in 9.9mL of LB broth and grown for 3-4 hours in a shaking incubator. Following the incubation cells were centrifuged and the supernatant was removed. The pelleted cells were then re-suspended in CaCl2 and cooled on ice for 30 minutes. Our plasmids were then added to the microcentrifuge tubes containing the DH5α cells and the tubes were placed in a water bath at 42oC for 60 seconds. This heat shock created pores in the cellular membrane thereby allowing the entry of plasmids. Our cells were then placed on ice for 5 minutes to close the pores in the membrane. Following this recovery, LB broth was added and the cells were placed in the shaking incubator for 60 minutes at 37 oC. After the incubation cells were then plated on agar plates and then incubated for 24 hours incubated.To determine whether our transformation was successful, antibiotics were added to our agar plates. The experimental plasmids contained antibiotic resistance and therefore only cells that had successfully been transformed with our experimental plasmids grew on the agar plates. These transformed cells were used to amplify our experimental plasmids.Miniprep:  Having amplified our plasmids, we next aimed to isolated them from our DH5α cells. In order to do this, a miniprep was performed using the E.Z.N.A. Plasmid DNA Mini Kit II (Omega Bio-tek Inc, Norcross, Georgia). DH5α cells were grown in LB and treated with RNase. Cells were then lysed, cellular debris was pelleted and the lysate was passed through a HiBind DNA mini column. Using the HiBind column, DNA was trapped as proteins while eluting proteins, membrane molecules and any other unwanted substrates. A wash solution containing ethanol is then spun through the column. The ethanol aided in precipitating the DNA. DNA was eluted using distilled water. The isolated plasmids were then visualized using gel electrophoresis. Plasmids were run on a 1.5% gel at 125V for 40 minutes. Molecular size of the plasmids were visualized using 1 kb Invitrogen 0.9% Ethidium bromide/Agarose and 1 kb Ready 1% TBE/Agarose DNA ladders.Digest:Having visualized that plasmid isolation was successful, we used the remaining product of our miniprep to perform a restriction digest of our experimental plasmids. The restriction digest was performed using restrictions enzymes SpeI and PstI for plasmids Pc1-1, Pge1-1 and Pge1-2, enzymes Xbal and PstI for plasmid J04450, and enzymes SpeI and PstI for plasmid J04500. To each chitinase minicentrifuge tube we added 30.5 uL of ddH2O, 5 uL of cutsmart buffer, 1 uL each of restriction enzymes and 12.5 uL each of Pc1-1, Pge1-1 and Pge 1-2. To each vector minicentrifuge tube we added 30.5 uL ddH2O, 5.0 uL cutsmart buffer, 1 uL each of restriction enzymes and 12.5 uL each of J04500 and J04450. We then gently pipetted each minicentrifuge tube followed by a centrifuge at 14,000 RPMs for 10 s. 20 uL from each tube was visualized using gel electrophoresis. Fragments were run on a 1.5% gel at 125 V for 40 minutes. Molecular size of the fragments were visualized using 1 kb Invitrogen 0.9% Ethidium bromide/Agarose and 1 kb Ready 1% TBE/Agarose DNA ladders. The remainder of our restriction digest was stored at -20oC for ligation. Ligation:  To deactivate our restriction enzymes, tubes were placed in a water bath at 80oC for 20 minutes. To ligase our parts into J04550 and J04500 we added 6.5 uL of each digested chitinase inserts (Pc1-, Pge1-1 and Pge1-2) to 2 uL of either the J04450 plasmid or the J04500 plasmid. We then added 1 uL of T4 DNA buffer and 0.5 uL of T4 DNA ligase. The ligation mixture was pipetted gently three times, spun down at maximum RPMs for 5 s and allowed to incubate at room temperature for 10 minutes. This gave us 6-0.6 mL centrifuge tubes containing our ligated plasmids.   The ligated plasmids were then transformed into 100 uL of competent DH5a cells and left for 24 hours. We then grew up these ligated cells for miniprepping to obtain the six new constructs. From there the isolated plasmids were visualized using gel electrophoresis. Plasmids were run on a 1.5% gel at 50V for 120 minutes. Molecular size of the plasmids were visualized using 1 kb Invitrogen 0.9% Ethidium bromide/Agarose and 1 kb Ready 1% TBE/Agarose DNA ladders.Proof of Concept:  We will take some cells from a streak plate and incubate them in 15mL of LB broth and 5uL chlor for 12 hours. In a 1.5 mL centrifuge tube, we will put 1.2mL of culture and spin at 10000 rpm for 3 minutes, and then remove the supernatant. We will repeat this step until we have a large pellet. Once we have a large pellet we will resuspend in 750uL of LB broth. Next we will make 100mL of standard LB chlor plates (makes 2 plates) and add 100mg of practical grade chitin. This will serve as an assay for the production of chitinase, as these plates should have a cloudy color with chitin, but if the chitin is degraded they should be more transparent. We will place 7 drops of 60uL of our cells on these plates, and look to see if there is any color change   To end we submitted all six of our biobricks to iGEM. We performed all of our protocols under aseptic conditions and were conducted at room temperature unless stated otherwise.   At the time of submitting our wiki, we are in the process of proving proof of concept. We made very tiny agar plates containing 10 mg of practical grade chitin and grow a specified volume of our Pge and Pc cells on them. This will be a great introduction to exploring our question of whether our cells can produce chitinase, which will eventually lead to the use of chitinase in the degradation of the chitin-rich blue stain fungus cell wall.ResultsFigure 1. DIY Shaking Incubator: Hova-Bator Incubator Atop Fisher-Price Electric Plug-In Baby SwingFigure 2. Failed Bacterial Transformation Plates Using NEB Top 10 E.coli May 11, 2014Notice the odd fringe of growth around the central core. Notice the colony shape is not circular or dense in the core.Figure 3. Successful Bacterial Transformation Plates Using DH5a E.coli May 12, 2014Notice no strange fringe around colonies. Colonies are concise and dense.Figure 4. Successful Ligation Transformation Plates Using DH5a E.coli June 3, 2014Ligated DH5a with chitinase cells in J04500 backbone (pictured on left); Ligated DH5a with chitinase cells in J04450 backbone (pictured on right)Figure 5. Gel Results of Digestion with Restriction Enzymes on J4450 Plasmids To Isolate Chitinase Protein in our DH5a E.coli cells with 1 Kb Invitrogen Ladder (June 14)Moving from right the first well is a track it 1kb invitrogen DNA ladder, the second and third wells consist of a construct made up of PcChia 1-1 in a pSB1C3 backbone. In well 2 this construct was cut by restriction enzymes PstI and EcoRI, causing the chitinase coding region to become detached from the backbone. In well 3 this construct was only cut at the EcoRI cut site, causing the plasmid to be linearized. Wells 4 and 5 followed the same process as wells 2 and 3 with the exception that the chitinase coding region in wells 4 and 5 is of the PgeChia 1-2 variety. Wells 6 and 7 contain the PgeChia 1-1 gene. Using the ladder, our chitinase genes look to be approximately 1000 base pairs (lanes 2, 4, and lowest band), the pSB1C3 backbone appears to be approximately 2000 base pairs (lanes 2, 4, and 6 second lowest band), and the linearized construct looks to be about 3000 base pairs (lanes 3, 5, and 7). These numbers match up with our theoretical values. 2 uL ladder4 uL loading dye + 20 uL plasmid digestpH TAE buffer solution at 8 and at Room temperatureFigure 6. Gel Results of Digestion with Restriction Enzymes on J4500 Plasmids To Isolate Chitinase Protein in our DH5a E.coli cells from the Vector (June 17)Moving left to right, lanes 1, 8 and 9 contain our two ladders, 1 kb Invitrogen 0.9% Ethidium bromide/Agarose (lane 8) and 1 kb Ready 1% TBE/Agarose (lane 1 6 uL of ladder; lane 9 12 uL of ladder). In well 2 this construct (PgeChia 1-1) was only cut at the EcoRI cut site, causing the plasmid to be linearized. In well 3 this construct (PgeChia 1-1) was cut by restriction enzymes PstI and EcoRI, causing the chitinase coding region to become detached from the backbone. Wells 4 and 5 followed the same process as wells 2 and 3 with the exception that the chitinase coding region in wells 4 and 5 is of the PgeChia 1-2 variety. Wells 6 and 7 contain the PcChia 1-1 gene. Using the ladder on the right, our chitinase genes look to be approximately 1000 base pairs (lanes 3, 5, and 7 lowest band), the pSB1C3 backbone appears to be approximately 2000 base pairs (lanes 3, 5, and 7 second lowest band), and the linearized construct looks to be about 3000 base pairs (lanes 2, 4, and 6). These numbers match up with our theoretical values. 2 uL ladder4 uL loading dye + 20 uL plasmid digestpH TAE buffer solution at 8 and Room TemperatureDiscussion - interpretationOur iGEM experiment purported that if we engineered an E. coli construct with a spruce/pine tree chitinase, then that transgenic E. coli will produce the tree chitinase. In doing so, our research will lend further support for a biological solution to a major ecological problem facing Canada and the USA.Optimization of DNA   To begin, our specific gene of interest, the chitinase, is not available in the iGEM registry. The cloned Chitinase genes were received from Dr. Bohlmann at UBC. The DNA needed to go through some additional changes before they were usable. These pieces contained the PstI cut site in their coding region, which could wreak havoc on our restriction digest by cutting the Chitinase at the wrong place with the enzymes. In addition, the biobrick prefix and suffix were not present, so the Chitinase parts could not be ligased to any of our other parts. Use of NEBCutter website was beneficial in determining that our sequence was problematic and what was required to fix the situation. BioBasics Inc optimized our DNA so that the illegal cut sites were removed and we were able to utilize this DNA for our experimental purposes.DNA Recombination Protocols   Transformation of iGEM’s NEBTop 10 E.coli cells proved extremely difficult. In the end, after several attempts, it was decided that we switch to DH5-alpha E.coli cells. This strain has worked under our laboratory conditions. DH5 alpha cells maintain plasmids well and can be made competent on a more consistent basis. Modifications of the transformation protocol included suspending cells in CaCl2 on ice for 15 min prior to removing the second supernatant, increasing the spin RPMs in order to collect a greater volume of pellet while making cells competent, increasing the heat shock 45 s, floating on ice for 5 min, increasing the volume of solution taken for growth on plates, addition of more luria broth media before growing colonies on plates and including control plates along with the experimental plates. Modifications to our miniprepping protocol included increasing our volumes of ingredients added to the culture tubes and ensuring that the dry spinning was achieved before adding the elution buffer. Restriction digest and ligation protocols were following specifically using addgene website and iGEM methods. For identification of plasmids or genes we used gel electrophoresis. We had issues with the integrity of our bands and ladders and consequently with our purported results. Part of the issue was that our plasmids floated out of the wells while loading which meant that we did not dry spin the centrifuge column enough. We corrected this step and had greater success as seen in Figures 5 and 6. As for the choice of ladders, it appears from Figures 5 and 6 that the 1 kb Invitrogen 0.9% Ethidium bromide/Agarose DNA ladders worked the best in our TAE buffer solution and in our high school laboratory conditions.Gel Results    Looking at our data, we believe that the length of the parts that were restriction digested with two enzymes should add together to equal the length of the parts that were restricted with one enzyme. By the ladder on the right of both Figures 5 and 6, our chitinase genes look to be approximately 1058 base pairs for PgeChia 1-1 and PcChia 1-1 and 1067 base pairs for PgeChia 1-2 (1), the pSB1C3 backbone appears to be approximately 2070 base pairs (2), and together the constructs look to be about 3128 base pairs for PgeChia 1-1 and PcChia 1-1 and 3137 for PgeChia 1-2. These lines match up with our theoretical values. The genes for the production of chitinase were the same ones tested in Kolosova, N., et al (3). Proof of Concept   We are currently in the process of attempting to prove the production of chitinase from our transformed DH5alpha cells in the presence of lab grade chitin agar plates. Results to be determined.Limitations   Accessibility to high tech equipment and critical chemicals was a hinderance with the wet lab component of our project. For example, we do not have a optical density machine, which made many of our calculations that required knowledge of how much plasmid our solutions contained unpredictable. Also, as we try to achieve proof of concept, we are missing some of the compounds that would help us make a different kind of agar plate that is optimal for testing for the breakdown of chitin. Furthermore, looking at our gels that we ran in the results section, although the lines look to be close to where they would be expected to be, they are not exact. We see two lines where the double digested parts are and one line where the single digested parts are, but these lines are not exactly where they should be in regards to the ladder. Seeing the rounded edges of the lines in our gel, we believe that our TAE buffer may be have been incorrectly made, which could cause our samples to run incorrectly. Significance of Corroboration   Despite our relative inexperience in the field of genetics and phytochemistry we were presented with the opportunity to work alongside Dr. Bohlmann and Dr. Kolosova, two researchers at UBC’s Michael Smith Laboratory and members of the TRIA project. Their coauthored paper on chitinase and their in depth knowledge of phytochemistry, the mountain pine beetle and the blue stain fungus proved to be a very valuable resource to our team as we set out to test chitinase production in transgenic E.coli4.   Our project, although parallel to the work done by Dr. Bohlmann and Dr. Kolosova, has provided iGEM with 6 new chitinase biobricks that will go on to aid future teams in their endeavors. In addition to our lab work we have also completed a high school level lab manual which combines tested lab protocols with high school level terms and directions, thus making synthetic biology more accessible to high school students. In the context of iGEM we have blazed a trail by creating six new BioBricks in a single iGEM cycle and providing iGEM with a multitude of high school level protocols to make synthetic biology more applicable and accessible at the secondary levelAreas for Future Studies   Our project is by no means ready to be put into a real world application. Firstly, the cell walls of the blue-stain fungus are not made exclusively of chitin. In Kolosova et al3 Phytochemistry, when the chitinase was tested on the blue-stain fungus, no antifungal activity was observed, as the cell walls contain many more compounds which are not degraded by chitinase. In future years, we may research these compounds, and see if we can alter our plasmid to also degrade them. Also, we currently are using the LacI promoter in our constructs. This promoter will not function if IPTG is present. For the forest ecosystem, this could be hazardous, as there are many beneficial fungi in forests which we would risk killing. For next year we hope to try and find a promoter which functions only when the tree is producing stress hormones such as terpenes or other indicators. This would help the tree fight off the infections with minimal, if any damage to the other fungi of the forest. Additionally, we need to find a method of delivery of our plasmid. The DH5alpha cells that we used are not able to survive in an environment such as a forest. Hopefully we could use a bacteria that is naturally present in the tree, to avoid introducing an alien species to the forest. Producing chitinase in E Coli is just the first step in combating the blue-stain fungus. Work CitedVancouver Referencing Guides1. http://store.biobasic.com/2. http://parts.igem.org/Part:pSB1C33. Kolosova, N., et al. Cloning and characterization of chitinases from interior spruce and lodgepole pine. Phytochemistry (2014).4. http://www.for.gov.bc.ca/hfp/mountain_pine_beetle/AppendicesPc Chia1-1 optimization     Project overviewPge Chia1-1 optimization    Kolosova et al.2014 PhytochemistryPge Chia1-2 optimization    Stage One Report The BC Experience and Lessons for GAER Final January 2007Protocol flowcharts Retrieved from "http://2014hs.igem.org/Team:CoBRA/ProjecSafety From 2014hs.igem.org    After reviewing the World Health Organization (WHO) Laboratory Biosafety Manual we have determined that in regards to table 1 and 2 the organisms and techniques we have used fall into group 1 for both tables. This classification thus dictates that we as a group have applied general microbiological techniques to an open wet lab setting. We as a group understand the importance safety in the lab have always held safety as a first priority. Before we even initiate the lab portion of our project we ensured that each and every wet lab member was briefed in sterile lab techniques and protocols to limit contamination as well as ensure the safety of each member, and the community.   It is important to recognize that proper biological safety extends far beyond the confinements of a lab, it extends into the surrounding community and environment. In order to ensure that our team protected these extending areas from potential biological contaminants we organized a system of safe and secure waste disposal and sterilization to guarantee that none of our bacteria, despite its inability to function outside the lab, made it into the open environment. Initially, before we entered the lab, we considered the various risks and hazards that could arise as a result of our lab work. These risks consisted of improper disposal or bacteria, chemicals, and other lab materials. In order to overcome this obstacle we devised a system to properly dispose of these materials with the guidance of Shawn Dodd, a past Cochrane High Alumni and Health Sciences major at the University of Calgary. Since our group has not had the opportunity to receive formal lab safety training we have taken it upon ourselves to ensure that we understand the various MSDS and WHMIS symbols as well as proper lab techniques. As per iGEM’s request we have also invested in an emergency spill kit so that we are prepared to handle and contain any hazardous spills should they occur. Disposable materials used in the project ware autoclaved before disposal to avoid contamination.   Should our project not go according to plan and our bacteria are released into the environment we have taken precautions to ensure that they do not thrive outside of a lab setting. By utilizing lab grade DH5α E.Coli cells we have effectively removed our bacterias ability to function outside of predetermined lab conditions. Over the years synthetic biologists have ‘trained’ specific strains of E.Coli to depend on lab conditions in order to survive, thus when these cells are suddenly exposed to external environmental conditions through improper disposal or accidental contamination they are unable to adapt fast enough to survive and die, thus leaving the environment unaffected. Despite the extremely low chance of malicious misuse with our lab chemicals and bacteria we have still taken precautions to limit any form of extreme misuse by storing our chemicals and bacteria in a locked room when they are not being used and having a teacher advisor supervising all lab protocols. This ensures that all team members are working with the highest degree of professionalism when in the lab.    Our BioBrick parts, although brand new to the registry do not raise any safety issues. With that being said we still recommend that future teams exercise basic biological safety when working with our parts as well as report any identified safety issues surrounding our parts to our team   Our institution does not have a biosafety group, committee or review board. The protocols and biosafety rules we followed were the GMT (good microbiological techniques) as outlined in the world health organization laboratory biosafety manual. (http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf) These were in line with our federal biosafety regulations. (http://canadianbiosafetystandards.collaboration.gc.ca/cbsg-nldcb/assets/pdf/cbsg-nldcb-eng.pdf) None of the parts that we used in our project this year are classed above BSL 1.   We are going to use this question as an opportunity to discuss future potential risks and impacts of our successful project. If our project were to become commercially viable, something that is still years away, we have plans for several safety features included in the bacteria itself. First of these would be a limited lifetime, something that would be necessary to prevent our alien organism from persisting in the environment for too long. Secondly would be a kill switch, engineer our bacteria such that it is especially susceptible to a an as yet undetermined substance. These plans however, at the present stage, are still entirely unnecessary. Our plasmid has no secretion tag present, nor are our cells capable of surviving anywhere other than in laboratory conditions.   With these safety features in mind there are still several potential issues that may arise. Primarily that the chitinase that our bacteria is engineered to produce will affect organisms other than its intended target, the Blue Stain Fungus. Between now and commercial viability however much more research will be done into this particular form of chitinase and which organisms it could potentially affect. Furthermore there is the potential, however slim that our biological machine will work better than anticipated, including a possibility of elimination of the Blue Stain Fungus from this ecosystem entirely. However much thought has been put into eminimating this slim possibility. Retrieved from "http://2014hs.igem.org/Team:CoBRA/SafetSponsors From 2014hs.igem.org Thank you:Dr. Joerg BohlmannDr. Janice CookeDr. Natalia KovalevaPatrick WuLisa OberdingHimika DastidarDavid LloydNo Pictures from: Magdalina Pop, Shawn Dodd Retrieved from "http://2014hs.igem.org/Team:CoBRA/Sponso1278GenetiX Tec CCMPrepa Tec Campus Ciudad de M?xicoHigh SchoolBiodetection of Anoxia in Lake XochimilcoLake Xochimilco in Mexico City faces a condition of extreme pollution which endangers the endemic species; many of which are near extinction. Oxygen levels depletion in the lake directly affect the flora and fauna, making it less hospitable or even deadly. Our goal is to produce a biosensor that can easily and inexpensively detect anoxia in different regions of the lake. Using an oxygen promoter in addition with the biological markers RFP and GFP we could theoretically detect low dissolved oxygen levels in water samples. In addition, we intend to use a second construct with an Iron promoter to detect iron concentrations that also endanger the sustainability of living organisms in the lake. Once we identify critical regions of the Lake, our report could incentivate Civil Council and authorities to propose concrete legal initiatives to reduce pollution in the identified areas and start remediation campaigns.1302BBa_K1278999http://2014HS.igem.org/Team:GenetiX_Tec_CCMBBa_K1278000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164020140http://2014hs.igem.org/files/presentation/GenetiX_Tec_CCM.pdfhttp://2014hs.igem.org/files/poster/GenetiX_Tec_CCM.pdfHigh SchoolLog in   Team:GenetiX Tec CCM From 2014hs.igem.org Welcome to our wiki We are high school students at Tecnológico de Monterrey, Mexico City Campus, a team with juniors and seniors working together. What made us a team was our passion for science, we all wanted to innovate, to create, to surprise and engine something useful. Our goal is to prove that if you plan on doing something, no matter what, you can achieve it with the right focus. Biodetection of Anoxia in Lake Xochimilco Lake Xochimilco in Mexico City faces a condition of extreme pollution which endangers the endemic species; many of which are nearing extinction. Oxygen levels depletion in the lake directly affect the fauna, making it less hospitable or even deadly. Our goal is to produce a biosensor that can easily and inexpensively detect anoxia in different regions of the lake. Using an oxygen promoter in addition with the biological markers RFP and GFP we could theoretically detect low dissolved oxygen levels in water samples. In addition, we intend to use a second construct with an Iron promoter to detect iron concentrations that also endanger the sustainability of living organisms in the lake. Once we identify critical regions of the Lake, our report could incentivize the Civil Council and authorities to propose concrete legal initiatives to reduce pollution in the identified areas and start remediation campaigns. Contents HomeThe Project BiosensorEutrophicationLocalization ResultsHuman Practices Travesuras 2014Travesuras 2013Conference Data SafetyNotebook Biobricks The Team Members SponsorsAknowledgements The Project The main idea of our project is to achieve the detection of anoxia and Iron concentrations in water systems. What we propose is to construct an easy way of monitoring the levels of O2 and Fe in the lake by using biosensors. By using modified bacteria E. coli for this, we will try to find a cheaper, easier, and faster way to detect the problem of anoxia and iron concentration in some aquifers of Mexico City. We will have to analyze samples of water at different depths to know where the problem is worse and what probable native species could be more affected. Our purpose is the identification of adequate dissolved oxygen levels for a stable support of life and the identification of iron concentration below threatening levels in order to know if the lake has the physical and chemical properties to support wild-life naturally. With the use of biosensors, specialized for detecting concentrations of oxygen above a 2% dissolution, we used modified E. coli with an oxygen promoter (BBa_K258005) that will detect the low concentrations and a reporter of (GFP or RFP) that will indicate the activation of our promoter (Figure 1). The Iron promoter reacts inside an environment with a concentration of iron ranging from 1 ppm and on (A. Quintero,2007). The acceptable levels of iron in drinkable water are lower than 0.5 ppm (WHO, 1996). To achieve the objective using E. Coli we will construct different types of modified plasmids for our bacteria to express the biosensors based on iGem biobricks. The idea is to use sensitive promoters: one for oxygen, and another one for iron; those promoters will lead to an expression of GFP or mRFP. This will provide a visual signal to indicate the presence or absence of these elements. Biobrick parts BBa_K258005 (O₂ prom), BBa_I765000 (Fe prom), BBa_E1010 (GFP) and BBa_J04650 (mRFP) were selected for the construction of the biosensors (Figure 2). These were transformed into E. coli strands DH5-a, TOP 10, and NEB 10-b for storage and subsequent plasmid growth and isolation using a Zymo Research® DNA extraction kit. Once we measure and identify critical regions of the Lake, our report could go directly to the Citizen Council for its consideration. The competent authorities should be able to propose concrete legal initiatives to reduce pollution in the identified areas and start remediation campaigns that re-establish the local aquatic environment to a stable, liveable, friendly ecosystem for the inhabitant species. Biosensor A biosensor is an instrument for the measurement of biological or chemical parameters. They usually combine biological and physical-chemical components. Generally, they consist of three parts: The biological sensor: It may be a tissue, a culture of microorganisms, enzymes, antibodies, nucleic acid chains, etc. The sensor can be taken from the wild or be a product of synthetic biology.The transducer: Its function is to bind the other two elements and translate the signal emitted by the sensor.The detector: It can be optical, piezoelectric, thermal, magnetic, etc. The most common example is a biosensor that measures blood glucose. It uses an enzyme that processes glucose molecules, releasing an electron for each molecule. Said electron is collected at one electrode and the electron flow is used as a measure of the glucose concentration. The caged canaries used by miners to detect the presence of lethal gases can be seen as an early example of biosensors (Wikipedia, 2014) By using modified bacteria E. coli for this, we will try to find a cheaper, easier, and faster way to detect the problem of anoxia and heavy metals in some aquifers of Mexico City. We will have to analyze samples of water at different depths to know where the problem is worse and what probable native species could be more affected. Some of the benefits of using biosensors instead of other sensing methods, as observed by Ajit Sadana and AzoSensors, are: A fast response in time.Fast and continuous measurement.High specificity because of its shape-specific recognition.Simplicity in its use.Capability of measuring concentrations ranging from 10-18 to 10-19 M, so we need low sample requirements.Capability of real time measurements. Eutrophication Eutrophication is the process by which the increased availability of one or more limiting growth factors needed for photosynthesis cause excessive plant and algal growth. Some of these factors are the amount of carbon dioxide, sunlight and nutrient fertilizers. The elements coming from the nutrient fertilizers that especially affect the photosynthesis rate are nitrogen and phosphorus. (Chislock , 2013) Plants require many different nutrients or components for the realization of photosynthesis. Nitrogen and phosphorus are the first components depleted in the water even though there is a greater amount of other needed substances.  While performing photosynthesis about 8 times more nitrogen is needed than phosphorus. Thus, phosphorus limits eutrophication if nitrogen is more than 8 times abundant as phosphorus, while nitrogen is the limiting factor when its concentration is less than 8 times abundant as phosphorus. Erosion of surrounding areas is also an important cause of eutrophication because the nutrients of the ground are not retained by the roots of plants and trees that should be there. So deforestation is an environmental element that strongly affects this process.  (UNEP, 1) The process of eutrophication of an aquifer occurs naturally over centuries as they are filled with sediments, abundant in nutrients (figure 1). However, this process has been recently much accelerated due to the contamination produced by human activities. The discharges into aquatic systems bring a lot of limiting nutrients for eutrophication, including nitrogen and phosphorus. These polluting human residues thrown up into water systems come from point and non-point pollution sources. (Chislock, 2013) Figure 1. Natural eutrophication      The term “point source” is referred to as any single, discernible source from where the polluting agent is originated, such as a discharge pipe from a factory, sewage plant. The other term “non-point source” means that the pollution does not come from a single determinate source. This type of pollution happens when water moves across the land and pick in its way human-made pollutants that can be deposited later on in water bodies. (Harvey, 1)     There are different levels of eutrophication according to how severe or advanced the process is. The first and harmless classification of eutrophication is the oligotrophic, where there is a low concentration of nutrients in the water and thus less biologic production. Then we have the mesotrophic where there are intermediate levels of nutrient concentrations and there is a moderate biologic production that doesn´t affect severely the aquatic environment. The real problem begins when we get to the eutrophic level where there is an elevated concentration of nutrients and a very high biologic productivity.  Another classification is reserved for where the nutrient levels reach extremely dangerous concentrations that take the aquifer´s condition to a critical state; it is called hypertrophic and is almost always caused by the cultural eutrophication. An important indicator for the eutrophication level is chlorophyll. The total amount of chlorophyll represents about 1% of plant biomass, so in this way the total biomass can be estimated allowing the determination of the degree of eutrophication. (Mazzeo, 1) Table expressing the characteristic values for each of the eutrophication classifications. (UNEP) Eutrophication brings a lot of complications to aquifers. The enormous creation of dense blooms of noxious, foul-smelling phytoplankton reduces water clarity and harms water clarity. These blooms limit light penetration to the water body. This limiting of sunlight to littoral zones causes the die-offs of the great amount of plants and algae that grew up without control due to eutrophication. When these dense algal blooms eventually die, microorganisms start the decomposition of organic matter and severely deplete the available dissolved oxygen, causing hypoxia or even anoxia. These hypoxic environments are cause of dead zones for most of the inhabiting organisms for the lacking oxygen. (Chislock 2013) The normal levels of dissolved oxygen in water for the maintenance of life are around 6mg/L. Environments are considered hypoxic when the concentration of dissolved oxygen goes below 2.8 mg/L. When the dissolved oxygen levels reach the hypoxic condition many species die. Depending on the size and other characteristics of the organisms, the limiting concentration for survival will have low variations. The hypoxic conditions can change in different lapses of time. They can occur just for a few moments (minutes/hours) or they can reach chronic states that last for weeks or even months, causing depletion of local species. (Cisterna 2008) It is important to supervise aquatic environments conditions´ to prevent the initiation of eutrophic conditions. Eutrophication can kill all life in natural environments. If some symptoms of eutrophication are detected in time it is possible to attack the problem and control it, or even eliminate it. Some methods for controlling eutrophication are: Covering sediments, preventing release of nutrients.BiomanipulationUsing chemicals such as copper sulfate to kill excess of algaeAerating the hypolimnion of a lake, reducing the release of nutrients from the sediments. (UNEP) Localization First of all, we have to know what eutrophication is, well the  eutrophication is the process of excessive growth of algae and weeds water in the water, caused by phosphates and other pollutants discharged to waters. In a eutrophic aquatic ecosystem two things happen: more oxygen is required to break down and increases the population of organisms known as primary producers: organisms that make photosynthesis, as macroalgae and lilies. These can reach atrophy processes exchange of oxygen and water flow. The liquid is cloudy and the lack of oxygen can devastate populations of various organisms. How does it affect the lake of Xochimilco? In hydrological Xochimilco area that is located south of the metropolitan area of ​​Mexico City's 189 miles of canals that have been contaminated by the contribution of sewage, domestic waste, industrial, agricultural and drainage system leaks. This has affected different species of living beings that inhabit the area, causing a decline in biodiversity (Juárez-Figueroa et al., 2003). It is estimated that close to urban centers or agricultural nutrient input to a lake can be accelerated by the activities human, a process known as cultural eutrophication, which is the case with Lake Xochimilco to be within the city of Mexico. This is precisely effluent mainly caused by plants sewage treatment, containing nitrates and phosphates runoff of fertilizers and waste animals and accelerated erosion of eutrophication .The eutrophication derived from crops by the recent addition of phosphates and nitrates, as a result of activities human, it is also a serious problem for lakes, especially Lake Xochimilco. During warm periods, the overhead produce dense growth nutrient vegetables such as algae, cyanobacteria, water lilies and duckweed. Oxygen dissolved in the surface layer of water is near die exhausted when large masses of algae, which fall to the bottom and are decomposed by aerobic bacteria. This can kill fish and other animals water they consume oxygen. If theexcess nutrients continue to flow a body of water, the water reaches the bottom be rotten and almost unusable for living things, because bacteria take over and produce anaerobic decomposing substances with poor odors, such as hydrogen sulfide and the methane. Factors The factors affecting the degree of eutrophication are: Climate: warm climates favor the process. Shallow bodies of water and / or low flow are more conducive to the development process  Drainage Area: little tree cover subject to abundant rainfall tions favors erosion and entrainment of nutrients into the water body  Geology: In drainage areas where sedimentary rocks predominate vapor no greater phosphorus runoff. Clay soils drain poorly and also favor runoff and result in nutrient supply. The causes of eutrophication include: a) Natural:  atmospheric inputs: precipitation. resuspension of bottom sediments. release from anoxic sediments. breakdown and excretion of organisms. nitrogen-fixing microorganisms. b) Anthropogenic:  Discharges of industrial, agricultural, municipal and waste treatment plants. Deforestation increases erosion and reduces the recycling of nutrients in the watershed, increasing their income to the water body.Fertilizer applied in excess.Sewage farms (silos, drums).Septic tanks.Detergents with large amounts of phosphorus.Contribution of pollutants from rainwater.Sewer system of cities and towns do.Measures to control eutrophication include: Control of nutrient inputs: Waste treatment before being poured into the body of water.Restricting the use of phosphate detergents.Control of land use.Prepantanos: remove nutrients from waste water that are fixed in the biomass of algae and macrophytes.Physical and chemical  waste water treatment: chemical precipitation and filtration. What we found.... Elements found in the lake of Xochimilco Sample12345678MO mg/LO154251881515552952575Phosphorus mg/L5.85.67.66.723.017.115.014.2pH6.27.26.77.68.07.07.07.0Temperature °C3.83.84.83.86.97.07.07.0Precipitation21.020.022.023.021.519.821.054 Results Construction of the proposed biosensors was not completed successfully due to what appear to be flawed ligations and/or low efficiency competent cells. The necessary methodology and procedures have been laid down for further work on construction, development and improvement of the biosensor system in the future. Further work include the completion of the biosensors, proof of concept growing bacteria in absence and presence of stimuli and application of biosensors to a range of samples from specific points distributed all along Xochimilco lake. Results of these tests could be then applied in the construction a map of the lake specifying the most critical points that need to be treated. Agarose gel showing the results from restriction reactions. We used a 1kb DNA weight ladder (5) for measuring our fragments. The restriction reactions from the O2 promoter plasmid (1,2) were done using Spel and Pstl. The oxygen promoter has a ~ 155 bp weight. The restriction reactions from the plasmids containing the GFP were cut with Xbal and Pstl (3,4). GFP can be identified by the weight of 761 bp. The iron promoter plasmid is cut with Spel and Pstl with a weight of 1067 bp (6). Finally RFP plasmid was cut using Xbal and Pstl with a weight of 747 bp (7,8). We can notice that the oxygen and iron promoters are cut with the same restriction enzymes, as the GFP and RFP also are also cut in the same restriction sites. We decided to use the higher bands in 1,2,6 corresponding to the oxygen and iron promoters as the receptor plasmids for our subsequent ligation reactions. The lower bands located in 3,4,7,8 were cut and used as inserts inside the oxygen and iron promoters in the ligation reactions. Figure 1. The agarose gel shows the unsuccesful ligations, represented on the right. Human Practices Travesuras 2014 GenetiX, as a student group, participated in the event Travesuras 2014, which had place the 26 of April 2014 inside the installations of Tecnologico de Monterrey, Campus Ciudad de Mexico. The GenetiX station was located in the high-school chemistry laboratory where 4 groups attended, composed around of 25 children each, with ages that ranged between 7 and 16 years. Inside the station there were nine of our members present involving children into science in a fun and enriching way for them. These experiments included de polymerization of “Slime” with color and “Silly Putty”. Before initiating the experiment, a quick explanation of the process of polymerization was given. For the polymerization of the Slime, the kids had the opportunity of adding in plastic cups 10mL of polyvinyl alcohol and 3 mL of a borax solution in addition with a drop of colorant. Later, the children agitated the mixture with wooden sticks until it took the slimy consistence expected. This procedure was made in groups of between 2 and 3 kids who shared the slime. A polymerization of the silly putty was made in the same way in which the slime was made. In this case, 10 mL of white glue (which contains the polyvinyl acetate) and 20 mL of saturated borax solution. The children agitated the mixture until it obtained the desired consistency. Later, they were able to play with it also in groups of 2 or 3 children. All the experiments were supervised by the members of GenetiX in order to prevent any accident and solve further doubts from the children regarding the experiment. Finally a DNA demonstration took place. For such, a banana was cut into pieces and got liquefied in a blender with distilled water and 2.5 gr of NaCl. It was liquefied until a dense liquid was achieved and then poured into a coffee filter inside a glass flask. We left the mixture in filtration for about 10 min until the flask was half-full. Afterwards 5ml of liquid soap was added to the pulp in order to break the cell walls and membranes and free the DNA. Also 3 gr of meat tenderizer and another 3 grs of sodium bicarbonate were added into the mixture. Once the ingredients were added, the sufficient amount of cold ethanol was added in order to form a thin layer above the banana mixture. Since the DNA is insoluble in ethanol, a whitish layer appears above the banana mixture. Once the process ended, the final mixture was shown to the children and a brief explanation of what DNA is was given. Travesuras 2013 GenetiX firstly participated in the Christmas themed Travesuras 2013. In this event, kids from other schools raging from Elementary schools to Middle Schools attended to be part of various activities made by us, students from the Tecnologico de Monterrey, surrounding the topic of Christmas. We were mostly involved in helping prepare the decoration of the main stations. Some of the activities that we participated in was the making of cardboard spheres and decoration of said materials, including decorations such as Christmas trees, hanging colored paper in patterns and the working tables as well. We also helped to cover the floor for decoration and prevent further stains in the real floor. We were tasked with watching and guarding the kids during the whole event and also to entertain them. Conference As part of our Human Practices, on June 14th 2014, we gave a presentation for students and teachers from Tecnológico de Monterrey, Colegio Lestonnac and other Middle School and High School level institutes. As students and teachers who are interested in Science were invited, their participation was very dynamic. There, we explained basic concepts of Synthetic Biology and Biotechnology to the audience. Our main topic was Synthetic Biology and its present and future applications. But, in order to transmit our message clearly, we had to start from the basics; that is why, one of our contents (the first one to be presented), were the characteristics of living beings. In this point, we stated that a living being has complex and organized systems, have a metabolism, need to maintain homeostasis, can grow, can reproduce, respond to stimuli, and are adaptable. We gave the definition of Synthetic Biology as the design and construction of biological parts, devices or systems, or the re-designing of existent natural biological systems in order for them to be useful, and we also explained the techniques for the creation of synthetic organisms, such as acceleration of heavy particles, electroporation or heat shock.  In order to complement the concepts, we presented some examples of synthetic organisms and their functions, such as water pollution-absorbing plants, trees that change their color in case of a chemical attack and goats that can produce spider web. Afterwards, we explained our participation in the iGEM contest, described our project to the audience (who found it interesting and useful) and performed an experiment which consisted in extracting DNA from bananas to stimulate their scientific curiosity. Data Safety   Important Definition: Biohazard: An agent of biological origin that has the capacity to produce deleterious effects on humans, i.e. microorganisms, toxins and allergens derived from those organisms; and allergens and toxins derived from higher plants and animals. Biosafety: The containment principles, technologies and practices that are implemented to prevent the unintentional exposure to pathogens and toxins, or their accidental release Biosecurity: Control of accidental and deliberate release of biohazardous material Safety Questions. Would any of your project ideas raise safety issues in terms of Researcher Safety: All team members had to participate in a general health and safety induction, where we learned handling biological material, aspects on chemicals, guidance in waste disposal of sharps, trace chemicals, and biohazardous material and the general protocols of the lab we work in. At all times while working in the lab, we were supervised by our advisor, part of the High school Science Department, or lab technician from the university’s School of Life Sciences Laboratory. The lab we work in is classified as BSL 1 (biosafety level 1), according to the European Union Directive 2000/54/EG. Work inside a BSL 1 lab does not involve the use of potentially harmful materials to the researchers if they act corresponding to the general precautionary measures. Researcher should wear a lab coat, safety glasses and gloves and one must not drink, eat or smoke whilst working at the bench. The safety degree of the worn protection should depend on the chemicals and microorganisms handled. An important example is handling antibiotics and DNA coloring agents with gloves and safety googles. Most importantly, everybody should always be aware of what he is doing, with what kind of biological parts and chemicals he is working and how to handle them safely.Public Safety: We used different bacterial strains throughout our project. E. coli 10 beta, Top 10 and DH5 alpha; which are non-toxicogenic, disabled, non-pathogenic, non-colonising, laboratory-adapted K12 strains, which are widely used in research and present no hazard to human health. Environmental Safety: In the lab, waste must be contained in a biohazard box with an autoclavable biohazard bag. Liquids must be inactivated either via chemical methods (e.g., with bleach) or using an autoclave. Solids that have been in contact with biohazardous materials should also be treated by autoclaving and then transfered into a different bag to indicate that the waste has been deactivated. Broken glass and needles should be disposed in a sharps container. Full and sealed sharps containers can be added to solid waste. References: Chart, et al (2000). An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5a and EQ1. Journal of Applied Microbiology, 89, 1048-1058. URL: http://ors.uchc.edu/bio/resources/pdf/3.6.1.A_colipath.pdf Escherichia coli K-12 Derivatives Final Risk Assessment. Biotechnology Program under the Toxic Substances Control Act (TSCA). URL: http://epa.gov/biotech_rule/pubs/fra/fra004.htm Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?Did you document these issues in the Registry?How did you manage to handle the safety issue?How could other teams learn from your experience? No extra safety issues were detected during the construction of our biosensor.  The secondary ecological consequences will have to be assessed once the system is applied. Is there a local biosafety group, committee, or review board at your institution? What does your local biosafety group think about your project? The school has a person in charge of Health and Safety for each of the Laboratories. The technician was in constant contact with the team, supervising that the rules were being followed. We also adhered to our countries legislation in the Chamber of Deputies (the Law of Biosecurity for Genetically Modified Organisms, Nueva Ley DOF-18-03-2005 http://www.diputados.gob.mx/LeyesBiblio/pdf/LBOGM.pdf), the SAGARPA and the Law of Science and Technology from CONACyT. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? In the assembly and distribution kit the iGem could include safety equipment such as a Laboratory Health and Safety Manual, gloves and googles samples. In the case of our project, we could include two resistance markers so the bacteria, if accidentally released, had less probability of subsisting. Notebook Protocol 1 Preparation of culture media (1L). 1.- 5 g of yeast 2.- 10 g of sodium chloride. 3.- 10 g of triptone. 4.- Add water till 1 uL volume. Protocol 2 Competent cell´s preparation 1.- Centrifug your media culture tube (25 mL) at 4,000 rpm during 15 min. 2.- Pour the supernatant and add 10 mL of cold MgCl2 solution. Centrifuge them at 4,000 rpm at 4 °C during 15 min. 3.- Pour the supernatant and resuspend the pellet in 40 mL of cold CaCl2 solution. Keep it in ice during 20 min and place them in eppendorf tubes. Centrifuge them at 4,000 rpm at 4°C during 15 min. 4.- Pour the supernatant and resuspend the pellet in 40 mL of cold 85mM CaCl2/15% glycerlol v/v solution. Centrifuge again at 2,100 rpm at 4°C during 15 min. 5.- Pour the supernatant and resuspend in 2 mL of cold 85bmM CaCl2/15% glycerol v/v solution. 6.- Aliquote in tubes of 100 uL previously frozen and store at -80°C. Protocol 3 DNA extraction from Buffy coat: 1.- Centrifuge 5 mL of culture medium at 1,500 rpm for 10 min at 4°C. 2.- Carefully transfer 600 uL of buffy coat to a microtube. 3.- Add 1 mL of RBC buffer and mix throughly by vortex. 4.- Centrifuge at 4,500 rpm during 30 s and pour carefully so your pellet is not wasted. 5.- Repeat steps 2 and 3 adding just 500 uL from RBC buffer. 6.- Once separated, add 300 uL of lysis buffer to the pellet and mix by vortex. 7.- Add 150 uL of protein precipitation solution. 8.- Centrifuge at 11,000 rpm during 5 min; then, transfer the supernatant to a new microtube. 9.- Add 700 uL of cold isopropanol and mix by inverting until you see a white formation within the tube. 10.- Centrifuge at 11,000 rpm during 4 min and pour carefully taking care of the pellet at the bottom. 11.- Add 500 uL of cold ethanol 70% to wash and then centrifuge at 11,000 rpm during 4 min. 12.- Pour and let the ethanol evaporate at room temperature. 13.- Resuspend the DNA pellet with 12 uL of TE buffer; incubate all night at 4 °C. Protocol 4 Electrophoresis: 1.- Prepare 1L of TAE 10X (48.4g of Tris base[ tris (hidroxymethyl) amminomethane], 11.4 mL of glacial acetic acid (17.4 M) and 3.7 g of EDTA then fill with deionized water to 1 L. 2.- Prepare a 50 mL solution of 3% agarose using TAE buffer (without using water). 3.- Heat the gel in the microwave in intervals of 30 s, 20 s and 10 s until the agarose is completely dissolved, taking care that it does not boil so much. 4.- Let cool down the gel (55-60°C) and pour in a base with the comb already on its place to form the charging wells. 5.- Wait until the gel solidifies. 6.- Remove the comb and place the base with the gel inside the electrophoretic chamber. 7.- Add TAE buffer until the gel is covered. 8.- Take 5 uL of each sample from the PCR and 1 uL of charging gel in a new PCR tube, mix well. 9.- Charge the samples inside the wells of the gel, adding a weight marcker. 10.- Close the electrophoretic chamber and run at 120 V during 30 min. Follow the displacement ofthe samples. 11.- Take out the gel from the chamber very carefully and place in the UV light to see the DNA strands. Protocol 5 Competent cell transformation: 1.- Place 1 uL of the plasmid in a microtube, gently add 100 uL of your competent cells. Take another tube and place only 100 uL of competent cells, so you have a control group. 2.- Mix gently with a pipette and incubate during 30 min in ice. 3.- Ab ruptly place your cells in 42°C dry bath during 2 min and replace in ice whwn finished. 4.- Add 900 uL of LB medium and incubate at 37 °C for 30 min. 5.- Inoculate with 200 uL of your transformed cells in LB-Amp. 6.- Incubate overnight and store at 4 °C of freeze in 15-25% glycerol. Lab archives// Mar 12, 2014 In order to grow our bacteria, we inoculated with 100 uL of E. Coli DH5-alpha in 50 mL of LB medium, and 100 uL of E. Coli TOP in 50 mL of LB medium. We left both samples incubating overnight Lab archives// Mar 20, 2014 We inoculated E.Coli NEB10-beta in 50 ml of LB medium. Lab archives// Mar 27, 2014 We prepared the medium in which our bacteria will grow, for that we dissolved solid LB with agarose, then we add it into 10 plates, we waited a few minutes for it to solidify and finally we added 500 uL of our antibiotic that is chloranphenicol to each of those plates. Lab archives// Apr 01, 2014 we transformed luciferaze from DNA to cells by incerting them with change of temperature Lab archives// Apr 02, 2014 We made a litter of agar and 500 L of growth medium, and we sterilized them along with crystal media disperssion balls Lab archives// Apr 03, 2014 Today we prepared buffers for home made mini preps and did a cell re culture. Lab archives// Apr 07, 2014 We added 700 mL of water in a beaker and then we prepared 23 grams of powdered agar that we added slowly while we moved the beaker. This in order to help the dissolve the powder. After that, we added other 300 mL to the solution and finally we passed it to an Erlenmeyer matrass. Also we prepared 500 mL of growth medium. First, we measured and added 5 grams of tryptone, 5 grams of yeast and 10 grams of sodium chloride. Those ingredients were added slowly to a graduated cylinder with 350 mL of wáter while the flask was moved. After finishing the addition we added other 150 mL of water. The solution was then transfered to another Erlenmeyer flask. Both Erlenmeyer flasks and a glass bottle with crystal media diperssion balls were sterilized with temperature and pressure. Lab archives// Apr 02, 2014 300 ul of ampicillin at 100mg/ml chloramphenicol 3 Plates with agar Promoters: BBa_K258005 (pO2), BBa_176500 (pFe) BBa_J52008 (luc-3) The plates were impregnated with ampicillin promoting bacteria in the following manner |pO2 | Plate | 150 ul Ampiciline | K258005 | ------------------------------------------------------ |pFe | Plate | 150 ul Ampiciline | 1765000 | ------------------------------------------------------ |Luc-3 | Plate | Chloranphenicol | J52008 | Apr 08, 2014 **/Today we realized the mini prep for the oxygen promoter. /** --500 ul of Ampiciline //this is for the oxygen promoter --50 ul of oxygen promoter --45 ml of LB medium --at room temperature 15 minutes at 1500 rpm in the centrifuge. --500 ul of chloramphenicol // this is for the red liciferase --50 ul of red luciferase --45 ml of LB medium --The new LB medium was prepared with //LB medium --10 g yeast --5 g salt --15 g Tryptone MINI PREP First 400 ul of Genomic Lysis Buffer to the 40 ml overnight [bacteriaand oxygen promoter] Then the mixture is transferred to a Zyne-Spine Collumn in a Collection Tube. Centrifuge 10,000 rpm /1 min Transfer the Zyno Spin Column to a new Collection Tube. Add 200 micro liters of Pre Wash Buffer. Centrifuge 10,000 rpm /1 min Add 500 uL of of g-DNA Wash buffer to the spin column. Centrifuge 10,000 rpm /1 min Transfer the spin column to a clear micro centrifuge tube. Add 50 uL of DNA Elution Buffer . Incubate 2-5 minutes to at room temperature. Centrifuge at top speed per 30 seconds. Then store at -80 °C Apr 22, 2014 We prepared 2 overnight cultures (45ml of LB medium): GFP E0040 pSB1A2 with 100 uL of ampicillin. M-cherry (RFP) BBa_J04450 pSB1C3 with 500 uL of chloramphenicol. Apr 24, 2014 2 Transformations: Bacteria NEB 10 Beta, DH5alpha 2 Mini Preps: GFP, RFP //Transformation and preps:-- Centrifugation at 2500 rpm for 15 min.--1ul DNA in 100 uL of bacterial culture (luciferase). --Filtering GFP, RFP bacteria. --Added 400 ml of Lysis Buffer Genomyc each crop by columns. --Centrifugation at 10,000 * g for 1 min. --Last column to another tube and DNA Prewash + 200 uL Buffer. --Centrifuged at 10,000 * g for 1 min. --Add 500ul of DNA Wash Buffer, centrifuged at 10,000 * g for 1 min. --Transfer to clean tube, add 50ul of DNA Elution Buffer. --Incubate for 2-5 min at room temperature, centrifuged at maximum speed for 30 sec. Apr 28, 2014 Today was prepared LB media. It also was done the sterilization of 1 mL and 200 uL tips, water and ependorfs. After the sterilization of the LB media was done, we cultivated in it the bacteria carrying the oxygen promoter, RFP, and GFP. Then we left the bacteria growing in the incubator at 37°C overnight. May 05, 2014 Plating bacteria with GFP, RFP, promoter Oxygen and iron promoter plasmids. Everyone in their respective environments and specific antibiotics (ampicillin and chloramphenicol). Tubes with bacteria was added and placed them in LB medium incubator to grow more product if necessary. May 08, 2014 We did a centrifuge at 4800 rpm for 20 min for the plasmids had been in overnight which had oxygen promoter, GFP and RFP to rush after we separate the aqueous phase. May 19, 2014 We did inoculation of GFP, RFP and Oxigen Prmoter May 20, 2014 We started minipreps preparation of GFP, RFP and Oxigen Promoter. We added 1.5 ml of culture to each Eppendorff (4 for each promoter) and we centrifugated it at 13.2 rpm for 10 minutes, we did the same procedure 2 times. Also, we sterilized two Erlenmeyer flasks and 40 Eppendorffs. May 20, 2014 //Mini prep preparation 1. We added 1.5 mL of the cultures to their corresponding Eppendorffs and centrifugated them at 3400 rpm during 10 minutes other three times. 2. Once we had a pellet we discarded the supernatant and we added 300 uL of TEG buffer (made of 100 mM of Tris-HCl pH8, 2 mM EDTA and 20% glucose) 3. We added 700 μL of NS solution (0.2 M NaOH, 1% w/v SDs) and we mixed by inversion. 4. We added 400 μL of 3 M sodium acetate pH 5.3, and we mixed by inversion. 5. We centrifugated the Eppendorffs at 3400 rpm during 15 minutes. 6. We transfered the supernatant to other Eppendorffs and we added 50 uL of cold isopropanol and mixed by inversion. 7. We centrifugated the Eppendorffs at 3400 rpm during 15 minutes. 8. We discarded the supernatant and washed each pellet with 70% ethanol and we let it dry for 15 minutes. 9. We resuspended the pellet in 500 uL of TE buffer and then we added 10 mg/mL RNAseA. 11. We precipitated the DNA with 100 uL of ethanol in each Eppendorff. 12. We centrifugated for 1 minute at 3400 rpm. 13. We got rid of the supernatant. 14. Finally, we resuspended the pellet in 50 uL of TE buffer UNAM procedures// Digestion |BSA | 1 uL | |DNA | 2 uL | |Buffer (2) | 1 uL | |Buffer Xba I+Pstl | 3.3 uL | |fe Xba I+Spef | 3.3 uL | |H20 | 5.4 uL | Gel agarose 1 kb NEB Ampyciline -> Pentneryl | ---------------Bristol - Myers Squibb | ---------------1 gram/ 3 mL | ---------------inyectable |pO 2 | 3.3 uL |1 kb marker 5.4 uL Homemade Mini prep Preparation 1.- Overnight bacterial culture of 5 mL in falkons of 50 mL 2.- Centrifugate in an eppendor of 1.5 mL and throw supernadant 3.- Add 152 uL of Buffer 1 + RNAse (11 micro-liters /1 buffer micro-liter ) **Buffer 20% Glucouse 4.- Mix via Buffer and add 150 uL of Buffer 2 *Wait 4-5 minutes* (1.5mL)Buffer 2 -> 880 uL H2O,20uL NaOH 10 N ---------------------------------------100 uL SDS 10% 5.- Mix via inversion and add150 uL Buffer 3 ------------------------------Sodium Acetate 3M pH 5 6.-Centrifuge 5 minutes. 7.-Take the supernadant to antoher tube. Add 1 mL ETOH Absolute 8.-Centrifuge by 5 minutes and decant 9.-Wash with 500uL ETOH 70 % 10.-Centrifugate 1 minute 11.-Decant and dry at 37 celsius centigrades (30 minutes) 12.-Resuspend in 15 uL of H2O Ligation Inserto 7mL Plasmid 3mL Buffer 10x 1.5mL Ligase 0.5 mL H2O 3 mL Total = 15 ml E.Coli cells transformation. To transform E.Coli cells with an exogen DNA, 200 uL of competent cells were taken they were mixed with 1-20 ug(Micrograms) of DNA that was incubated on ice for 30 minutes. This makes posible the absorbtion of the exogen DNA to the cells surface, after that, the cells are incubated at 42Celsius Degrees during 90 seconds, for then incubate them during 1 minute again in ice. THis termal shock makes posible the entrance of the plasmid to the inside of the cell. 600 uL of LB medium were added and then incubated during 60-90 minutes at 37 Celisus Degres, to do posible the segregation of the exogenic plasmid during the celular division (3 to 5 generations). Finally, Alicuote of 100-200 uL were cultivated by dimention in the LB agar medium, which ere incubated at 37 Celsius Degrees. After 15-20 hours of incuvation colonies could be observed Ligations 1# Grow h mL in overnight 2# Do minipreps 3# EcoRI and PstI restrictions. --for Fe EcoRIand SpeI O2+ GFP ---1,2,3 - 931 Fe+GTP ---4,5,6 -1,828 O2+ RFP ---7,8,9,10,11,12,13 -890 Fe+RFP ---14,15,16,17,18,19 -1,787 GFP -> 761 bp EcoRI Po+I // 728 bp XbaI SpeI RFP -> 714 bp Xba - SpeI // 747 bp EcoR - Ps+I pFe -> 1,067 bp EcoRI - SpeI pO2 -> 178 bp EcoRi SpeI // 145 bp Xba - Ps+I O2 + GFP 1/1 - 1/2 - 3/3 FO 2 + GFP 4/1 - 5/2 - 6/3 O2 + RFP 7/1 - 8/2 - 9/3 - 10/4 - 11/5 - 12/6 - 12/13 Fe + RFP 13/1 - 14/2 - 15*16/3 - 17/4 - 18/5 -19/6 Digestion 2 --- BSA --- 1uL 4 --- DNA --- 2uL 3 --- (M) Buffer // Enz (0.3 uL each one)--- 1uL 5 --- EcoRI --- 0.3 uL --- PstI --- 0.3 uL 1 --- H2O --- 5.4 uL Total == 10 uL rxn GFP 761 bp RFP 742 bp pFe 1,067 bp pO2 -15.5 bp pO2 + GFP 931 bp pFe + GFP 1,828 bp pO2+ RFP 890 bp pFe + RFP 1,787 bp Gel 1 = 8/9/10/Lodder 1kb/11/12/13 Gel 2 = 15/16/17/Lodder 116/18/19 Gel 3 = 1/2/3/4/Lodder 1kb/5/6/7 Biobricks Biobrick name Developer TeamDescriptioniGEM Contest BBa_K258005: Oxygen promoter-Vitreoscilla hemoglobin(VHb) promoter iGEM09_METU-Gene The expression of this promoter is regulated by the presence of oxygen. Made withthe purpose to characterize the response of the promoter to changes in oxygen availability in the environment. IGem 2009 BBa_I765000: Iron Promoter iGEM07_Colombia_Israel The expression of this promoter is regulated by the presence of iron in its environment. IGem 2007 BBa_E0040: Green Fluorescent Protein Antiquity Extracted from the jellyfish Aequeora Victoria. This reporter protein emits a green fluorescent color when exposed to ultra violet light. IGem 2004 BBa_E1010: monomeric Red Fluorescent Protein Antiquity This reporter protein emits a red fluorescent color when exposed to ultra violet light. IGem 2004 The Team Members Jessica A. Flood G. A.K.A.: Jess Principal Instructor Jessica Flood studied Chemical Sciences at UDLA-P, México (Universidad de las Américas Puebla) and later she achieved a Master degree in Science with a specialization in Biological Chemistry at the University of Toronto, Canada. Her main focus is bioinorganic chemistry, her research being done in copper chemotherapeutic agents and interaction of metal ions with proteins and enzymes. Diego Hernández Molas A.K.A.: Molas Semester: Fifth Semester President I am an 18 years old Mexican high school student. Since I can remember I´ve always loved science and been amazed of how the world works; I’m constantly asking myself what are the possible causes of different physical phenomena around me. When I was a kid I used my saved money for buying scientific artifacts such as chemistry kits, a microscope and a telescope. My love for science first began with biology. I was simply astonished of how live makes its path through all adversities and how the diversity of all living species inhabit the world. It was at secondary school when I first formally learned chemistry. Since the first day I got involved with it and started thinking in a much smaller scale how all the world worked. I studied also chemistry by my own apart of my school classes because I was hungry of more knowledge, I couldn´t simply wait for my next day´s chemistry class. It was in high school when I participated in the regional chemistry Olympics of Mexico City and got really advanced knowledge in chemistry. In the iGEM group I am the president of the group. I began recruiting team members with my fellow teammate Gonzalo Escalante. For the team I drew out the idea of the project we were about to begin and then we began with all the research. In the team I do mainly lab work and research, but I am also very involved in almost all the other working areas. Gonzalo Escalante Sanjurjo A.K.A.: Gonzo Semester: Fifth Semester Vice President For some time I was looking for a student group focused on science, which didn’t exist. As there were no groups dedicated to this I decided to start our own along with Diego Hernandez, this is how the group called GenetiX emerged. Together we looked for more people and began to think of a project for the iGEM competition. Right now I’m the group vice-president and what I have done in my group is: I got a sponsor called Hidrosina who gave us 2000$ for our project, also I've worked in most of the administrative and practical aspects of the project. Emilio Adame Valencia A.K.A.: Grandpa        Semester: Fifth Semester I am a 17 year old Mexican high school student. I am one of the most recent members of GENETIX, where I am currently developing strategies for the promotion of our team at Tecnológico de Monterrey and other educational institutions by creating social media profiles that show our progress along with the production of the uniform by supervising the creation of our shirts (regarding materials and embroidery) and by establishing connections with authorities inside the campus. The reason I am working with GENETIX is because, even though I want to study Law, I have always found Science very interesting, I believe in the team and I care for its members. L. Daniel Aragón B. A.K.A.: Coffee Monster     Semester: Sixth Semester I graduated from high school in May, I’m 18 years old, and I aspire to become a great scientist. Although I’ve already been admitted to one of Mexico’s top universities, my goal is to leave my country and become a global citizen, dedicated to solve the most interesting and fascinating scientific challenges. I’ll be starting college next August, and although at first I had decided to apply for a degree in Biotechnology Engineering, I think I might switch to physics in the near future, only time will tell. As a member of GenetiX, I worked almost in every part of the project; whether I was helping at the lab in the university or coding for the wiki in my room, I tried my hardest to fulfill my coworkers’ expectations. I also had the honor to lead (although, I don´t think I could have done anything without all the feedback I got from my teammates) the Division of Human Practices, a task I enjoyed most sincerely. iGEM is a great project and I hope I can be part of it once again very soon. Diogo Miguel Burnay Rojas A.K.A.: Mike Semester: Fifth Semester I am an 18 year-old Mexican student. Since I was a kid I had always loved the special effects on movies and as well I have always been attracted to videogames, I have always like to be on the laboratory instead of only learning theory, I am planning to study Biomedics because it combines my love for computers and my interest of being on the laboratory . In Genetix, my current role is to create animations, 3D modeling and creating games Bernardo Esaú Castillo Montes A.K.A.: Black Semester: Sixth Semester I'm a 17 years old high school student. I got the live for the sciences because of my dad (he is a chemical engineer),I also love computers "Talk qwerty to me :D". Currently I am part of GenetiX team in the ITESM High School, my work is to help in the lab, code for the wiki and also mantain the comunication with the other iGEM teams in Mexico. I want to study Nanotechnology because I want to change the world innovating in a new field of science. I am an active inventor in Instructables, and I upload constant help codes to the page stackoverflow. Carlos Martínez Fornos A.K.A.: Charlie Semester: Sixth Semester I am a 19 year-old Mexican student. As long as I can remember, sciences were always my favorite subjects. When I was in elementary school I participated in the First Lego League International Tournament for Robotics in Minneapolis University in Minnesota. With my team, we won the second place in the project category. Even though bioengineering is one of my favorite areas of study, I plan to acquire a degree in physics in order to become a nuclear energy engineer. In GenetiX, my current iGEM team, my work has been focused mainly on research, creative contribution and laboratory work, though I have also participated in Human Practices. Luis Alfonso Soriano Pérez A.K.A.: Poncho Semester: Fifth Semester Hi everybody! I am 17 years old and I study High School. I am part of the laboratory team and the division of Human Practices. I am interested in science in general and I want to study chemistry to work in the laboratories of production and research of medicine. I have participated in several math contests since I was in elementary school, and I got a place between the best 20 in the International Science Contests of ITESM this year. I entered GenetiX because I am interested in seeing how we can modify microorganisms to improve our life and knowledge. Yael del Carmen Suárez López A.K.A.: Yayis Semester: Sixth Semester I’m a 17 years old mexican student, since I was a kid I have been interested in biological sciences. I became a diver when I was 9 years old, and I think that’s when my love for biology was born but I really started to develop a great scientific curiosity until High School; I want to study Biotechnology because I think that it’s amazing and really interesting that you can use microorganisms to help the improvement of other people’s lives. In Genetix, my team for the iGEM, I worked mainly in the lab and doing theoretical work, as well as in Human Practices. Sponsors Acknowledgements Lamberto Álvarez-Palacios M. Sc. Tec High School Head of the Science Department Monterrey Institute of Technology and Higher Education, Mexico City Campus Luis Vaca Dominguez M.D. Ph. D. Alicia Sampieri García B. Sc. UNAM, Cellular Physiology Institute     Jorge Santos Welti Chanes Ph. D. Biotechnology Doctoral Program Director Monterrey Institute of Technology and Higher Education, Monterrey Campus Daniel Rodriguez GoNext Project   OUR COMPLETE PAGE: varediadesign.co.nf      Retrieved from "http://2014hs.igem.org/Team:GenetiX_Tec_CCM1248CAPS KansasThe core of the team are high school students participating in the CAPS Program, but this year we are expanding to an after school CAPS BioClub as well.High SchoolSynthetic Production of Alkanes Derived from Yeast Pyruvate KinaseHumans rely on carbon resources for nutrition and energy. Biofuels derived from corn-based ethanol and the microbial degradation of cellulose are not fully sustainable, due to the competition of food supply and expense of input energy. Cyanbacteria are bacteria with similar features as microalge in the sense that they are known to fix carbon dioxide into alkanes through the collective processes of photosynthesis, glycolysis, and fatty acid biosynthesis. The CAPS iGEM Team 2014 continues metabolic engineering of these pathways by expressing yeast-derived pyruvate kinase, known to be a key regulator of glycolysis, within the cyanobacteria Synechocystis PCC 6803 in an effort to increase alkane production. Assays for the production of pyruvate, fatty acids, and alkanes will be used to characterize our system. In addition, we will be determining various environmental conditions that increase the effectiveness of alkane production such as CO2 and O2 limitation.1203BBa_K1248999http://2014HS.igem.org/Team:CAPS_KansasBBa_K1248000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=160120140http://2014hs.igem.org/files/poster/CAPS_Kansas.pdfHigh SchoolLog in   Team:CAPS Kansas/Project From 2014hs.igem.org " Page Discussion View source History teams Log in   Team:BV CAPS Kansas/Project From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHigh School iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFutureOld Home TeamHome 1 2 3 4 BV CAPS iGEM Tweets Thanks! Project Abstract - The Effects of Increased Pyruvate Kinase Expression on the Production of Alkanes in Cyanobacteria Humans rely on carbon resources for nutrition and energy. Biofuels derived from corn-based ethanol and the microbial degradation of cellulose are not fully sustainable, due to the competition of food supply and expense of input energy. Cyanbacteria are bacteria with similar features as microalge in the sense that they are known to fix carbon dioxide into alkanes through the collective processes of photosynthesis, glycolysis, and fatty acid biosynthesis. The CAPS iGEM Team 2014 continues metabolic engineering of these pathways by expressing yeast-derived pyruvate kinase, known to be a key regulator of glycolysis, within the cyanobacteria Synechocystis PCC 6803 in an effort to increase alkane production. Assays for the production of pyruvate, fatty acids, and alkanes will be used to characterize our system. In addition, we will be determining various environmental conditions that increase the effectiveness of alkane production such as CO2 and O2 limitation. HomeWelcome TweetsHS iGEM TeamWho we are StudentsAdvisors Project Project Overview Problems SolutionsNotebook Methods AchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEM Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/ProjectSolutions From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Solutions Attempted solutionsRenewable energy is a growing industry. This industry is anticipated to supplement and, ultimately, replace non-renewable fuels. The United States Energy Independence and Security Act of 2007 proclaimed a goal to produce 36 billion gallons of renewable fuel, with 16 billion being cellulosic ethanol, by the year 2022. This was the first major step in the advancement of biofuels.Biofuels are energy produced from renewable resources that have a balanced carbon dioxide emission-absorption trade-off. The optimal materials to make biofuels are plant biomass, vegetable oil, and especially municipal and industrial wastes. The major classifications of biofuel are biodiesel, biogas, and bioethanol. Biodiesel is made by the emulsification of vegetable oils and other fats. Commonly, biodiesel suplements petroleum-based fuel. The second class, biogas, is the decomposition of organic matter. This creates a mixture of methane and carbon dioxide that is used to generate steam and electricity. Finally, there is bioethanol. This is made by fermenting the sugars in corn or other agricultural residues. Bioethanol can be used in internal-combustion engines. Currently, the most common biofuel is corn-based ethanol. This first-generation biofuel accounted for 2.7% of transportation fuel in 2010. Ethanol, however, is quite expensive to produce and not nearly as efficient as gasoline or diesel. Another concern is the use of land to produce corn for fuel, rather than food. Second generation biofuels, those derived from lignocellulosic biomass (dry plant matter), are the most abundant carbon fuel source. Unfortunately, they, like first generation fuels, require land, energy, and nutrient investment. A major barrier to production is the high energy required to extract trapped sugars during fermentation. Even then, there is a high percentage of pentoses (five carbon monosaccharides) that are even more difficult to ferment. A low cost, low input way to create renewable biofuel is still being sought after. Third-generation biofuels are a source of hope. Many of these utilize photosynthetic microbes, like cyanobacteria. This method requires only sunlight and adequate growing condition. The microbes do not need much space or nutrient input. However, biofuel output through this method is relatively low. The possibilities here sparked our interest. Our SolutionsSince alkanes play a major role in the composition of fuels such as diesel and jet fuel, the engineering of these becomes beneficial. We propose that metabolic engineering with the use cyanobacteria strains Synechococcus PCC6801 and Synechocystis PCC 6803 and modification of the metabolic pathway glycolysis to produce alkanes (shown in the tinkercell below). These alkanes will be derived from the up regulation of pyruvate kinase. The gene for the pyruvate kinase will be takes from rabbit muscle and inserted into cyanobacteria due to the high retroactivity during production of pyruvate kinase. With the obtainment of the rabbit muscle pyruvate kinase, it will be inserted into the cyanobacteria to up regulate the production inside of the photosynthetic bacteria. This can be supplemented to decrease the dependence on non-renewable fuel sources, since this fuel is derived from a carbon-neutral source. Therefore, we plan to increase the production of alkanes by reducing the bottleneck of limited resources. By placing an overactive system in cyanobacteria, we will create more resources to create alkanes. We’ll used a broad-host range plasmid from Hawaii’s 2008 iGEM team (BBa_K125000) to be the back bone for our two highly active pyruvate genes. One is derived from rabbit muscle (BBa_K977000) and the other is a yeast mutant (BBa_K977001). The Alkane Pathway Explained Step by Step: Hexokinase, alongside ATP, phosphorylates (adds a phosphate group to) a molecule of glucose, yielding ADP Glucose 6-Phosphate Phosphoglucose Isomerase isomerizes (changes form of) Glucose 6-Phosphate into Fructose 6-Phosphate Phosphofructokinase, alongside ATP, phosphorylates Fructose 6-Phosphate, yielding ADP and Fructose 1, 6-Biphosphate Fructose Biphosphate Aldolase cleaves Fructose 1, 6-Biphosphate into Glyceraldehyde 3-Phosphate and Dihydroxyacetone Phosphate Triosephosphate Isomerase isomerizes Dihydroxyacetone Phosphate into another Glyceraldehyde 3-Phosphate Glyceraldehyde Phosphate Dehydrogenase, alondside NAD+ and H2(PO4), dehydrogenizes (removes hydrogen from) Glyceraldehyde 3-Phosphate, yielding NADH/H+ and 1, 3-Biphosphoglycerate Phosphoglycerate Kinase, alongside ADP, dephosphorylates 1, 3-Biphosphoglycerate, yielding ATP and 3-Phosphoglycerate Phosphoglycerate Mutase converts 3-Phosphoglycerate into 2-Phosphoglycerate Enolase pulls a molecule of H2O from 2-Phosphoglycerate, yielding Phosphoenolpyruvate (PEP) Pyruvate Kinase, alongside ADP and H+, dephosphorylates PEP, yielding ATP and Pyruvate Pyruvate Dehydrogenase, alongside NAD+, dehydrogenizes Pyruvate, yielding NADH/H+ and Acetyl Coenzyme-A Acetyl CoA Carboxylase, alongside ATP and a carboxyl group (H(CO3)), carboxylizes Acetyl CoA, yielding Malonyl Coenzyme-A Beta-Ketoacyl-ACP Synthase and Acetyl CoA react with Malonyl CoA, yielding CO2 and Acetoacetyl-ACP 2NADPH and Malonyl-ACP react with Acetoacetyl-ACP, facilitating hydrocarbon chain elongation, yielding CO2, NADP+, and Acyl-ACP Acyl-ACP Reductase, alongside NADPH, reduces (changes the oxidation state of) Acyl-ACP, yielding NADP+ and Acyl-AH Aldehyde Decarbonylase decarbonylizes (removes a carbonyl group (CO) from) Acyl-AH, yielding an Alkane HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Solutions" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/SolutionOutreach From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM OutreachAttributionsFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! OutreachWe held two sessions (morning and afternoon) of a four day middle school bioscience camp for 50 students. It was themed "Life: an expedition". The camp was aimed at introducing students to the diversity of life and experience the latest technologies in molecular biology using state of the art equipment. Students had an opportunity to work with organisms inside and out in their native environment. Other CAPS alumni volunteered as helpers at the camp. We taught them about DNA, synthetic biology, and ecology through hands-on activities. They learned:Laboratory equipment use including micropipetting, centrifugation, and vortexingDNA extraction Transformation of bacteria and practicing sterile techniques and streak platingGel electrophoresis of DNADiscuss current iGEM project and the establishment of an on-going club for next yearWe have also made Facebook and Twitter pages that are consistently kept up to date with everything that we have accomplished. This allow us and the other iGEM teams to keep up with are work as well as see the great things all the other teams are doing. Finally, we designed a new way to teach students 3A assembly. It is a paper activity that really forces students to grasp the new concept before they hop into a complicated lab. HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Outreach" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Outreac/Future From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Future Our plan for future projects!In the future, the 2015 iGEM team will be completing the wet lab experiments. Next year, we can make more parts with various promoters and ribosomal binding sites. To characterize the parts better, we will be using a pyruvate kinase assay and gas chromatography. We also want to examine optimal conditions of cyanobacteria, including experimentation with CO2 limitation to reroute Acetyl-CoA usage in the cell to the alkane pathway. Furthermore, we plan on making a more in-depth and accurate TinkerCell model to show the amount of resources directed into specific pathways in the cell and how we are trying to change that usage. We would also like to expand our community outreach by expanding a BioBuilder.HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Future" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/FuturTeam From 2014hs.igem.org Media:Example.ogg teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Team Students Austin Chick Austin Chick, a.k.a. Masta' Mix, has a strong passion for the world of biology. He spends the majority of his time in the lab whether it be learning the next big experiment or messing around with the 3D printer. In the short time when he is not in the lab, he spends his time stacking pears at Hy-Vee, playing video games, and watching romance movies with his girlfriend. The Masta' Mix is truly one of a kind. Muriel Eaton Muriel "Chaperone" Eaton fell in love with science through Science Olympiad in middle school. She will be a freshman at K-State this fall to major in biochemistry. When not doing science, Muriel is thinking about science. She also enjoys tearing up the floor with her expert DDR moves, dressing up her cat, reading of the marvels of chaperone proteins, cruising in her Camaro, and watching the exploits of NCC-1701. Her motto is "if you can't change it, don't complain." Chloe Ortbals Chloe Ortbals or better known as McFlow. From her Western blotting skills and to her mastery of her pipetting skils she has it all on lock down. In her free time she hangs with friends, listens to music, works at AMC and goes running on long and tiring distances. She loves animals (her favorite is a yellowlab mutt) and takes an interest in research of the cancer type. Chloe is the McFlowy. Aiswariya Ramaswamy Hello! My name is Aiswariya Ramaswamy. I go to Blue Valley Northwest, and I will be a junior this fall. I enjoy dancing, tennis, track and field. I participate in many clubs including KAY Club and Science Olympiad. My interest in life science began at a young age, and I especially like learning about biology. When I heard about iGEM, I was really excited to join and learn more about biofuels. In the future, I would like to do something in the medical field.   Nithin Saripalli My name is Nithin Saripalli. I go to Blue Valley Southwest and I am a sophomore. I am a tech enthusiast and enjoy working with technology when I am not doing science and homework. I love science because I like to know how the world around me works. I like biology because it is the study of life and it opens up a lot of opportunities. I am interested in iGEM because I like working with science. I like biotechnology and iGEM allows me to participate in Biotechnology. I would like to be in a career in Biotechnology. I do not know what college or exact career I will be in, but I still have time to decide. Alexis Vance This woman of many words is a sophomore at Blue Valley High (the Originals). This is what she has to say for herself:I have done competitive horseback riding for 9 years, I own a 5 year old Hanoverian Gelding named Encore. I love science and unhealthy amount. I wish to go into Biochemical Engineering. pGLO is my favorite plasmid.I hope IGEM will help me gain important lab experience and make important contacts in the Bio engineering world.   Tiffany ChaoTiffany Chao is a 12th grader that just graduated from Blue Valley West High School. She also has attended CAPS for four semester where she learned about biology and iGem. Tiffany is also called "Chao Chao" because she has many other friends named Tiffany. Altough she is learning about science research and is on the iGem Team, Tiffany will be going to Johnson County Community College for her undergrad and the University of Kansas to study pharmaceutical.   Team Austin Chick - Blue Valley Northwest '15Muriel Eaton - Blue Valley North '14 attending Kansas State UniversityChloe Ortbals - Blue Valley West '15Aiswariya Ramaswamy - Blue Valley NortwestNithin Saripalli - Blue Valley Southwest '14Alexis Vance - Blue Valley High'16Tiffany Chao - Blue Valley High '14(Left to right, 1st row to 2nd row) Mentors Aron Fenton Dr. Fenton researches Allosteric Regulation at the University of Kansas Medical Center. The results from his study will be used to direct future mutagenesis studies as a way of "tracing" which residues in the protein communicate the allosteric signal. To the same goal, his lab is also initiating D/H-exchange mass spectrometry experiments that will identify what regions of the protein have altered flexibilities due to the allosteric signal. Qingling Tang Ms. Tang is the chief laboratory technician in Dr. Fenton’s lab. Ms. Tang's ready smile brightens up the lab and her willingness to work brings extra positivity to the team. Instructors Eric Kessler Mr. Kessler’s interest in biology can be traced to his youth on his family’s annual canoe trips where he was obsessed with catching turtles. He has earned three undergraduate degrees, in Education from UMKC, and Psychology and Zoology from the University of Texas. These studies were followed by a Masters in Biology from Emporia State University where he completed research on the retreat-site selection of woodland snakes. He began his foray into things molecular through participating in the Age of the Genome Woodrow Wilson Fellowship 2002 and has attended all three high school iGEM competitions. He enjoys photography and spending time outdoors with his family. Joseph Whalen Mr. Whalen began his adult life in biology as a commercial fisherman in Alaska. His interest in the outdoors led him to complete an undergraduate degree in Environmental Science from the University of Kansas. From there he worked as a liaison between research and high school education as a GK-12 Fellow, and then completed three years teaching science at Blue Valley High School. Before returning to CAPS he completed his Masters in Molecular and Cellular Biology at Oregon State University, where he studied genome defense mechanisms in the fungus, Fusarium graminearum. Outside of school, Mr. Whalen enjoys rock climbing, and traveling with his wife and friends. Kelley Tuel From a young age, Mrs. Tuel has enjoyed nature, the outdoors and figuring out how things work. Her love of all things living and amusement by high school students prompted her to earn undergraduate degrees in both Biology and Education from Kansas State University. Mrs. Tuel obtained her MS Degree in Biology from Emporia State University. Her graduate work was on the molecular genetics of Halobacterium sp. NRC-1, a salt-loving Archaean, and its instructional use in high school classrooms. During her research, she isolated and named two previously undescribed mutant strains of Halobacterium. Each mutant processes a novel transposon which disrupted the buoyancy vesicle operon (gvp). Mrs. Tuel teaches Veterinary Medicine at the Center for Advanced Professional Studies. HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Team" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/TeaFrom 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Welcome Welcome to the BV CAPS page!This is the BV CAPS iGEM team’s second year participating in the iGEM jamboree. Our group is from Overland Park, Kansas where the state flower is the beautiful sunflower. Our program called Center for Advanced Professional Studies (CAPS) contains students from the five different high schools in the Blue Valley School District. At CAPS, students are able to learn in depth about a specific career path and are able to experience real-world problems. This year we are trying to overexpress pyruvate kinase in cyanobacteria to increase the production of alkanes which is one of the important biofuels.iGEM 2014 High School Jamboree000:00:00:00    Days                 Hrs               Min             Sec    We can't wait to see you all at the competition!Best viewed in Google Chrome   (Download)  HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansahttp://2014hs.igem.org/wiki/images/6/6a/Hawaiilogo.jpgAttributions From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Slideshows 20142012 Glycolysis Poem Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Attributions Academic and Corporate Supporters:Integrated DNA Technologies – making primersQiagen – plasmid extraction kitNew England Biolab – competent cells, restriction enzymes, Gibson Assembly kitPast teams:University of Hawai’i, Manoa 2008 – plasmid backbone/shuttling vectorUniversity of California, Davis 2012 – wiki formatMentors:Dr. Aron Fenton, KUMC – provided laboratory space, materials, equipment, and experienceQingling Tang, KUMC – laboratory technician, helped make the primers for site-directed mutagenesisTeachers:Eric Kessler – images, team coordinator, twitterJoseph Whalen – solicitor, guru of laboratory protocols and equipmentKelley Tuel – advisor, extremophile expert, presentation coachTeam:For the most part, we worked on everything together. Each member was "in charge" of a certain aspect. They oversaw the completion of their section by soliciting the help of others or doing it themselves. Alexis– PreziAustin– wet labChloe– wet labMuriel – general editing, wikiNithin– wikiHomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Attributions" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Attribution/Methods From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks!MethodsInserting the BBa_K977000 pyruvate kinase gene into BBa_K125000 vector required a number of different steps and techniques.Because the vector already contained a usable promoter, the main procedure involved four different methods.1. Using site-directed mutagenesis to remove a naturally-occurring PstI restriction site in the pyruvate kinase gene.2. Using PCR to “flank” the pyruvate kinase gene with the BioBrick Standard Restiction Enzyme sites.3. Using 3A Assembly to insert the new part into the BBa_K125000 vector.4. Transforming the cyanobacteria.Site-Directed MutagenesisSite-directed mutagenesis is a method for altering the sequence of DNA and can be used to produce both point and frame-shift mutations. In this case, it was possible to take advantage of codon redundancy and alter the DNA sequence without altering the amino acid sequence. It was necessary to do sitedirect mutagenesis because the pyruvate kinase gene contains in its sequence two PstI restriction sites. (CTGCAG)Because PSTI is part of biobrick assembly, it was necessary to use site-directed mutagenesis to remove these sites prior to cleaving and ligating the part. Otherwise PstI would chop the gene to pieces whenever we (or anybody else) tried to assemble anything with it.Primer design was very simple, as the primers were identical to the gene except that they contained the desired mutations. Again, the primers were about 20 bp to ensure that they would only attach to one region along the gene.Forward primerTemplate strand- 5’- ctcatctccctgcaggtgaagcag 3’ Primer -5’- ctcatctccctccaggtgaagcag 3’ (Restriction sites are in red, point mutation is in blue)Reverse primerAgain, the reverse primer necessitated the use of the reverse compliment of the original sequence. Template strand 5’- CTGCTTCACCTGCAGGGAGATGAG -3’Primer 5’- CTGCTTCACCTGCAGGGAGATGAG 3’Click here for the official Standard Registry Procedures regarding site-directed mutagenisis.PCRIn order to get the pyruvate kinase gene to conform to Biobrick Standards, it was necessary to add the four Biobrick restriction sites.This was done with a variation on PCR annealing and extension per the instructions given by the Registry. Theoretically, this method was simple. All it required was forwards and reverse primers that consisted of the BioBrick prefixes and suffixes and the first twenty base pairs of the gene.Forward primer5’- GTTTCTTGGAATTCGCGGCCGCTTCTAGATG TCAAAGTCCCACAGTGAAGC -3’ Reverse PrimerTo orient the sequence 5’-to-3’, it was necessary to use the reverse compliment of the last twenty base pairs of the gene. 5’- GTTTCTTCCTGCAGCGGCCGCTACTAGTA TTATTACGGGACGGGCACCACGCGCA -3’(Red is the tails, green is the gene) After this, it would be possible to proceed with PCR as normal, and though the region of restriction sites would “hang off” in the first round of PCR, they would be added to the gene as “flanks” in subsequent rounds as the extension stage would transcribe the flanks on as well as the gene’s DNA. However, a number of challenges presented themselves.At 51 and 55 bp, the length of the primers far exceeded the typical 40-45 bp. The long “free ends” that contained the restriction sites threatened to rip the section of matching sequence away from the template strand. It proved impossible to shorten these primers while still including the standard restriction site. Nonetheless, the primers were ordered and the experiment was attempted. After running the results on a gel, the length of the resulting fragments suggested that the PCR was successful.As for the exact procedure, the Procedure offered by the registry was used. Upon creating the part, we used 3A assembly to insert the part into the BBa_K997000 vector (It’s completely standard, so look it up!) and transformed the cyanobacteria.In the future, we might repeat this experiment with yeast, and though the sequences would be different the procedure would be essentially the same.Other Standard iGEM Protocols for Preparing the our Vector and Tranforming our Part into our Cyanobacteria ChassisPreparation of Competent CellsTransformationPlasmid Mini PrepRestiction DigestLigation3A AssemblyTinker CellA program called TinkerCell was utilized to help visualize the steps taken through glycolysis and the fatty-acid pathway to create alkanes. The image created in TinkerCell allows one to see each step in the modification of a molecule. Beginning with glucose and ending with alkanes, the image shows what the molecule becomes, which enzyme catalyzes the reaction, what type of reaction occurs, what is required for the reaction to occur, and the other products of the reaction.Asssay for Pyruvate Kinase ActivityPhotosynthetic microbes, like cyanobacteria, hold the potential to produce biofuel with only sunlight and adequate growing conditions. These microbes do not need much space or require nutrient input to create fuel. However, biofuel output through this method is relatively low, which sparked our interest.Whether it is diesel fuel or gasoline, most of the energy comes from alkanes. A cell makes alkanes by the fatty-acid pathway. A main resource needed for the fatty-acid pathway, acetyl-CoA, is produced by glycolysis. We hypothesize that if we increase the resources required for the production of alkanes, then the cell will produce more alkanes.By overexpressing the catalytic protein pyruvate kinase, we hope to increase pyruvate within the cell. This should allow for an increase in activity within the alkane fatty-acid pathway because of pyruvates conversion to acetyl-CoA. PEP phosphorylates ADP  1 molecule pyruvate + 1 molecule ATPTo determine the amount of pyruvate activity produced by our cyanobacteria (engineered for overexpression of pyruvate kinase), we must assay for pyruvate kinase. A cocktail of magnesium chloride, ADP, EDTA, bicine (buffer), NADH and lactate dehydrogenase is made. Added to the purified pyruvate from the cyanobacteria plus PEP, the lactate dehydrogenase will utilize the NADH to reduce pyruvate to lactate. The amount of lactate produced indicates the output of pyruvate from the cell and can be read using a spectrophotometer at 340 nm. Using the spectrophotometer readings and a mathematical formula that takes into account dissociation rate of PEP into pyruvate into lactate acid, our initial pyruvate amount is determined.Assay for AlkanesWhether it is diesel fuel or gasoline, most of the energy comes from alkanes. A cell makes alkanes by the fatty-acid pathway. A main resource needed for the fatty-acid pathway, acetyl-CoA, is produced by glycolysis from pyruvate. Click here to review the alkane pathway. We hypothesize that if we increase the resources required for the production of alkanes, then the cell will produce more alkanes.Once it is determined that we have sufficient pyruvate production from our cyanobacteria (as determined by the pyruvate kinase assay), we will do lipid profiling by gas chromatography to verify the lipid produced is the substrate needed for the cell to produce alkanes. Gas chromatography takes the initial compound we wish to analyze (lipid produced from our cyanobacteria with over-expression of pyruvate kinase) and binds it to the liquid on the walls of the gas chromatography column. The lipid reacts with an added inert gas. This process yields a peak in the spectrum specific to the lipid in question. We anticipate that the lipid produced by our cyanobacteria will be the substrate for the cell to undergo the fatty-acid pathway to produce alkanes.The next step will be to confirm that alkane production has been completed by the cell. Working with our mentor, Dr. Fenton, we will also use gas chromatography to verify the alkane produced.HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Methods" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Method/Problems From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! ProblemsHumans and other organisms are dependent on carbon for food and energy. With the development of agriculture 10,000 years ago, our populations grew, we migrated across the globe, and our demand for resources increased. Subsequent industrialization in the 1800’s led to an exponential increase in human population that was fuel by the discovery and use of fossil fuels. At that time, the Swedish scientist, Svante Arrhenius, in an effort to explain the ice ages, realized that atmospheric carbon dioxide could influence surface temperatures like a greenhouse. Specifically, he concluded that carbon dioxide could act as an atmospheric insulator allowing the penetration of incident solar radiation while retarding its subsequent radiation as heat from Earth’s surface. He also predicted that increases in carbon dioxide emissions resulting from the burning of fossil fuels could influence global climate. He was honored with a Nobel Prize in Chemistry in 1903. Although Earth’s temperature is known to be influenced by natural cycles associated with variation in our planets orbit around the Sun, the consensus of current climate scientists is that human actions are responsible for the current rise in Earth’s temperature. Recently, the International Panel on Climate Change (IPCC) reports that a doubling of atmospheric carbon dioxide could result in an increase in the average global temperature of between 2 to 4.5oC. Soon, we will reach an atmospheric concentration of 400 ppm CO2 which is 120 ppm higher than pre-industrial levels of 280 ppm. The Earth’s temperature has increased over 1oF in the past century, and the last decade was the warmest on record since thermometer-based observations began. Global warming is now known to increase the probability of extreme weather events, and result in rises in sea level due to the melting of glaciers and the polar ice caps. The adaptability of organisms across the globe is also being tested by these abrupt changes. With an understandable unwillingness to relinquish our current standard of living, we are increasingly testing ourselves as our population has surpassed 7 billion individuals and could possibly double by 2050. Humans are an ingenious and creative species though, and recent acknowledgement of our problems has led to research into the development of renewable, clean fuel. HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Problem/Notebook From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Notebook June 20Austin, and Nithin were the only ones to meet today and when they got to caps time seemed to fly they took the poster that had edits from the day before and started changing and fixing it. In the middle of that Nithin went off to work on the images that would be placed inside of the wiki and having to use Photoshop to do most of the work it took a long time but they were able to get a lot accomplished but no recording was done today. June 19Austin, Nithin, and Aiswariya all meet up at caps and continued recording of the video since the day before they had been taught how to do it all by Mr. Rodgers and the Film class teacher. While they were recording Austin compiled the video in Final Cut Pro X. Then after a moment of recording they had to focus time on the poster and the wiki to finish everything up and make sure the team meets the requirements. June 18Austin and Nithin meet up since Alexis couldn't make it, but a nice surprise came their way and that was another team member showed up and this was the start of their recording. It took Austin a ton of takes with his team member biography and Nithin seemed to be able to knock it out like a breeze. Though Austin did seem to have a comfortable time saying Joe Whalen's biography. June 16Austin, Nithin, Alexis all meet up at caps in the afternoon to continue their work on the presentation and the arrangement of the presentation as it follow and since Austin had to leave early the meeting was a short one but they were able to start talk of how to record it. June 15Austin regrouped with a few people from the team, which included Nithin, Alexis and Muriel (wirelessly). Once everyone talked about what needed to be done and what was done and how everything was going to get accomplished they started in to working. Austin started by writing quick short hand sentences about the project while Alexis took those sentences and expanded upon them. While they were doing that Nithin was editing the wiki and off and on helping with the presentation. June 11Austin worked on mapping out the tinkercell which is the method that we have chosen to map out the pathway that we are going to use for the alkane production. He did this is three textbooks, the internet and tons of colorful sharpies to take a piece of paper and map them out with all the reactions, inputs, outputs, enzymes and this was all done on a sheet of printer paper then transferred into tinkercell June 6The iGEM team meet today again to start continue talking about wither we were going to be a site team or if we would have enough representation to go to Boston. The majority thought we should be a site team. Then the leaders of the project began to talk more about the project and help everyone gain an understanding of it. Shortly following that Mr. Kessler got us all to do our team member biography. May 30Today marked another day when the team meets up at Bioclub and worked on the wiki, the wiki workers were comprised of Nithin, and Muriel. The other worked on a team song, outreach, and fundraising where they started getting ideas for a smaller presentation where they could send it to a potential donator for them to get an idea of the project. May 26Today Nithin and Muriel spent time looks at last year’s wiki and content so they had a base reference point and then proceeded to create pages, and images for the wiki that could be used as headings. May 23Today was our meeting without mentor Dr.Fenton! We had a lot of fun going up there but it was just Austin, Chloe, Mrs.Tuel, Mr.Kessler, and Mr. Whalen. We talked a lot about the pathway and the different steps that we would have to go through to complete the project. He also was able to answer some of our question about the plasmid that we have, the production rates, and other confirmations. May 20Today the team meet up again at bioclub to talk about more in-depth process like the phosphorylation and other reactions that happen in the metabolic path way and Austin felt like he over described it at every single step but even if they didn't fully understand it they got the main picture. May 8This was brainstorming session where the team got together as a whole to find ways to fund raise, update each other on the wet lab and how it should all play out if everything goes as planned, and to make sure everyone has their forms in so Mr. Kessler can send them off to Boston so we can access out wiki. March 4This was one of our first bioclub meet ups where each one of us began signing up on the igem site and wait to be approved, and Austin gave an excellent speech for everyone on the simple parts of the project which helped gather interest and make things just slightly more clear for what we’re doing. February 24Potential team members from the research and molecular class visited our mentor, Dr. Fenton, to discuss the course of the project and review basic metabolism. December 11Mr. Kessler started talking about fundraising and all the people that he could send things off to and how he does it. He also handed out a packet on what a good poster looks like but Austin still thinks that his Anthrax poster was the best that they have ever seen. Though most people did generally agree that the poster should tell the overall story. HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Notebook" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/NotebooSafety From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Safety Here at CAPS, we make safety our number one priority when we plan and execute our experiments. Situational awareness and communication is essential for any success. When using cyanobacteria or chemicals, we always work in sterilized environment. It’s important not to contaminate our sample with ourselves or surroundings and not to contaminate us with our sample! At all times, we wear our personal protection equipment (PPE) which consists of disposable gloves, lab coats, and eye protection. We follow a safety handling protocol suggested by one of our mentors to safely and efficiently handle cyanobacteria. A mentor or qualified adult is always present to be an extra set of eyes. We know what we are going to do before we set foot in the lab. Researcher SafetyEveryone on the team obtained laboratory safety training through the Machinery of Life/Foundations of Molecular Medicine and Bioengineering CAPS Class and had experience with bacterial culture (in E.coli) and genetic transformation (inserting pGLO into E. coli) prior to beginning the project. During the work, our team took the following safety precautionsLab coats, gloves and goggles were worn at all times. We were supervised in both the CAPS lab and at Dr. Fenton’s lab down KU Med. All equipment (both disposable and autoclaved) was sterile. The bench and other work surfaces were sterilized with water and bleach before and after lab work. Because cyanobacteria can pose a risk if ingested, there was no food or drink in the lab. There were no lit burners or other fire during lab work. Organism and Part SafetyBecause our project utilizes the mixotrophic cyanobacteria Synechocytis PCC6803 and E. coli (two BSL-1 organisms) safety risks may initially appear to be minimal, almost nonexistent. Neither of our organisms are pathogenic. However, upon closer examination, our project contains elements that call for considerable thought towards safety. First, we recognize that cyanobacteria are autotrophs and require little other than water and light to survive. Therefore, if we did not practice sterile technique, it would be possible for cyanobacteria to be dripped or spilled and survive. Cyanobacteria produce enzyme-inhibiting toxin that can affect the nervous system, immune system, and liver. These toxins have killed waterfowl and marine mammals that drank from algal bloom-contaminated water. However, the worst cyantoxins, cyclic petides, alkaloids, and ketides are not produced by our chassis, Synechocytis 6803. Alkanes are biologically inactive and impose a small but present risk. Many of the alkanes that are gases or liquids at room temperature (most notably, ethane, methane, propane and butane) are flammable. These gaseous alkanes are not toxic and would only pose a danger if we breathed them in such high concentrations (which would have to be twelve to sixteen percent) that we suffered from oxygen deficiency. Environmental Safety However, the main risk deals not with toxins themselves but with the dangerous of cyanobacteria to the environment and the risks our altered Synechocytis PCC6803 might pose to the ecosystem. Besides the inherent risk of introducing any genetically modified organism into the world outside the laboratory, we recognize that our Synechocytis 6803, like any cyanobacteria, could become the culprit in an algal bloom. Per iGem safety suggestions, we consider the worst-case scenario. Harmful algal blooms (HABs) are essentially proliferations (often as dense as millions of bacteria per milliliter) of cyanobacteria in salt or freshwater. The water may appear entirely green and become so turgid that animals that hunt by sight cannot see more than a centimeter or two. The cyanobacteria may consume all the oxygen in the water, leaving none for other organisms, and when they die, they sink to the bottom of the body of water, and can suffocate organisms there. Though some algal blooms can be natural (for example, those that are believed to be related to El Nino) many are the direct result of human eunutrition. Runoff from fertilizers puts high levels of phosphates and nitrogen into water, creating an ideal environment for cyanobacteria growth. If our alkane-producing cyanobacteria were to be the culprit in an algal bloom, the effects might be worse than if an unaltered cyanobacteria proliferated in the bloom. Because alkanes are a major component of gasoline and diesel, the effects might be similar to an oil spill. Furthermore, certain bacteria and archea are able to metabolize alkanes, possibly leading to an out-of-control proliferation of these organisms. Ethical SafetyWe recognize that our yeast pyruvate kinase gene was isolated by Dr. Fenton and that we only get to use it per his permission. Accordingly, we have kept him informed on our work with it and the possibility of this gene becoming readily available in the Standard Registry of Biological Parts. HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Safety" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Safet/Achievements From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEM Achievements Team Accomplishments This year, we tried to further develop the project from last year. The new factor was our support came from the new bioclub rather than the research class. Many of our team could not attend this year, so we decided to be a virtual team (our first year going virtual). We are fortunate to have contact with a research mentor to guide and lead us through this process while Dr. Fenton is also being introduced to synthetic biology. We have added two new parts relating to energy production to the registry for which few protein coding sequences exist. This link provides information showing the protein coding sequences. Now we have increased the available parts relating to energy production by 20%. Our team has learned the protocol for site-directed mutagenesis and for PCRing prefix and suffix to parts.This year we are extending the Hawaii team's work with cyanobacteria. We are using cyanobacteria as a chassis which is a unique one; most teams work with E. coli. Cyanobacteria is not even in the chassis registry, as shown in this link One of our team members, Mason, has learned to use Tinker Cell to show the pathway of glycolysis to alkane production.For the JudgesThe wiki this year is more dynamic relative to last year's wiki. There are extensive code editing and features that are more appealing.This year we used a modeling program called Tinker Cell. It shows a step-by-step pathway from glycolysis to alkane production. The new parts that we added to the registry, Rabbit Muscle and Yeast PK, are natural. They are important in tackling possibly the world's most important problem which is clean sustainable energy.Three of our team members helped teach a middle school bioscience camp again! This year the camp expanded to two sessions reaching to 50 kids. We taught them DNA extraction, bacterial transformation, micropipetting, gel electrophoresis and more. To spread the word of our work, we have created a Twitter account, CAPS iGEM, and a Facebook page, CAPS iGEM. We have also created a Facebook group, High School iGEM 2014, for everyone to join. This Facebook group is designed to communicate, share, and socialize amongst all the other teams during the iGEM jamboree!HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Achievements" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/AchievementGallery From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEMThanks! Fun Who says that science can't be fun?Cyano Machine To the tune of Yellow Submarine by Muriel Eaton (with help from my friends Lennon and McCartney) In the lab where I was born,Lived cyano-bacteria,And he produced biofuels, From conversion of pyruvate, So we expressed an active gene, Till the bottle neck was reduced, And produced lots of alkanes, In our cyano powered world, We all live in a cyano powered world, Cyano powered world, cyano powered world, We all live in a cyano powered world, Cyano powered world, cyano powered world, And our enzymes are all aboard To make a standard biobrickAnd the sun begins to burnWe all live in a cyano powered worldCyano powered world, cyano powered worldWe all live in a cyano powered worldCyano powered world, cyano powered worldFull speed ahead Mr. Kessler, full speed aheadPCR it is, Sgt.Cut the gene, drop the geneAye, aye, Sir, aye, ayeCaptain, captainAs we make a biomachine (bio-‘chine)The world is using fuel clean(World of us, fuel clean)Sky of blue and cyano of green(Sky of blue, cyano of green)In our cyano powered world(In our cyano, powered, aha)We all live in a cyano powered worldA cyano powered world, cyano powered worldWe all live in a cyano powered worldA cyano powered world, cyano powered worldHomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Gallery" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Galler/References From 2014hs.igem.org teamsPageDiscussionEditHistoryMoveWatchLog in iGEM Main iGEMHS iGEM AttributionsOutreachFun Facebook HS iGEMCAPS iGEMiGEM Twitter CAPS iGEMiGEM HQ Glycolysis Poem SafetyProject Project OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture TeamHome 1 2 3 4 BV CAPS iGEM TweetsTweets by @CAPSiGEM ReferencesGeneral References on BiofuelsAnne Ruffing (2014) “Metabolic Engineering of Hydrocarbon Biosynthesis for Biofuel Production”. InTech. 263-298. C. Dellomonaco, F. Fava, and R. Gonzalez (2010) “The path to next generation biofuels: successes and challenges in the era of synthetic biology” Microbial Cell Factories. 9(3) . C. Martin, D.. Nielsen, K.Solomon and K. Jones Prather (2009) “Synthetic Metabolism: Engineering Biology at the Protein and Pathway Scales” Chemistry & Biology. 16(3) , 277-286. D. Savage, J. Way, and P. Silver (2008) “Defossiling Fuel: How Synthetic Biology Can Transform Biofuel Production” ACS Chemical Biology. 3(1) , 13-16. Jay Keasling on Biofuels - a collection of links L. Jarboe, X. Zhang, X. Wang, J.. Moore, K. Shanmugam, and L. Ingram (2010) “Metabolic Engineering for Production of Biorenewable Fuels and Chemicals: Contributions of Synthetic Biology” Journal of Biomedicine and Biotechnology. Michael Brenner et. al. (2006) “Engineering Microorganisms for Energy Production” Office of Biological and Environmental Research of the Department of Energy. Pamela Peralta-Yahya1 and Jay Keasling (2010) “Advanced biofuel production in microbes” . Biotechnology Journal. 5(2) , 147-162. R. Radakovits, R. Jinkerson, A. Darzins, and M.. Posewitz (2010) “Genetic Engineering of Algae for Enhanced Biofuel Production” American Society for Microbiology. 9(4) , 486-501. S. Lee, H. Chou, T. Ham, T. Lee and J. Keasling (2008) “Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels” Science Direct. 19, 556-563. Cyanobacteria and Pyruvate Kinase ReferencesA. Schramm, B. Siebers, B. Tjaden, H. Brinkmann, and R. Hensel (2000) “Pyruvate Kinase of the Hyperthermophilic Crenarchaeote Thermoproteus tenax: Physiological Role and Phylogenetic Aspects” Journal of Bacteriology. 182(7) , 2001–2009. Ana Ramos et al. (2004) “Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis” Microbiology. 150, 1103–1111. Aron Fenton and Aileen Alontaga (2009) “The Impact of Ions on Allosteric Functions in Human Liver Pyruvate Kinase”. Methods in Enzymology. 466, 83-107.D. Gong, Z. Gong, Y. Guo, and J. Zhu (2002) “Expression, Activation, and Biochemical Properties of a Novel Arabidopsis Protein Kinase” Plant Physiology. 129, 225–234. H. Huang, D. Camsund, P. Lindblad and T. Heidorn (2010) “Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology” Nucleic Acids Research. 38(8) , 2577–2593. H. Knoop, Y. Zilliges, W. Lockau, and R. Steuer (2010) “The Metabolic Network of Synechocystis sp. PCC 6803: Systemic Properties of Autotrophic Growth” Plant Physiology. 154, 410–422. Hsin-Ho Huang and Peter Lindblad (2014) “Wide-dynamic-range promoters engineered for cyanobacteria” Journal of Biological Engineering. 7(10) . Jiro Hattori et al. (1995) “Pyruvate kinase isozymes: Ancient diversity retained in modern plant cells” Biochemical Systematics and Ecology. 23(7–8) , 773–777, 779–780. M. Malcovati and G. Valentini (1982) “AMP- and Fructose 1,6,-Biphosphate-activated pyruvate kinases from Escherichia coli”. Methods in Enzymology. 90, 170-179.Open Wetware Synthetic Biology Course Website with information on Algal Biofuels http://openwetware.org/wiki/CH391L/S13/Algal_Biofuels S. Nagarajan, D. Sherman, I. Shaw, and L. Shermana(2012) “Functions of the Duplicated hik31 Operons in Central Metabolism and Responses to Light, Dark, and Carbon Sources in Synechocystis sp. Strain PCC 6803” J. Bacteriol. 194(2) , 448. T. Dandekar, S. Schuster, B. Snel, M. Huynen and P. Bork (1999) “Pathway alignment: application to the comparative analysis of glycolytic enzymes” Biochem. J. 343, 115-124. Takakazu Kaneko et al. (1996) “Sequence Analysis of the Genome of the Unicellular Cyanobacterium Synechocystis sp. Strain PCC6803. II. Sequence Determination of the Entire Genome and Assignment of Potential Protein-coding Regions” DNA Research. 3, 109-136. Thomas P. Howard et al. (2014) “Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli” PNAS. 110 (19), 7636–7641. V. Knowles, C.Smith, C. Smith, and W. Plaxton (2001) “Structural and Regulatory Properties of Pyruvate Kinase from the Cyanobacterium Synechococcus PCC 6301” J. Biol. Chem. 276, 20966-20972. Vicki L. Knowles and William C. Plaxton (2003) “From Genome to Enzyme: Analysis of Key Glycolytic and Oxidative Pentose Phosphate Pathway Enzymes in the Cyanobacterium Synechocystis sp. PCC 6803” Plant Cell Physiol. 44(7) , 758–763. Wolfgang H. Nitschmann and Gunter A. Peschek (1986) “Oxidative Phosphorylation and Energy Buffering in Cyanobacteria” J. Bacteriol. 168(3), 1205. X. Liu, S. Fallon, J. Sheng, and R. Curtiss III ( 2011) “CO2-limitation-inducible Green Recovery of fatty acids from cyanobacterial biomass” PNAS. 108(17) , 6905–6908. Y. Guo et al. (2012) “Beta-Cell Injury in Ncb5or-null Mice is Exacerbated by Consumption of a High-Fat Diet”. Eur J Lipid Sci Technol. 114(3), 233-243.Synthetic BiologyCaltech Synthetic Biology Journal Club http://openwetware.org/wiki/Caltech_Synthetic_Biology_Journal_Club R.Shetty, D. Endy and T. Knight Jr (2008) “Engineering BioBrick vectors from BioBrick parts” Journal of Biological Engineering. 2(5). Tom Knight (1996) “Idempotent Vector Design for Standard Assembly of Biobricks” PNAS. 93(20), 10891-6. DiagramsPathway Diagram01 http://2014hs.igem.org/wiki/images/8/8c/Pathway_Diagram.PDF Pathway Diagram02 http://2014hs.igem.org/wiki/images/c/cc/Image3.png Jansson, Christer. "Figure 1." Earth Science Division. Lawrence Berkeley National Laboratory. Web. 22 May 2014.Ruffing, Anne M. "Figure 3." Intech. InTech, 20 Mar. 2014. Web. 22 May 2014. HomeWelcome TweetsHS iGEM TeamWho we areStudentsAdvisors ProjectProject OverviewProblemsSolutionsNotebookMethodsAchievementsReferencesFuture Safety Safety Fun Fun Outreach Outreach Attributions Attributions iGEM Main iGEM HS iGEMRetrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/References" Retrieved from "http://2014hs.igem.org/Team:CAPS_Kansas/Project/Reference1295RAMNOTIREN CALGARYCentral Memorial High School, AB, CANADA http://schools.cbe.ab.ca/b823/High School1012BBa_K1295999http://2014HS.igem.org/Team:RAMNOTIREN_CALGARYBBa_K1295000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165820140High SchoolLog in   Team:RAMNOTIREN CALGARY/AboutCMHS From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM TEAM Members Official Page About US About Calgary Calgary is one of the largest cities in Western Canada, expanding 726.5 km2 and has a population of over 1 million people. It was founded in 1875, when the RCMP erected Fort Calgary (then Fort Brisebois) to protect the western plains from whiskey traders. When the Canadian Pacific Railway reached the area in 1883, Calgary grew into an important commercial and agricultural center. Because of its strong agricultural and ranching history, Calgary has been nicknamed “Cowtown”; a nickname that is made relevant every summer during the world-famous Calgary Stampede. (an annual agricultural fair founded in 1905) Currently, Calgary's economy is dominated by the oil and gas industry, and major attractions include Canada Olympic Park (the site of the 1988 Winter Olympics), Calaway Park, and the Lilac Festival. Calgary is also home to the Calgary Flames, our National Hockey League (NHL) team. Famous Calgarians include Naheed Nenshi, the first Muslim to become mayor of a major Canadian city, as well as the current Prime Minister of Canada, Conservative leader Stephen Harper. About Central Memorial High School Central Memorial High School is a diverse place for students all around the city to explore and express their passions and talents. Central offers several programs such as National Sport Academy, Performing and Visual Arts, Advanced Placement, and Leading-edge CTS programming. Students are drawn to Central because of its dynamic and diverse learning experiences and unique variety of programs. The academic program is spirited and challenging at all levels. Central Memorial offers personalized learning opportunities ensuring each student is able to experience a variety of rich learning experiences that fit his or her learning needs and goals. Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/AboutCMHSReferences From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM ReferencesA New Strategy in the War on Cancer. (2009, October 1). . Retrieved , from http://www.ted.com/talks/david_agus_a_new_strategy_in_the_war_on_cancer.htmlTargeted Cancer Therepy. (n.d.). . Retrieved , from http://www.cancer.org/acs/groups/cid/documents/webcontent/003024-pdf.pdfhttp://new.ted.com/playlists/63/a_cure_for_cancerhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC205287/ http://biotech.szbk.u-szeged.hu/RG_Jegyzet/pBADg3_man.pdf http://www.wisegeek.com/what-is-endostatin.htm Papers:Hooke, A., Oeschger, M. P., Zeligs, B., & Bellanti, J. Ideal Target Organism for Quantitative Bactericidal Assays. Infection and Immunity.Anti-angiogenesis mediated by angiostatin K1–3, K1–4 and K1–4.5; Anti Angiogenesis mediated by angiostatin. Endothelium and Vascular DevelopmentNussenbaum, F., & Herman, I. N. Tumor Angiogenesis . Insights and Innovations, 2010.Antiangiogenic peptides and proteins; https://d2l.cbe.ab.ca/d2l/le/content/306178/viewContent/3812058/View Recombinant Human Angiostatin by Twice-Daily Subcutaneous Injection in Advanced Cancer Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/ReferenceProject/Content From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM PROJECT Overview Human Practices Safety References AttributionsOur Idea Forty percent of Canadians are expected to develop cancer in their life time of that forty percent twenty five percent are expected to die from their cancer. Imagine what could be accomplished if all of those people were focusing their attention in their futures instead of the now? This question is exactly what motivated the Central Memorials IGEM team to create a bacterium that could target and kill cancer cells. We decided to combine the developing cancer treatment option of anti angiogenesis with synthetic biology to create a biological machine that would cut off the blood supply feeding cancer cells, effectively starving them. This method of killing cancer cells inspired us to title our project "Choking Cancer". The Sequence Our sequence consists of three parts: a lactic acid promoter, which is part BBa_K822000 on the IGEM registry, an anti-angiogenic factor, and a secretion tag. The lactic acid promoter activates the start codon, which promotes the production of angiostatin in the cell. A secretion tag will then be used to get the angiostatin out of the bacteria so that it can begin its inhibiting work on the blood vessels surrounding the tumor. The Link between Cancer Cells and Lactic Acid In the early stages of carcinogenesis, the tumor moves away from the blood supply and therefor its nutrient and oxygen supply. Many would assume that this would cause the tumor to experience hypoxia and nutrient shortages unfortunately this is not true. Cancer cells promote angiogenesis so that they can sustain their rapid proliferation and have a sufficient source of nutrients and oxygen. They also have a mutation that allows them to consume high amounts of glucose through a glycolysis pathway that, instead of sending glucose to the Krebs cycle, converts pyruvate to lactate. The large amounts of lactate secreted by tumor cells, as a direct result of the abnormal production of pyruvate, leads to the build up of lactic acid in the tumor surroundings. This build up of lactic acid is why our team chose to use a lactic acid promoter in our sequence. In doing so, our sequence will only become active in the presence of lactic acid and therefore near the tumor. Why the Lactic Acid Promoter Was Chosen The Central Memorial team chose to use bio brick BBa_K822000 (lactic acid promoter) because of the link between lactic acid and solid tumors described in the above paragraph. They wanted to choose a promoter that had been characterized and proven to be successful when used in sequences in they way BBa_K822000 has been. In doing this it allowed the team to focus less on how they were going to activate the start codon but instead on how they were going to develop the sequence, assays and protocols; as well as completing sufficient research on cancer and angiogenesis. Angiogenesis Angiogenesis is the process in which new blood vessels are formed from pre-existing blood vessels. Angiogenesis occurs in a healthy body during wound repair, to restore blood flow to tissues after injury. In women, it occurs during the monthly reproductive cycle as her body rebuilds the uterus lining, and during pregnancy, as angiogenesis helps to build the placenta and create blood circulation between mother and fetus. When the body is healthy, angiogenesis is managed by a series of “on” and “off” switches. The “on” switches are regulated by angiogenesis, stimulating growth factors such as Angiogenin, Angiopoietin-1, placental growth factor and vascular endothelia growth factor (VEGF) to name a few. The “off” switches regulated by angiogenic inhibitorssuch as Angiostatin (plasminogen fragment), platelet factor 4, thrombospondin -1 (TSP-1) and -2, and Troponin. If either Angiogenic stimulators of inhibitors are in excess, abnormal vascular growth occurs in the body. The Link between Angiogenesis and Cancer Cancer has a mutation that allows it produce small activator molecules of angiogenesis (research into what these activator molecules are is ongoing however it is thought that they are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are both possible activator molecules). These activator molecules promote angiogenesis so that the cancer cell can continue to grow. If the cancer cell did not have increased blood flow to it, it would not be able to sustain its rapid proliferation. Research has been done that shows that without an increased blood supply, cancer cells cannot grow to more the 0.5 cubic millimeters. Therefore, if increased angiogenesis is the tipping point between a harmful cancer cell and a harmless cancer cell, it makes sense to tackle the problem using anti-angiogenic factors and synthetic biology. Angiostatin - Our Anti-Angiogenic factor Angiostatin is a cleaved portion of the protein plasminogen, and has been proven to inhibit angiogenesis. When angiostatin is cleaved from plasminogen, it is cleaved as a four-kringle domain. However, the most effective inhabitation of angiogenesis happens in only the first three Kringle domains. Our team’s sequence consists of angiostatin containing the first three-kringle domains. Angiostatin inhibits 38kDa, which specifically blocks the growth of endothelial cells. There was a slight modification made by ourteams in their cleaved portion of plasminogen. We changed amino acid 308N to 308E, which prevents glycosylation. In preventing glycosylation, a sugar chain is not added, and therefore the body does not recognize and destroy the protein immediately after it is secreted into the body. Once the angiostatin is secreted out of the bacteria into the body, it will cut off the blood supply feeding the tumor. Secretion tag The secretion tag will allow the angiostatin to be secreted out of the bacteria. It is important that angiostatin is secreted out of the bacterium so that the already heavy immune response to bacteria can be decreased. Secreting the angiostatin out of the bacteria allows for only the protein to leave the bacteria, while the waste and toxins remain inside. This is a better option than lysing the bacteria, which causes all of the bacteria’s waste to be released into the body along with the angiostatin. This would catalyze a sever immune response in the body, and possible sepsis. Please check back in 2015 to see how we will create the secretion tag.Progress from January to June Creating a bacteria sequence takes a lot of work and patience. A problem must be found and an idea developed. This idea would reveal how synthetic biology could be used to come up with a construct that would help bring the solution (to the problem) into closer reach. Central Memorial came up with the problem and theoretical solution by:1. Finding a problem- this was cancer for our team 2. Doing some research into the problem- Which lead to the discovery of a developing treatment option that focused on angiogenesis 3. Researching this developing treatment option- in doing further research on anti angiogenesis Central Memorial found a way it could be combined with synthetic biology. They also found more information on cancer cells and how they are created. Including possible ways the cancerous tumor could be detected in the body by a bacterium. 4. Combing all the research- in doing so the team was able to come up with a sequence that consisted of a lactic acid promoter, which was chosen because in their research the team found that tumor cells secrete high amounts of lactic acid. When combing all the research they also came up with the idea to synthesize an anti-Angiogenic factor that would cause the blood vessels from reaching and feeding the tumor. The anti-Angiogenic factor they found was angiostatin. Their synthesized angiostatin contains 3 kringle domains cleaved from the original protein plasminogen. The original amino acid number 308N changed to 308E in order to prevent glycosylation and the protein being destroyed immediately after it was secreted. 5. Talking to the experts- Communicating with experts in synthetic biology, molecular biology and IGEM alumni allowed Central Memorials team to understand potential problems with their proposed idea, how these problems could be solved and future direction they should take. Potential problems, solutions and future direction are outlined below. • Lysing the bacteria to get the angiostatin out would generate a strong immune response- The solution to this problem would be creating a secretion tag that would secrete only the angiostatin out. • Muscles secrete lactic acid- the solution to this was for Central Memorials team to create a proof of concept idea. They would prove that bacteria with an anti Angiogenic factor such as angiostatin could be used to treat cancer cells. However in the future the sequence created could be inserted into a virus. This virus would contain a kill switch so that it would only be active and secreting angiostatin in the presence of a certain antibiotic. That way it could be used in conjunction with other treatment options. • Bacteria in the body would create a sever immune response in an already sick body- A possible solution to this problem could be to insert the bacteria into the Nano tubes NASA is creating. These tubes will allow the angiostatin to be secreted out via the secretion tag, however the Nano capsule will contain the bacteria and keep the body from having an immune response to it. • When the bacteria are injected into the body, how will it remain localized around the tumor? -This problem lead the team to an article explaining how a team of scientist at the University of California Berkeley, University of California and San Francisco have set out to create a tumor killing bacteria. This bacteria when injected into the bloodstream, would travel to the site of a tumor and insert itself into the cancer cell. Once inside the tumor it would produce a cancer- killing compound. In theory this cancer-killing compound could be our sequence. To read more about this bacteria click on this link.6. Assays are designed and protocols carried out- Centrals team designed two assays that would be carried out the fallowing year to prove that their sequence would work. The first being one would show that the lactic acid promoter is active in the presence of lactic acid. Creating a circuit containing lactic acid and RFP would do this. To see exactly how this would be done proceed to the Assay section. The second assay performed would show that the angiostatin is in fact an inhibitor of angiogenesis. Adding the bacteria to a kit plate that mimics angiogenesis would allow us to do this. The kit plate would change in some way to show that the inhabitation of angiogenesis took place when angiostatin was lysed from the bacteria onto the kit plate. To see exactly how Centrals team plans to carry this assay out proceed to the assay section. The third and final assay will be performed to make sure that the angiostatin is produced in the presence of lactic acid. Adding the fully formed circuit to the kit plate that mimics angiogenesis with lactic acid will do this. The bacteria will cause the angiostatin gene to be turned on in the presence of lactic acid. If there is lactic acid on the kit plate then it will turn on the promoter causing the lactic acid promoter to turn on the angiostatin gene. When the angiostatin is lysed out of the bacteria it will cause the kit plate to change in some way are a result of its ability to inhibit angiogenesis. To see the specifics of this assay proceed to the assay section. With the angiostatin in it to the In Vitro Angiogenesis assay kit we purchased will do this. This kit promotes angiogenesis or the formations of blood vessels. When this factor is active circles are formed on the kit plate, after adding angiostatin to the kit plates we hope the circles will disappear. If they do disappear then we will know that the angiostatin was successful. In order to ensure that it is in fact our protein causing the circles to disappear we will perform a few different tests involving the In Vitro Angiogenesis kit plate. First we will use the kit plate and add bacteria that do not contain our circuit. Hopefully the circles in the kit will stay intact, if they do we will know that it is not our bacteria causing the circles to disappear. Next we will add bacteria containing our angiostatin that have not been lysed. We hope that if all goes well then the circles will remain in the plate. This tells us that our factor is not somehow leaking out of the bacteria. Finally we will add our lysed bacteria to the kit plate (we will lyse the cells by adding detergent) and see if the circles disappear, if they do then our transformation of angiostatin into the bacteria was successful. The final essay performed will be performed in order to make sure that our circuit works correctly is quite simple. We will again use the kit plate we purchased that has an Angiogenic factor and put it into a well along with a concentration of lactic acid. This will be repeated five times with five different concentrations of lactic acid. If all goes well the circles should remain. In the next we will keep everything the same. However this time we will add bacteria. The next well will contain bacteria with our circuit inside however they have not yet been lysed and therefore the circuit will not be active. Again the kit plate and lactic acid is added to the well, and it is repeated five times. In the last well we will add our lysed bacteria and follow the same step we took for the previous sections. The well with the lowest concentration of lactic acid should have the greatest amount of circles however the amount of circles should be reduced compared to the wells where our circuit was not active. As the concentration of lactic acid increased the number of circles should decrease. 7. Carrying out protocols so that they will be done properly next year- This is done so that Centrals IGEM team can carry out all the procedures at the beginning of the year and get the circuit made by the end of December. They carried out a competent cell protocol, transformation method, growing a bacteria colony and feeding them, removing plasmids from bacteria and running a gel to verify plasmid size. To see these protocols in detail proceed to the protocol tab. Cancer is a huge topic of conversation and with ongoing research progressing researchers knowledge of this disease and how scientist could put an end to it; Central Memorials team hoped that in completing the above steps they could spark a new topic of conversation. A conversation that one-day may lead to the end of this horrible disease. Assays In different concentrations of lactic acid different amounts of red fluorescent protein should be produced.This kit promotes angiogenesis or the formation of blood vessels. When this factor is active. circles are formed on the kit plate; after adding angiostatin to the kit plates, we hope the circles will disappear. This would show us the success of the angiostatin. In order to ensure that it is in fact our protein causing the circles to disappear, we will perform a few different tests involving the In Vitro Angiogenesis kit plate. First we will use the kit plate and add bacteria that do not contain our circuit. Hopefully the circles in the kit will stay intact, if they do, it will confirm that it is not our bacteria causing the circles to disappear. Next we will add bacteria containing our angiostatin that have not been lysed. If the cirlces remain in the plate, it confirms that our factor isn't leaking out of the bacteria. Finally, we will add our lysed bacteria to the kit plate (we will lyse the cells by adding detergent) and see if the circles disappear, if they do then our transformation of angiostatin into the bacteria was successful.The final essay performed will be performed in order to make sure that our circuit works correctly is quite simple. We will again use the kit plate we purchased that has an Angiogenic factor and put it into a well along with a concentration of lactic acid. This will be repeated five times with five different concentrations of lactic acid. If all goes well the circles should remain. In the next trial, everything will be kept the same; however, this time we will add bacteria. The next well will contain bacteria with our circuit inside. The circut, however,has not yet been lysed and therefore it will not be active. Again the kit plate and lactic acid is added to the well and it is repeated five times. In the last well, we will add our lysed bacteria and follow the same step we took for the previous sections. The well with the lowest concentration of lactic acid should have the greatest amount of circles however the amount of circles should be reduced compared to the wells where our circuit was not active. As the concentration of lactic acid increased the number of circles should decreaseWhat We Plan To Do Our team plans to build three constructs. The first is a lactic acid promoter ligated with a red fluorescent protein. The second is a lactic acid promoter ligated with their angiostatin and the third is a lactic acid promoter ligated with angiostatin and a constituent promoter. In the first assay a circuit containing a constituent promoter will cause the angiostatin to be continuously expressed in the bacteria. We will then lyse it and then run them in a western. On the western, a dot or a line should appear which shows that angiostatin is being expressed in the bacteria. The second assay is performed to test the lactic acid promoter. A circuit comprised of the lactic acid promoter ligated with red fluorescent protein is inserted into the bacteria. These bacteria are then grown in different concentrations of lactic acid and then lysed. Next they are run on a western, the brightest line on the western shows at which concentration the lactic acid has maximum expression. The third assay is executed to test the lactic acid promoter with the angiostatin. It is carried out to make sure that angiostatin is not toxic to the bacteria, or if it is toxic at what concentration is it toxic to the bacteria. The team also wants to see at what concentration of lactic acid could they get the maximum amount of angiostatin produced. To gather this information the team would put bacteria containing the ligated circuit containing a lactic acid promoter and angiostatin into different concentrations of lactic acid. The bacteria would be grown in these different concentrations of lactic acid and then lysed. Running a western would show Central Memorial at what concentration of lactic acid they got maximum induction of angiostatin (this would be the brightest line on the gel).The fourth assay carried out is done to show the concentration of lactic acid needed to induce enough angiostatin to inhibit angiogenesis. This is done by growing bacteria containing ligated circuits of lactic acid promoter to angiostatin in different concentrations of lactic acid. The bacteria are then lysed and added to a kit plates containing vascular growth cells. The plate with tubes formed out of these vascular growth cells show angiogenesis and the plates without tubes formed shows that growth of the vascular growth cells was inhibited. The well with the fewest tubes formed would show the concentration of angiostatin needed to inhibit angiogenesis and at what concentration of lactic acid the concentration of angiostatin was produced. The final assay performed would be carried to find a secretion tag capable of secreting the amount of angiostatin needed to inhibit angiogenesis. This would be done by putting a bacteria containing a circuit a ligated circuit of lactic acid promoter angiostatin and a secretion tag (several circuits would be created to put into bacteria containing different secretion tags) these bacteria would be grown in physiological concentrations of lactic acid (concentration of lactic acid produced by tumor cells), lysed and then run on a western. The secretion tag that secreted the concentration of angiostatin closest to that of the optimal amount needed to inhibit angiogenesis would choose. The optimal amount of angiostatin needed was determined in assay number four. The bacteria grown in the concentration of lactic acid producing optimal amounts of angiostatin (found in assay four) would be lysed on the far right corner of the western so that the secretion tag trials (which were run on the same western) could be compared to it. Scroll Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/Project/ContenParts From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM Part NameTypeDescriptionLength (bp)BBa_K822000Regulatory Lactic Acid Promoter344BBa_J04450Reporter Red Fluorescent ProteinN/ARetrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/PartMembers From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM TEAM Members Official Page About US Description Mouse over or touch our faces to find out more... Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/MemberHumanPractices From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM PROJECT Overview Human Practices Safety References Attributions For the human practices portion of our project we put up a survey online, getting 136 people to respond to the questions listed below: 1. Have you or someone close to you ever been affected by cancer? 2. Would you be comfortable having bacteria injected into your bloodstream if it could potentially treat or cure cancer? 3. Would you still want to undergo this treatment if it meant suffering side effects such as fatigue and/or flu-like symptoms (i.e. nausea, vomiting, diarrhea, etc.)? 4. Assuming similar success rates, would you prefer to undergo chemotherapy, or this bacterial treatment option? 5. Would you choose this treatment option if the duration of the treatment was the same as that of traditional forms of treatment, such as chemotherapy? 6. If this form of treatment took twice as long as traditional treatment options, but with higher success rates, would you still opt for this treatment? 7. During treatment, would you be comfortable limiting/cutting out all physical activity? 8. Would you support your tax dollars going towards developing this type of treatment? 9. Identify your age range: 14-18, 19-24, 25-32, 41-51, 52+ We turned our survey information into pie charts and the results are as follows: We found that openness for the funding for this type of project had an overwhelming 95% approval. The majority of the people who’d taken the survey were open to trying our type of treatment, especially if it proved to be more effective than chemotherapy. The one major problem most people had, related to the function of the lactic acid promoter in the body. People were concerned about how much physical activity they would have to limit for what periods of time. Scroll Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/HumanPracticeProtocols From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project ContentHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM Heat shock Background: Competent cells are thawed, put on ice for 30 minuets and then put in a 37 degree incubator for five minuets with the foreign DNA plasmid of choice (this plasmid was pipetted into the tube with the competent cells after they were thawed and prior to putting them in the ice bath). Competent cells are frozen with pores in their membranes, icing them for thirty minuets allows for the plasmid to stick to the cells membrane. When the competent cells are put in a 37-degree incubator the pores of the cell membrane become larger therefor allowing for the plasmid to get in. Putting the competent cells back on ice after heat shocking them causes the pores to become smaller keeping the plasmid inside the cell.Procedure:1. Thaw the competent cells that your team made earlier (about 100 μL for each transformation). Remember to keep the cells cool while thawing them. 2. Add between 1 μL-20 μL DNA of choice to the thawed tube of competent cells and mix the DNA by gently pipetting the tube up and down.3. Once the DNA has been mixed in, leave the tube on ice for thirty minutes 4. After the thirty minutes are up, heat shocks the cells for five minutes in a 37-degree water bath inside a 37-degree incubator. It is most effective if you heat the water to 37 degrees while you have the competent cells on ice. 5. After five minuets, place in an ice bath for five minuets 6. Take off ice bath and add 250 μL SOC broth (used to maximize the transformation efficiency of competent cells). 7. Incubate for thirty to sixty minutes in a shaking incubator, or ninety to one hundred and twenty minuets in a non-shaking incubator.8. After the incubation period, centrifuge cells to remove all but 100 μL of supernatant. (As 100μL is marked on the 1.5ml tubes it is recommended that you pipette 20μL of the supernatant out until you get down to 100 μL)9. Re-suspend the cell into solution by vortexing or generally tapping) 10. Place around 50μL of the remaining competent cells onto antibiotic plates (such plate could be one containing LB and chloramphenicol) 11. Let competent cells grow over night on the antibiotic plate at 37 degrees Celsius. Once you have transformed your DNA into the bacteria, you will need to grow up many colonies of the bacteria. This will allow you to have multiple copies of your DNA to play with. To see how to do this, proceed to the Feeding Bacteria protocol.Feeding the BacteriaBackground: Feeding bacteria is simply the term used to describe transferring the competent cells (e.coli) containing your DNA of choice into new tubes containing LB and an antibiotic. In this protocol, we used chloramphenicol as the antibiotic. However, you should use the same antibiotic that you had on the plates you put your competent cells on in the last step of the transformation protocol. In transferring the e.coli from one tube to another you keep them happy and healthy. They are happy because they are being given more food, and a home free of waste. You should feed your bacteria every one to two days. Procedure:1. In a sterile area, pipette 1000 μL of LB broth into a 1.5 ml tube. 2. Pick one colony of ecoli (grown in the incubator) from the antibiotic plates created in the transformation protocol and place it in the 1.5 ml tube. It is recommended you use a clean pipette tip to pick the colony off of the plate, to get it off the pipette tip swirl the pipette in the LB broth (in the 1.5ml tube) for 20 seconds. 3. Add 10 μL of chloramphenicol (or the antibiotic of your choice) into the 1.5 ml tubes4. Place the 1.5 ml tube in a non-shaking incubator for 48 hours. This will allow the bacteria to grow and multiply. The tube should be cloudy after 48 hours, telling you your ecoli have grown. 5. Pipette 500 μL of the ecoli (which was grown in the incubator over 48 hours) into a new 1.5 ml tube containing 1000 μL of LB.*Note*-After you have created a 1.5 ml tube of E.coli, you should be feeding or “rehoming” 500 μL of these bacteria every day. This means repeating steps 1-6 everyday.6. Add 10 μL of chloronphenical (or antibiotic of choice) into the 1.5 ml tube. 7. Repeat steps 1-4 so that you have a new colony of bacteria growing in the incubator.8. Steps 1-4 should be repeated every two days, while steps 5-8 should be repeated every day until you are completely done all of your assays. In repeating these steps every day or every two days you ensure you have lots of e.coli containing your DNA Trouble shooting: If you look into your 1.5 ml tubes and notice your bacteria no longer looks cloudy, but streaky, then it means that your bacteria ruptured. They were left in their old tubes too long, and they ran out of food, and too much waste built up for them to survive. You will have to go back a day, and feed the bacteria you rehomed two days ago. If you no longer have these bacteria, or they too have ruptured you will need to create new ones out of the colonies growing on the LB antibiotic plates. Also if for some reason you must leave your bacteria for more then 2 days without being fed, you should put them in the fridge. This will slow down the log cycle and keep the bacteria from multiplying rapidly. In preventing them for multiplying rapidly, you will get less in the tube and they will run out of food slower and produce less waste. This is what had to be done we went 3-4 days without feeding their bacteria. Once you have grown your DNA in the bacteria, you will want to centrifuge the plasmid out so you can cut and ligate it to use in the sequence you are trying to create.Agarose Gel ElectrophoresisReagents and Materials: 1X TAE Graduated Cylinder 125 mL flask Agarose Gel Pouring Tray Tape Gel rig SYBR SafeProtocol: Measure out 100mL of buffer Transfer buffer to 125 mL flask Weigh out enough agarose to make a 1% gel Transfer agarose to 125mL flask Melt agarose in microwave until solution is almost boiling, stirring every 15-20 seconds Allow agarose to cool (do not let it cool to the point where it is hard) Add 4 uL of SYBR Safe to the cooling agarose Assemble the gel pouring apparatus by inserting gate into slots. Allow gel to cool until flask can be handled comfortably. Place comb in the gel rig. Pour agarose into gel tray. Allow to solidify. While the gel is solidifying prepare the samples. Add your sample and 1 uL 10x Loading Dye, 4 uL of DNA and 5 uL of water Pour 1X TAE over gel so that gel is covered by a 3-5mm buffer Load samples into lane (Don't forget to load a 1kb+ ladder into one of the lanes) Hook electrodes to gel apparatus. Run the apparatus at 100V for 30 - 45 minutes (make sure to watch that the dye does not run off the gel) Record the results.Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/ProtocolFrom 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Content OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM Choking Cancer Every year approximately eight million people die of cancer, and an additional fourteen million are diagnosed with the disease. The World Heath Organization predicts that without immediate action these numbers will increase by eighty percent by the year 2030.Cancer needs an abundant source of oxygenated blood to grow. To achieve this cancer cells release angiogenic factors that promote vascular growth. Without these factors, cancer is unable to survive. Consequently, current treatments focus on targeting/inhibiting the angiogenic factors released by cancer cells. Targeted therapy has also been a successful treatment option.Our project aims to combine these two therapeutic approaches to create a synthetic biological machine that seeks out cancer cells and inhibits angiogenesis. We plan to design a bacterium that in the presence of cancer cells secretes an anti-angiogenic factor. This will inhibit the growth of the capillary system that is crucial for cancer cell survival. Click here to see the introductory video we used to familiarize ouselves with what we would be researching. Here is a record of our. We also want to thank everybody who helped us along the way! Tweets by @CentraliGEM Our Sponsors Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARProject/Safety From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM PROJECT Overview Human Practices Safety References Attributions1. Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety.Our project mainly raises environmental issues. We are working with antibiotic resistant genes and as a result, we have to be cautious in how we dispose of the hazardous waste to avoid the creation of superbugs. As a result, we collect all our waste together and dispose of them according to our school board's rules.Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?Unfortunately, we did not have an opportunity to create any new BioBricks this year and as a result, we can't assess the safety concerns surrounding it. However, we made a centrifuge which powered by a dremel drill, has a speed of 30,000 rotations per minute. To avoid any physical injury caused by the centrifuge, we put up shields around the machine and we wore goggles whenever we were near it.3. Is there a local biosafety group, committee, or review board at your institution?Our school board has a person in charge of hazardous materials and removal; but because he is not located at our school, we have trained personnel who can handle hazardous waste. As part of that, we follow proper CBE administrative regulation regarding disposal of hazardous waste.4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?We would recommend that if your school board does not a biological materials waste management plan, the team should discuss it with their university mentors to avoid any breach of safety.Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/Project/Safetttributions From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM PROJECT Overview Human Practices Safety References AttributionsAdvisor and Instructor SupportAll research work was performed by the students of the Central Memorial iGEM team. Our teachers and advisors contributed support and ideas to the students and facilitated a safe and efficient laboratory environment. Special thanks to Dr. Ankush Garg, whose experience in genetics facilitated the evolution of our research. And thanks to Mr. Klemmer for his supervision over all our experiments and labwork. Thanks to Ms. Wyshynski securing our trip to Boston by getting our forms in order and gathering finances for us. Lastly, we would like to acknowledge Ms. Muhammad for her expertise in biology and her assistance with our wetlab preparationsOutside of Central Memorial Research and Technical SupportWe would like to thank and acknowledge the support of various individuals for their support. This includes Mr. David Lloyd's contributions and feedback and just overall guidance on the format of our wiki, our presentation, and reviewing our human practice procedures. Thanks to Ms. Himika Dastidar for contributing her time to listen to our competition presentation in depth and giving us advice on what we can do better. Similarly, thanks to Ms.Lisa Oberding for providing us with SBOL and figure support. Special thanks Kim Willoughby for her expertise and support in our research as she is a microbiologist. Special thanks also to Mr. Patrick Wu for all his advice he gave us in creating our wiki; not to mention the fact that he always responded to our requests as quickly as possible.Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/AttributionNotebook From 2014hs.igem.org teamsPageDiscussionView SourceHistoryLog in 1 2 3 4 HomeTeam MembersOfficial ProfileAbout CMHS Project OverviewHuman PracticesSafetyReferencesAttributions PartsNotebookProtocolsiGEM NOTEBOOK Week 18 Week 17 Week 16 Week 15 Week 14 Week 13 Week 12 Week 11 Week 10 Week 9 Week 8 Week 7 Week 6 Week 5 Week 4 Week 3 Week 2 Week 1 Week 18 Today we effectively got the plasmids out of our bacteria by using our home made centrifuge. The white mass appeared at the bottom of our 1.5 ml tube. We centrifuged the plasmids by using a kit we purchased earlier this year. We followed the protocol given to us with the kit. When the white mass appeared a proud moment was shared between group members. We have overcome so many obstacles to get to this point. Week 17 The Math department came through and managed to find us a drill that fit our 3D printer part. It also runs at a speed high enough to ensure the plasmid will be centrifuged out of the bacteria. This is exciting and means that we can finally get the next stage of our project going! At todays meeting we split into two teams. One team set up the centrifuge and the other team started to dissect the protocols. We hoped that by dissecting the protocols and going through them with a fine toothcomb they would be able to make more sense of them. Lucky for us both teams were successful and we finally have an idea on how we are going to carry out the next part of our project. Week 16 We have run into a few problems and as a result our project is at a standstill: The first of these problems being we don’t have a centrifuge and therefore we can't get the plasmids out of our bacteria, which is the next step in our process. Currently we are trying to locate a 3D printer piece that will fit on a dremmel drill. The 3D printer piece will allow us to attach 1.5 ml tubes to the dremmel drill. When the drill is running it can get up to 10000 RPM, as speed fast enough for us to centrifuge our bacteria with. However we can't find a 3D printer piece that will fit onto the drill we have. Therefore we have recruited one of the math teachers at our school to see if he has a drill that will fit our piece. The second problem is our group is having a very hard time comprehending the protocols. Our group is comprised of only relatively smart students with a passion for biology. Notice I say passion for biology not a PHD in biology. The reason we are having so much trouble understanding the protocols is because we have lost our leading teacher for the time being. However the team is determined to get through this situation. In fact we have planned a meeting that will just be focused on reading the protocols dissecting them. Hopefully after we do this we will be able to figure out what we need to do next and how it’s going to get done. We are also running out of pipette tips and therefore we have to be really stingy with them. When we re-home our bacteria, we have to make sure the tip don’t touch anything but the LB broth when we are filling our nine 1.5 ml tubes with it. This tip is then discarded and a new one is used for the chloramphenicol making sure that the new tip doesn’t touch anything but the solution as we fill the nine 1.5 ml tubes. It is only when transferring the bacteria from one tube to the other do we change tips. This is because we don’t want to cross contaminate the bacteria and get different types growing in the same tube. Currently we are trying to find a teacher who has the time to take some more pipette tips down to the u of c to get autoclaved, however this is no easy task. A student can’t do it as the CBE (Calgary Board of Education) won't allow it. Week 15 We have returned to the books and are frantically trying to get everything in order for the conference at the U of C; we created a presentation. The reason this is so important is because the conference is going to help better prepare us for the competition in Boston. We need to figure out where our research is lacking and if the presentation conveys our idea and information to an audience well. We are also working on cementing the assays we intend to carry out this year and into the beginning of the next school year. It was a fast paced meeting and we didn’t leave a lot of room for chatting and laughs. We went to improvement meetings and talked about our ideas. We are grateful for today. Week 14 We just started re-homing the lactic acid promoter, red fluorescent protein, and competent cells. They take over your life and currently all they do is eat and produce waste. Or at least that’s all they seem to do. Either way it is exhausting to move them every day. However , It is amazing to have begun the microbiology processes. Soon, we will be centrifuging the plasmids out of the bacteria so that we can cut and ligate them. This process usually takes about 10 minutes but after many trials, it takes about 30-45 minutes to re-home all the specimen. Our leading teacher just had a baby and we are very happy for his family. Week 13 We finally got started on some of the actual molecular biology! Although it was pretty basic stuff (or at least basic to those who have made a career out of this) it was super cool. We rehydrated the lactic acid promoter and the red florescent protein and using the heat shock method transferred the proteins into the competent cells that David Lloyd gave to us. For those of you reading this and don’t speak the molecular biology language what I said above means that we got our bacteria to express traits that are not genetic to the strain. However we added proteins to the bacteria so that they could express the traits we desired but we managed to get it finished. Week 12 Today we meet with our mentor David Lloyd; it was a great and productive. David helped us figure out the assays we are going to perform with the bacteria we have made. We learned that we would need to run two very important essays in order to verify a proof of concept with our bacteria. We will need to put the bacteria containing our ligated circuit of the lactic acid promoter and red fluorescent protein and into different concentrations of lactic acid. In these different concentrations we will see different amounts of red fluorescent protein expressed. In seeing this we will prove that our circuit works. Once we have proven this circuit to be accurate we can then ligate our lactic acid promoter to the angiostatin protein. We will test this circuit by putting it into the kit plate containing green circles and different concentrations of lactic acid. The plate with the highest concentration of lactic acid should contain the least amount of green circles, as the highest amount of angiostatin should have been released. The green circles disappear in the presence of angiostatin.We also began to discuss the presentation how to get ready for both the u of c and the competition in Boston. David talked to us about how we should engage the audience as well as making a presentation that flows smoothly and is easy to follow. He especially emphasized that we need to make sure to connect everything in the presentation back to our main idea and goal. David also told us to make sure that we don’t throw the less scientific things into our presentation at the very end. This would look rushed, messy and de value all of the hard work we have done of things like human practices. Overall this was an extremely useful meeting and we are now finally ready to start on some of the actual molecular biology. Week 11 Today was a very slow day as we had a low turnout and so, we got to walk the runway and pretend we were bio superstars! At our meeting we did a photo shoot so that we could add some fun photos to the web page. It was a blast; we got to wear the purple lab coats all the teachers at our school use and all of us were looking pretty snazzy. It also got me really excited to actually start creating our bacterium! Week 10 At this meeting we went through some papers which helped us find a section of cleaved Angiostatin which would be between the kringle domains 1-3 because of its anti-inflammatory factors. We talked about protein domains. There have been trials that have shown that this mutation within the DNA can prevent glycosylation--> adding a sugar chain to protein which targets it for degradation, so breaking up and getting rid of the bacteria. We talked about negatives of Angiostatin and positives. The exciting part is that we have the version of the section of Angiostatin protein that we want, we fixed and re wrote it to have the mutations we want, and talked about recombinant DNA and how we can cut the codes that we want and put it into our vector to create our bacteria. Week 9 We spent the meeting sifting through research on Angiostatin- specifically how it is cleaved, its shape and its DNA sequence- with Mr. Garg. It was a HUGE help having Mr. Garg as he was able to explain some of the more technical parts of the papers we were looking at.In one of our first meetings, we established committees to try and get the project moving forward in a more efficient manner. Our business committee were tasked with try to get companies to sponsor us. We have to start contacting sponsors as soon as possible. Everyone is worried that we have left it to the last minute. Over the past few weeks, we have been searching for companies that would be interested in sponsoring us and compiling a list of names, addresses and phone numbers we could send out or sponsor letters too. At today’s meeting, we began to call companies so we could try and find out whom sponsor letters should be addressed to. Everyone we spoke to was very friendly and we only got hung up on once. Week 8 Upon our research, we came up with three proteins that we could get bacterium to secrete to promote anti angiogenesis. The most promising one was Angiostatin as opposed to Platelet factor four which is essentially just a blood clotting protein and Thrombospondin, which is very complicated and has been a challenge for everyone who has tried to research it. Consequently, we have decided to go ahead and focus our attention solely on Angiostatin for now. At the end of today's meeting, we decided to begin to research Angiostatin further; specifically if the protein could be secreted. This is crucial because if it cannot be secreted then we cannot use it in our bacteria. Week 7 At today's meeting, we worked on our abstract. We also went over some papers that brought up the idea of TNF, IL-12 and Avastin as possible proteins but we had to look further into them to find out if they were a protein or synthetically made. We also discussed where it would be better to work out bacteria towards retracting cancer or stopping it and what were the pros and cons. Week 6 We discussed more about what protein and promoter we wanted to use, as well as how to transport the bacteria into the cancer cell. Some question that needed to be answered was what are the downfalls of the bacteria going everywhere in the body? And are there any vaccines, cure or drugs that assist angiogenesis? We talked about Angiostatin and its positives and negatives. The positives were that it is already used as a cancer therapy, has no resistance, and can be used in plasma. The negatives are you can’t use it if you have high blood pressure and whether or not it could be cleaved. We had to look into the cleavage of Angiostatin and whether or not it can be activated when secreted into the blood and if it should be cleaved before or after. We discussed if it will clot in the blood, the length of the amino acids if it were to be pre cleavage, and more about the side effects. We also talked about the human practice aspect and the online survey we had set up and its results. Overall we decided we needed to look more into the public’s openness for treatment, their openness for funding and the concern about physical activity and what the rate of physical activity would be. - Traelyn Week 5 It is time to get the human practice section underway! At today's meeting we created a list of eight survey questions that we are going to take to the public. These survey questions are supposed to help us determine how accepting the public will be of our idea. We are trying to figure out if someday, given the option, individuals would choose to have bacteria inserted into their blood stream instead of undergoing chemotherapy to fight cancer. Here is a list of the survey questions:1.Have you or someone you know ever been affected by cancer?2.Would you be comfortable having bacteria injected into your blood if it could treat cancer?3.Would you support your taxes going to this cause?4.Would you be okay with side effects such as fatigue/flu-like symptoms?5.Would you be okay with this form of treatment if the duration was the same as traditional treatment options? 6.If this form of treatment took twice as long but had a higher success rate, would you still opt for this form of treatment?7.Assuming similar success rates, would you prefer chemotherapy or would you use the bacteria?8.During treatment, would you be okay with no physical activity?We also discussed how we should explain the project to other people so that they understand what we are trying to do. This was not an easy task. To make matters even more difficult, we would have a very limited amount of time to explain our project. Near the end of the meeting, a couple of the other group members managed to find a succinct way to explain the project. Now the only thing that is left is to try and figure out how we are going to bring our survey to a large population of people. - Maggie Week 4 Now that we have officially decided that we want our bacterium to fight cancer by promoting anti angiogenesis, it is time to focus our attention on finding a protein that will do this. What will happen is this: once we find the protein that can promote anti angiogenesis, we will insert it into the bacteria and get the bacteria to secrete the protein in the presence of lactic acid. Now is that cool or what? We are currently trying to gather a list of all the possible proteins that could promote anti angiogenesis, and while this is a tedious task, we are getting through it. Once the list is put together, we have to go though it and try to weed out the most promising proteins. [EDIT] We talked about what our genetic circuit would be, discussing what bio bricks were and how we would need a promoter and a gene bio brick. These would code for out protein which at this point we had not decided what anti-angiogenesis protein we would use. We discussed the idea of using target therapy. - Traelyn Week 3 Now that we have decided on the type of biological machine we want to create, it is time to start doing some research. The group talked and we have decided we would like our bacterium to choke (for lack of a better word) cancer cells. We would do this by creating bacteria that contains an anti angiogenesis inhibitor, thus causing the blood vessels to retract and the cancerous tumour to starve.We also watched a TED Talk (http://www.youtube.com/watch?v=C_5Z31mUmtc) During this meeting that better explained anti angiogenesis and the success different scientists have had in treating cancer with anti angiogenesis drugs. It is truly amazing what some have been able to do! For example, there was this one experiment done on a dog with a Malignant Neurofibroma tumour growing on its shoulder that had spread to its lungs. Milo the dog had only a few months to live, however after being given a cocktail of anti antigenic drugs, the growth of the tumour slowed. In the end, the scientists were able to extend Milo's life by six times what the veterinarian had predicted. Now that is incredible!After finishing the TED talk, we ended the meeting. Everyone was supposed to go home and do some more research on anti angiogenesis. - Traelyn Week 2 After some discussion, the majority of the group decided we needed to come up with some different project ideas. It turns out that our seemingly brilliant idea to create a biological machine that can help celiacs digest gluten wasn't so brilliant. An IGEM team in 2011 engineered an organism to do just that, making it a completely unoriginal idea (and we want our idea to be original). So sadly, we had to go back to the drawing board and start thinking of some new ideas. Finally, after some very roundabout conversations, it hit us. We wanted to create a bacterium that could detect and help fight cancer! - Jamie Week 1 Today was my first IGEM meeting and man oh man was it exciting! Synthetic biology is probably one of the coolest things I have ever heard of, and I can't believe I'm getting the chance to divulge deeper into this incredible science. I mean, looking at what groups are trying to accomplish with synthetic biology is mind blowing. I especially liked the bacteria Lethbridge's IGEM team created; what a great idea to try and get a bacterium to produce insulin for people who can't. I hope that our team can come up with an idea for a bacterium that could potentially help people that are suffering from medical conditions. So far we have discussed looking into creating either a biological machine that could break down gluten or an exothermic bacteria that could help melt ice. - Jamie Scroll Retrieved from "http://2014hs.igem.org/Team:RAMNOTIREN_CALGARY/Noteboo1287Guelph CentennialCVICentennial Collegiate and Vocational InstituteHigh School001http://2014HS.igem.org/Team:Guelph_CentennialCVIhttp://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165020140High School1286HSAAHNU AnhuiHigh School Affiliated to Anhui Normal University Wuhu, Anhui, China ahsdfz.netHigh School801BBa_K1286999http://2014HS.igem.org/Team:HSAAHNU_AnhuiBBa_K1286000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164920140High SchoolLog in   Team:HSAAHNU Anhui From 2014hs.igem.org Team link goes here In-page anchor goes here Wikipedia iGEM 2014 HS Wiki other team's link { "title" : "Main | HSAAHNU" } Retrieved from "http://2014hs.igem.org/Team:HSAAHNU_Anhui/Team From 2014hs.igem.org TeamAlmost all team members “disappeared” during the division season, due to time limits or similar reasons. Eventually only one student on the original team roster, namely me, Minsheng Liu, stayed. Luckily, a student from Shenzhen Foreign Language School, Yuying Zhang, aided me designing our project.Later I conducted basic wet experiments under the supervision of a graduate from Anhui Normal University. However, due to financial and time reasons, we decided to give up the experiment.Members 顾涛 Gù Tāo Team Leader & Instructor Tao Gu is a biology teacher of High School Affiliated to Anhui Normal University. 杨如意 Yáng Rúyì Advisor Professor Yang is the department head of College of Environmental Science and Engineering of Anhui Normal University. 郭富裕 Guō Fùyù Instructor Fuyu Guo is a graduate of College of Environmental Science and Engineering of Anhui Normal University. He is the supervisor of our wet experiments. 刘闽晟 Líu Mǐnshèng Student Minsheng Liu is a grade 11 student of High School Affiliated to Anhui Normal University. He is responsible for the project design and wiki development. 张钰莹 Zhāng Yùyíng Yuying Zhang is a grade 10 student of Shenzhen Foriegn Language School. She is responsible for the project design. { "title" : "Team | HSAAHNU"}Retrieved from "http://2014hs.igem.org/Team:HSAAHNU_Anhui/Tea1293HUNGENIOUSRadnoti Miklos Experimental Grammar School Tisza Lajos krt. 6-8. H-6720 SZEGED http://www.rmg.sulinet.huHigh SchoolDeveloping a protective probiotic bacteria against Crohn's disease801BBa_K1293999http://2014HS.igem.org/Team:HUNGENIOUSBBa_K1293000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165620140High SchoolLog in   Team:HUNGENIOUS From 2014hs.igem.org Treatment of Crohn's Disease by Probiotic E.coli Bacteria Introduction Thanks to Our Sponsors Video Presentation Welcome! This is the first Hungarian iGEM HS team's homepage. During the time we were searching for ideas, we found out that Crohn’s disease makes a lot of people’s lives difficult with its serious effects in the gastrointestinal tract. Thus we have made a resolution to engineer a genetically modified probiotic bacteria, which could reduce the disease’s symptoms by producing a substance called N-acetylglucosamine from natural chitin resources. These bacteria could be transplanted into the human intestines, by the means of yoghurts or capsules. With this method it would be much easier to treat patients with Crohn’s disease in the future. Retrieved from "http://2014hs.igem.org/Team:HUNGENIOUShttp://igem2.mabite.info/images/brclogo.gif1254TP CC-SanDiegoTorrey Pines High School Canyon Crest AcademyHigh SchoolEngineering E. Coli Capable Of Extracellular Secretion Of Mycotoxin Detoxifying EnzymesMicrofungi that produce harmful mycotoxins flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure causes 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to be secreted to extra-cellular space by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to these proteins will have an immense commercial, industrial, agricultural, and health impact.1951BBa_K1254999http://2014HS.igem.org/Team:TP_CC-SanDiegoBBa_K1254000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161020140http://2014hs.igem.org/files/presentation/TP_CC-SanDiego.pdfhttp://2014hs.igem.org/files/poster/TP_CC-SanDiego.pdfHigh SchoolLog in   Team:TP CC-SanDiego/Journal.html From 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM Meeting Notes Lab Notebook Protocol Meeting Notes Notes Taken During our Meetings Lab Notebook E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes Protocol The Process to Complete our Project Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDiego/Journal.htmlFrom 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM See Statistics Statistics Statistics that are quite relevant to the nature of this experiment Toxicity of aflotoxin is 10 times that of hydrocyanic acid and 68 times of arsenic. This disease is the third-leading cause of cancer death globally according to WHO (2008), with about 550,000–600,000 new cases each year. Engineering Mycotoxins affect nearly 15-20% of all crops. Engineering E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes Microfungi produces harmful mycotoxins that flourish on improperly-stored nuts, grains, meat, and dairy. They especially thrive in developing countries, where the lack of advanced food storage and mycotoxin exposure cause 40% of the diseases. To lessen the problem, our team engineered E. coli strains using synthetic biology tools to produce chimeric mycotoxin-degrading fungal enzymes, Aflatoxin-Detoxifizyme (ADTZ) and Zearalenone Hydrolase (ZHD101), which are designed to secrete extracellularly by fusing with secretion signal peptides from alpha-amylase and beta-lactamase. In this study, we have successfully generated synthetic genetic materials to produce four chimeric mycotoxin-detoxifying enzymes. The levels of extracellular secretion is also characterized and analyzed. The project will allow a mass production of detoxification enzymes in a cost effective way, preventing the squandering of harvested crops, and limiting mycotoxin-related diseases. Increased access to these proteins will have immense commercial, industrial, health, and agricultural impacts. Sponsors Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDieghttp://2014.igem.org/wiki/images/2/23/NEW_LOGO.pngOutreach.html From 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM Form an iGEM Team Collaboration Lectures How to Form an iGEM team How to form an iGEM team from scratch This year TP_CC-San Diego made the video, "How to Form an iGEM Team From Scratch", that focuses on how to make an iGEM team, get a mentor, do research, and overcome some of the obstacles along the way (e.g. financial). The video provides various tips and advice to create and self-support and support a science team like iGEM from the members of the team who previously were a part of iGEM.The purpose of the video is to propagate research to many schools through setting them up for iGEM.We have currently emailed this video along with an introduction to iGEM and an offer to guide interested students in the process of forming a team to 300 schools in California . For the underprivileged ones, we offered them assistance in setting up a team or just getting the students into any sort of research.We hope to see new inspired teams from California, and hope to facilitate interested students’ pursuit for research and scientific discoveries. Collaboration Education is collaboration This year our team is a collaboration between two neighbor schools - Torrey Pines High School and Canyon Crest Academy. Since Torrey Pines had a successful year last year and had experience, this year we reached out to our neighbor school and introduced them to iGEM. Lectures were hosted at Canyon Crest Academy, and laboratory works were done at Torrey Pines High School and then the Simpson Joseph Laboratory at University of California, San Diego. Students from both schools worked together on various aspects of the project and split up the work. The collaboration allowed us to explore different points of view and combine the abilities of all the members to form a well rounded and efficient team. Lectures Pursuing knowledge In the beginning of the year, many students who were interested in participating in iGEM were unfamiliar with the details about synthetic biology. We contacted UCSD graduate and Ph.D students from biochemistry labs and the mentors from the Hasty Lab at UCSD provided us various lectures on synthetic biology. These lectures mainly focused on teaching the students basic lab skills and the science behind the lab processes. Students also studied from DNA Science: A First Course, Second Edition by David Micklos and Greg A. Freyer. The lectures were open to anyone in the neighborhood, including non-iGEM members interested in learning about synthetic biology and students from different high schools. Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDiego/OutreacResults.html From 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM Data And Analysis Conclusion and Future Directions PCR Amplification Amplifying vectors and synthesized genes to have desirable tails. By adjusting PCR settings, we were able to amplify the vector and inserts successfully. Successfully amplified inserts and vectors with the desired ends were used for Seamless Cloning. Transformation After seamless cloning; E. Coli uptake of assembled plasmid For AA, AZ, BA, and BZ, at least 10 colonies were grown for each LB+Amp agar plate that cultured the four different types of transformations. (Note: AA refers to alpha-amylase-ADTZ, AZ refers to alpha-amylase-ZHD101, BA refers to beta-lactamase-ADTZ, and BZ refers to beta-lactamase-ZHD101.) Colony PCR PCR of colonies as template DNA After the transformation in an LB+Amp agar plate, numerous colonies were selected individually for Colony PCR. The columns with the bands superficially signifies that the desired plasmids were constructed. The colonies that were reported positive were plated separately and re-verified. AA: 2, 6, 13, 14, 19 || AZ: 1, 2, 4, 5, 6, 7 || BA: 6, 7, 8 || BZ: 2, 3, 5, 6, 7 Sequence Verification Sequenced colonies vs. Theoretical plasmid Using NCBI's Align BLAST, the sequencing data was compared with the theoretical plasmid data. If there was a 100% match, we declared these colonies to be successful. All the mutations that occurred in our set of colonies were deletion or addition mutations. AA13, AA14, AA19, AZ1, AZ5, AZ6, and AZ7 were 100% congruent. AA2 was determined to be a very likely because the error occurred in the region where the sequencing data had various "N"'s and the ab1 peaks were fluctuant. None of the beta-lactamase-attached colonies seemed to have the correct plasmids due to point mutations in the ORF. AA: 2 (possible candidate), 6, 13 (100%), 14 (100%), 19 (100%) || AZ: 1 (100%), 2, 4, 5 (100%), 6 (100%), 7 (100%) || BA: 6, 7, 8 || BZ: 2, 3, 5, 6, 7 SDS-PAGE gel 1 Visualization of presence and size of protein of interest We tested four parameters : pellet, supernatant, IPTG-induced, and non-induced. For both pellets and supbernatants, IPTG-induced was hypothesized to have a stronger band for SDS-PAGE, with visible difference in both the pellet and the supernatant. The first gel did show sharp difference between pellet and supernatant, but did not show a clear difference between IPTG-induced and non-induced. It implies that the plasmid was neither expressed nor secreted. Western Blot Visualization of presence and size of protein of interest The Western Blot of two AZ colonies and two AA colonies were studied, both IPTG-induced and non-induced. The bands show that the plasmid was expressed at the very least, since it showed a stronger band in the presence of IPTG in the pellet. However, for the supernatant no bands were visible for neither IPTG-induced, nor non-induced, which shows poor secretion. Possible explanations for this results include: 1. The construct and the attached signal peptides are not functional and do not secrete. 2. E. Coli's secretion system is generally less effective. 3. The proteins are stored in the periplasm and not excreted to the extracellular space. 4. The protein's structure (size or chemical properties) somehow interfered with the ability of it to be transported out. SDS-PAGE gel 2 Visualization of presence and size of protein of interest The SDS-PAGE was tried again. This time, only IPTG-induced supernatant was taken into account to compare amongst colonies and narrow down the list of colonies needing to be tested. Some colonies showed expression of proteins of the approximately accurate size in the supernatant, while the previous gel showed none. The ones that did were selected, and of those, a western blot of both IPTG-induced and non-induced was suggested. Conclusion And Future Directions The fight will continue. Two of the four final constructs, AA and AZ, were sequence verified 100%. The SDS-PAGE analysis initially showed inconclusive levels of expression and extracellular secretion of the alpha-amylase attached proteins. The Western Blot, however, confirmed that expression is present, but did not show any signs of secretion. A secondary SDS-PAGE was done for more induced colonies, and there were signs of secreted protein that are to be explored. Future directions include: 1. Successful colonies from SDS-PAGE Gel 2 need to be tested further with Western blotting and compared amongst pellet, supernatant, induced, and non-induced. 2. Try to succeed in the construction of beta-lactamase signal peptide containing recombinant plasmids, since we were not able to for now. Then compare alpha-amylase signal peptide and beta-lactamase signal peptide levels of secretion. 3. Do protein functionality assay of secreted ADTZ and ZHD with HPLC. 4. Try other signal peptides, such as the TAT-dependent pathways or SRP-dependent pathways. 5. Genetically modify plants directly instead of modifying E. Coli. 6. Try organisms with more pronounced secretion systems such as yeasts and fungi. Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDiego/ResultTheTeam.html From 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM Gha Young Lee Why I'm Doing iGEM: The creativity and fresh applications of synthetic biology that iGEM inspires from young scientists like us drove me toward iGEM. Also, as the team captain this year, I want to be able to spread this type of iGEM spirit to the community around me. Coolest thing about science: It fits and it works, but at the same time it is unknown and sublime. It is also a very effective tool that transforms an idea to something tangible valuable with the power of nature. Hobby: Listening to international music, diving, stargazing and... secretly dancing by myself when I’m feeling good. Career plans: I plan to pursue medicine with a focus on nano and biotechnology. Skills: math, physics, organization Favorite Quote: "I owe all my success in life to having been always a quarter of an hour beforehand." - Horatio Nelson Victoria OuyangWhy I'm doing iGEM: iGEM is a great program that allows us to apply our biology knowledge to solve problems that exist in the world. It also requires a much deeper understanding of specific biology concepts than is taught in class. Most importantly, I really enjoy the hands on lab experience that it involves because I can link together what I learned and apply the knowledge practically. Coolest thing about science: There is always something more to know. Hobbies: watching dramas, paintingCareer plans: I plan to major in an engineering field. Skills: math, biology, piano Favorite quote: “Optimism is the faith that leads to achievement. Nothing can be done without hope and confidence.” – Helen Keller Christina Baek Why I'm doing iGEM: I am fascinated by bioengineering and how it allows life to become one complex machine that can be modified and edited. iGEM also gave me a wonderful opportunity to gain more experience in the lab. Coolest thing about science: Science gives the best answers to the question "Why?".Hobby: violin, listening to music, sleeping, eating, walking in the rain, making candles, reading, drawing, thinking Career plans: undecided Skills: overthinking, circular thinking, doubting Favorite quote: Life is not a marathon. Anisa Malangone Why I'm Doing iGEM: I love learning, especially about science, and have had some great experience in a lab at UCSD with neuroscience, but I was also drawn to synthetic biology and iGEM was the perfect way to learn more. Coolest thing about science: How much is unknown, and changing and yet to be discovered as well as how different areas of science link together. Hobby: watching movies like UP, reading things by C.S. Lewis, playing word games like hink pink, collecting quotes with my best friend, going on adventures to different counties, listening to Simon and Garfunkel, hiking at Torrey Pines reserve, having long talks, and of course synthetic biologyCareer plans: MD (neurosurgeon) & or PhD (focusing on either neuroscience or bioengineering)Skills: cooking 30 minute meals in 20 minutes, memorizing 2,500 vocabulary words (and forgetting a plethora of them), seeing the little things in life, solving hink pinks, eating 6 times a day, being there for anyone at anytimeFavorite Quote: So we fix our eyes not is what is seen but what is unseen. For what is seen is temporary but what is unseen is eternal. 2 Corinthians 4:18 Brandon Read Why I'm Doing iGEM: I am doing iGEM to carry on the legacy left by our previous team leader, Tareq Younis, and to learn more about the synthetic biology field while practicing skills involved in participating in the jamboree. Coolest thing about science: The coolest thing about science is that it (in most cases) seeks to find the truth.Hobby: My hobbies involve being a personal pal for friends or online strangers who need help finding direction in their lives, playing League of Legends, reading the news, and seeing my S.O. as frequently as possible.Career plans: My career plans involve making use of any opportunity I see fitting as a challenging and rewarding path towards something greater.Skills: Skills? Please, I am dabes. Favorite Quote: "To hell with circumstances; I create opportunities."- Bruce Lee (rest in peace). Evan Kallenberg Why I’m Doing iGEM: I am doing iGEM because it is an amazing opportunity to perform in-depth lab research and provides insight into a field of study that I am very interested in pursuing.Coolest Thing About Science: The coolest thing about science is that science attempts to explain why and how everything in the universe happens. Also, for those who work hard, science can be the key to some of the most profound discoveries in the world. Hobbies: My hobbies include backpacking, rock climbing, reading, snowboarding, and spending time with friends.Career Plans: I am interested in pursuing a career in biochemistry or astrophysics (or both if possible).Skills: rock climbing, snowboarding, and rowing.Favorite Quote: "I believe every human has a finite number of heartbeats. I don't intend to waste any of mine." (Neil Armstrong) Edward Doyle Why I’m Doing iGEM: I joined on a whim, really.Coolest Thing About Science: In the whole of the universe, tucked away in a corner, there is a small sphere – a planet. The notion of this planet is to produce, grow, multiply, and kill the curious phenomenon of life. The life desires dreams, to imagine, for without dreams the possible is boring, yet not all dreams are possible. Experimentation serves to rein in imagination, science to navigate between the two poles, to reconcile what might be with what is. Hobbies: Reading, trumpet, music composition, handwork art, math, and physics.Career Plans: None. Skills: Parallel parking. Favorite Quote: “But then to what end,” asked Candide, “was the world formed?” “To make us mad,” said Martin. – Voltaire, Candide Michael Margolis Why I'm Doing iGEM: I’m doing iGEM because it is a great opportunity to do interesting science work with friends. Coolest thing about science: I think the coolest thing about science is how far reaching its application are, from helping eradicate diseases to allowing us to have computers and artificial intelligence.Hobby: A hobby of mine is playing the violin and viola. I love musical instruments and the expressiveness they allow. Career plans: I plan to become a pediatrician and hopefully do research on the side. Skills: Some of my skills include breathing and playing musical instruments. Favorite Quote: My favorite quote is one from Dr. Seuss: “Be who you are and say what you feel, because those who mind don't matter, and those who matter don't mind.” Daniel Sands Why I'm Doing iGEM: I’m doing iGEM because it is a great opportunity to do interesting science work with friends. Coolest thing about science: I think the coolest thing about science is how far reaching its application are, from helping eradicate diseases to allowing us to have computers and artificial intelligence.Hobby: A hobby of mine is playing the violin and viola. I love musical instruments and the expressiveness they allow. Career plans: I plan to become a pediatrician and hopefully do research on the side. Skills: Some of my skills include breathing and playing musical instruments. Favorite Quote: My favorite quote is one from Dr. Seuss: “Be who you are and say what you feel, because those who mind don't matter, and those who matter don't mind.” Dane Malangone Why I'm Doing iGEM: Initially I joined not only because I was interested in the material, but also because several friends recommended it to me. Coolest thing about science: I'm not sure if I can name just one thing about science; there's too many to choose from. However, a couple of my favorites include: Men on the moon or eradication of smallpox. Hobby: I enjoy reading. Career plans: I want to become an oncologist. Skills: I can camp and cook. Favorite Quote: "Teach thy tongue to say, 'I do not know,' and thou shalt progress." –Maimonides Alexander Wen Why I’m Doing iGEM: I joined iGEM because I saw it during club day and I thought it would be interesting. After we had meetings, I got hooked to the whole idea of synthetic biology and now I’m here doing iGEM.The Coolest Thing about Science: The coolest thing about science is that with research and experimenting you can unlock the secrets of how the world works. You are also able to use your imagination to change the way people look at life (through synthetic biology)Hobbies: My hobbies are playing baseball, playing piano, and baking. Career Plans: I hope to do something related to biology for my career. Since I was a toddler, I was always intrigued by the field of biology. So I’m going to go with my gut and pursue careers similar to my interests.Skills: I feel that I get along with a group of people, and I’m able to communicate with all types of people. I believe that my main strength is in the teamwork department.Favorite Quote: “Education is the ability to listen to almost anything without losing your temper or your self-confidence”-Robert Frost Ellen Ouyang Why I'm doing iGEM: How is manipulating the structure of living organisms to fulfill a decided purpose not appealing Coolest thing about science: How it explains the universe and the discoveries that seem magical. Learning about the process behind accepted facts of the world (and contradictions) is fascinating. New wondrous advancements exist when previously thought highly improbable.Hobby: Drawing, Reading, Playing instruments, Diving in the interwebs, Making paper models of icosahedrons Career plans: Interested in medicine Skills:Arts, Violin, Piano, Guzheng, Hand crafts, Filling out informational biographiesFavorite quote: All the world's a stage, and all the men and women merely players: they have their exits and their entrances, and one man in his time plays many parts, his acts being seven ages ~Shakespeare Nicki Nikkhoy Aidan Kahng Why I'm Doing iGEM: I am doing iGEM because I have always been interested in bioengineering yet haven't had much lab experience. Coolest thing about science: Nothing is everything. Hobby: Running, Table Tennis, Ultimate Frisbee Career plans: Skills: Not knowing what I'm good at. Favorite Quote: "War does not determine who is right - only who is left." -- Bertrand Russell Mokhshan Ramachandran Brian Choi Hobby: Online Chatting and watching Big Bang Theory What I want to major in college: Chemical or Biomedical EngineeringFuture job: Husband Abilities: singing in high pitch, eating 210 pieces of hot cheetos a day, knitting, passive aggression Interesting facts: I was casted in The Light in the Piazza as Giuseppe Naccarelli.Favorite Quote: "Hard work beats talent when talent does not work hard." Zilu Pan Why I’m Doing iGEM: I am doing iGEM because I think Synthetic Biology is a fascinating subject in that we are able to change the world and benefit lots of people by investing efforts into gene engineering; manipulating simple organisms may turn out to have a more profound impact than we have ever imagined.Coolest Thing About Science: The coolest thing about science is that everyday, scientists are discovering new things that could potentially benefit people around the world, providing powerful solutions to questions that were impenetrable in the past.Hobbies: My hobby is to do Chinese Calligraphy during my spare time and watch an episode of TV here and there. I also enjoy reading science articles in the news.Career Plan: I plan to pursue a career in the realm of finance and investments, discovering talents and investing in those that can potentially change the world means a lot to me.Skills: I know how to play piano, art and calligraphy. I know design and layouts due to my experiences as a yearbook editor. And of course, I have acquired many laboratory skills throughout my high school journey.Favorite Quote: “God has given you one face, and you make yourself another”-William Shakespeare Melinda Wang Why I'm Doing iGEM: I want to learn more about the field of synthetic biology and gain more lab experience. Coolest thing about science: How it can be used to help others by extending their lifespan or improving their lifestyle. Hobby: Drawing and writing. Career plans: A career where I leave a positive impact on society, or at least on the people within my sphere of influence. Skills: I know how to swim, dance, and play piano and clarinet.Favorite Quote: “One of the things I learned when I was negotiating was that until I changed myself, I could not change others.” - Nelson Mandela Julia Wisnia Why I'm Doing iGEM: I love biology, and the wonders of biological engineering excites me.Coolest thing about science: It is limitless. Hobby: Drawing and writing.Career plans: Brain surgeon.Skills: Working well under pressure Favorite Quote: "Be yourself; everyone else is already taken." - Oscar Wilde Previous Next Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDiego/TheTeaProject.html From 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM Background Idea How Impact Attributions Background The Project Background and Explanation AflatoxinB1-Detoxifizyme is produced by Armillariella Tabescens intracellularly. Its gene is analyzed to have no introns and has been recombined into a plasmid before. Extraction is rather difficult and commercialization has not been able to be done yet. Zearalenone Hydrolase is produced by Clonostachys Rosea. Its gene is also analyzed to have no introns and has been recombined into a plasmid before. Similar to ADTZ, commercialization has not been done yet. Natural E. coli secretes various proteins by signal peptides that recognize translocons. We focus on the SEC pathway that lets the protein fold in the periplasm first, as it usually results in a more accurate folding, correct formation of disulfide bridges, minimizes degradation, and is less likely to result in accumulation. Idea E. Coli Capable of Extracelluar Secretion of Mycotoxin Detoxifying Enzymes Our idea is to attach signal peptides to the N-terminus of the detoxifying enzymes. We used alpha amylase signal peptides and beta-lactamase signal peptides. The original proteins for these signal peptides are secreted out naturally by E. Coli, especially in strain K that we used for our project. They both follow sec-dependent pathways. In the case of the beta-lactamase signal protein, cases of successful fusion proteins secreted by the beta-lactamase signal protein have been shown. Successful continual excretion of the mycotoxin degradation enzymes can be purified in mass amounts periodically, or the chimeric E. Coli can be placed directly on crops to prevent and combat mycotoxin exposure. Since there is no current cure to mycotoxin exposure, enhanced prevention with successful secretion will be very influential. How The Process to Complete our Project In order to achieve our goal, we modified our vector and synthesized genes with reverse PCR and primers with specific tails. After the parts were assembled by Seamless Cloning. The parts were transformed in an LB+Amp agar plate, and colony PCR was performed for an initial verification step. After the initial verification, the plasmids were purified from the colonies that were declared positive and compared to the theoretical sequences. The final colonies were then grown, and each culture was separated into two categories - IPTG induced, and non-induced. During the log-phase, induced and non-induced cultures were then separated into cell pellets and supernatant. SDS-PAGE gels were run for each induced pellet, non-induced pellet, induced supernatant, and non-induced supernatant for comparison. Induced and non-induced were compared to verify the functionality of the vector, and the pellets and supernatants were compared for excretion. The whole expression and secretion were verified by Western Blotting with anti-his antibodies. Impact The solution to mycotoxins. Currently there is no cure for mycotoxin exposure and the symptoms that follow after, which is predominantly liver cancer. Nor is there an effective way of dealing with the amount of food that is affected annually. Improvements in the storage methods, for example, is ineffective in underprivileged communities and societies, and fungal infection of food is a persistent problem. Therefore, with the improved secretion, there will be an estimated 15-20% gain of crops, industrial and financial gain especially in third world countries, up to 28% less cases of liver cancer due to mycotoxins, and in general, significantly less chronic exposure. Attributions Teamwork, education, and assistance The TP_CC-SanDiego iGEM Team came up with the idea during its brainstorming meetings and performed the laboratory work. The team made group decisions about the direction of the project, collaboratively troubleshooted, and as a group, put together the data and presentation. The Simpson Joseph Laboratory of UCSD provided the students with the lab space, equipments, and various reagents that allowed the students to perform the wet laboratory work. The members of the Joseph Lab gave the team many tips and protocols. The Jeff Hasty Laboratory of UCSD provided the team and others with lectures and past iGEM experiences that helped them greatly. Dr. Chang-Ho Baek from Thermo Fisher mentored the team throughout the project, and gave them lectures and advices for troubleshooting when there were obstacles. Thermo Fisher provided the team with various reagent donations. Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDiego/ProjecSafety.html From 2014hs.igem.org Home Project Results The Team Journal Outreach Safety iGEM Safety Proper Precautions when performing our experiment Retrieved from "http://2014hs.igem.org/Team:TP_CC-SanDiego/Safet1291AUC TURKEYAtlantik Educational Institution Ahmet Ulusoy High School, Ankara, Turkey www.ahmetulusoy.k12.trHigh SchoolDegradEColorResponse systems possess great significance in the validation of designed systems in the current state of synthetic biology. This year we, team AUC_Turkey, focused on the improving of response system. In our appraisal, we recognized that it would perhaps be easier to degrade dye instead of producing fluorescent proteins or pigments. In our new response system, the module will primarily be transparent and will change color according to enzyme activity thus creating a distinction between the initial and secondary modules. The process will allow a relatively faster response through the cleavage of the enzymes. The usage of different types of enzymes will result in differentiation in the cleavage and through the examination of the color; it will be possible to make find an equivalent match on the scale. DegradEcolor will be a fast response system as an alternative to the most popular response systems1931BBa_K1291999http://2014HS.igem.org/Team:AUC_TURKEYBBa_K1291000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165420140High SchoolLog in   Team:AUC TURKEY/Results/Protein Analysis From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ResultsCloningProtein AnalysisFunctional Assay Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Results/Protein_AnalysisCloning From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ResultsCloningProtein AnalysisFunctional AssayCLONING THE TOP10 STRAINS TRANSFORMATION Plasmids containingthe BBa_K1291071 (Horseradishperoxidase composite part) and BBa_K1291299 (Lignin Peroxidase composite part) parts weretransferred into our T10 strains, which were preparedthrough the competent cell preparation protocol, using transformation protocols. Colonies were seen in agarplates that had the appropriate antibiotics after a 16 hour long incubationprocess.   PLASMID ISOLATION The colonies takenfrom plates were incubated in LB Broth containing AMP for 16 hours. DNA isolation was doneon the bacteria culture to checkthe efficiency. The adequacy of the amount of thenucleic acid concentration for digestion was met in the acquired samplesand it was seen that thesamples had peaks at OD260, which was captured by thenanodrop.   Digestion and DNA Electrophoresis Gelelectrophoresis was conducted on the samples so thatthe enzymes which were digestedwith the EcoRI and PstIcould be verified in terms of their wavelength. The acquired results showed that theplasmid transfer process was successful for the Top10 strainswhile the NEB10 strains, which were taken fromthe stock, were disfunctional thus were unusable.    CLONING THE BL-21 STRAINS Sincewe require a high concentration of proteins in our project, we decidedto use theBL-21 strain of E.coli instead of Top10 strains due to itsenhanced protein synthesis capacity. Plasmids containing the BBa_K1291071 (Horseradish peroxidase composite part) and BBa_K1291299 (Lignin Peroxidasecomposite part) parts were transferredinto our BL-21 strains, which were prepared throughthe competent cell preparation protocol, using the transformation protocol. The coloniessuccessfully acquired after a 16-hour long incubation process.   PLASMIDISOLATION                                                                                                                                                                                                                                          The chosen colonies were incubated for 16-hours in AMP containing LBBroth. DNA Isolation was done totest the efficiency of the transformation. A peek at OD 260 was observed and sufficientlevels of DNA concentrationwas acquired to continue todigestion.  Digestion and DNA Electrophoresis Gelelectrophoresis was conducted on the samples so thatthe enzymes which were digestedwith the EcoRI and PstIcould be verified in terms of their wavelength. The acquired results showed that theplasmid transfer process was successful for BL-21 Strains and it was possibleto continue to the nextstep.Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Results/CloninTeam/Acknowledgements From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube TeamParticipantsGalleryAcknowledgementsThe participation in theiGEM competition is a wonderful experience for us. In the past few month, wegot many new insights into synthetic biology, worked hard in and outside of thelaboratory and had a lot of fun together. Now, we are glad to meet new people at the iGEM 2014 High School Jamboree  Weare happily looking forward to learning more about the interesting projects ofthe other iGEM high school teams and gradeupourlanguageskills.While taking part in the iGEM competition, we received great support andtherefor would like to kindly acknowledge the following groups andorganizations:§  Prof. Dr.Esra Gündüzfor the great opportunity to do our lab work in his lab at the Turgut OzalMedicine Faculty,§  Our Sponsors BTC ,MathWorks,TurgutOzal Medicine Faculty and Atlantik Educational Instution  for the financial andmaterial support,§   Dr. Ayşe Çelik forhelp our modelling§  Prof.Dr.Mehmet Gündüzfor him supports .§  Yrd.Doç.Dr.Ömer FarukHatipoğlu for mentoring .§  Doc.Dr.Murat Yağmurcafor guidance.§  Burak Yılmaz for helpus with SENTEGEN Company.§  Prof.Dr.Sadık Çiğdemfor accepting our interview offer.§  Prof.Dr.CihangirTanyeli for gave us really important informations and guidance.§  Doc.Dr.ZeynelSeferoğlu for introducing new tips.§  Doç.Dr.HüsamettinErdamar for provide good contidions.§  Yrd.Doc.Dr.MuradiyeAcar for guidance §  Esin Demir for supportingus §  Alfa Paintingmanagers and workers for their interviews and giving good informations.§  Yrd.Doc.Dr. YaseminYüksel for suppliying colors.§  Doc.Dr.Kadir Demircanfor giving ideas for wikis.§  Doc.Dr.Orhan Başboyafor suppliying colors.§  OPET workers for freetea.§  Security officier forproviding security in faculty. Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Team/AcknowledgemenProject/Parts From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ProjectIntroductionMechanismModellingParts  Part Design:In ourproject there are 4 different parts. Two of them composite parts that arefunctional and other 2 parts are our enzymes that are proteins. We designed ourparts with Gene Designer 2.0 program. We found our enzyme sequences from NCBIPubMed Database. In our project we need that our proteins to be produced and weneed our enzymes so much because they do the all work. For more production wechose the strongest promoter there is which the strongest constitutive promoterJ23119 in Part Registry is. When we design our parts we received thisinformation that; scientists use inducible promoters for proteinover-production so in our design, right after the promoter and also in front ofour RBS, we added BamHI restriction enzyme site.After the RBS we added our enzymes sequences HRP-C and Lip-1 and after theenzymes sequences we added His-Tag ( 6 Histidinesequences). Because we planned to do Western-Blotting technique in order toshow that our proteins are produced successfully.   1)    HorseradishPeroxidase-C Part Design:  2)    Lignin Peroxidase-1  Part Design :                 Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Project/ParFrom 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Introduction Story of a color wizard Notebook The day by day process Participants Hardworking guys Assay Are those proteins working? Future Plans What we are looking forward to Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEhttp://2014hs.igem.org/wiki/images/c/c5/IGem_Logo.gifNotebook/Timeline From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Lab GarageNotebookProtocolsSafetySeptember16Itwasn’t easy for us to get all team members in one place who has adopted theidea of we will start to work for next year straight away right after the‘Jamoreee. September sixteen the date schools get opened, was the meeting dayfor team members who were at their hometown for the summer holiday. We delayedhaving new members alongside the idea of working with the experience of oldmembers still itching for iGEM.Actuallyour aim was to get started quickly thanks to the tons of new ideas for our nextproject from everyone in the team. But that was not the case since it isagainst the laws of nature! The alternative ideas left from last year wasalways a possible b plan for us, but still we were aware of the fact that if wewant to get some awards and get a high rate, these were not suitable at all. Westarted to think for new ideas...September 23We wanted make the best of the time wehad ideas from previous year alongside of new and fresh ideas. We decided towork separatly since we thought it was too early to make our meetings weekly.Some members prefered scaning new datas seeking for inspiration, unlike otherswho chosed to improved their fantastic ideas. Our ideas were not much butmodest. These are some highlights from these ideas: -Paper cleaning bacteria: This idea wasthe dream of our team since the beginning. The bacteria we produced wassupposed to clean the paper by simply destroying the ink on it. Incorrectoutputs must hurt our team. - Bran preventing bacteria: It isobvious that the bran produced by the outer layer of the headskin is one of thebiggest issues makes mankind suffer. Variety of products in the market anddozens of clinic researches seeking for a solution shows the importance of thisissue. We decided to prevent this brans made of deadskin with the bacterium weproduced. We gave up this idea because brans which are the main food resourceof the mites could go through some metabolic processes. - Bacterium to support Plants defense:This project started with the idea of protecting plants from invasions ofinsects and then aimed to program our bacterium in a way that they will makesure plant starts to defend itself earlier and stronger. - E.coli designed for X-tremeconditions: E.coli the worker of the synthetic biology they ara likely to geteffected by x-treme conditions and give unwanted bad results for the project.Increasing the durability of E.coli would encourage its usage in the worldofsynthetic biology. We aimed to create an alternative system which was goingto increse its durability with the part that we produce alongside of the gensof the project. We are convienced on the fact that using the genom of abacteria which was in the records book of guiness and resistant to x-tremeconditions such as “deinococcus radiodurans”.  November 16-We didbrainstormingJanuary 22-Everyonecame with 5 project ideas at leastMarch 3-We startedto net our projectApril 27-We decideour Project ‘’DegradEcolor’’1 Haziran-We sterilized our lab andmaterials4 Haziran-We bought our airlineticket.7 Haziran -We went to sponsorshipinterview.We agred with BTC construction company10 Haziran We made liquid culture11 Haziran-We prepare competent cells12 HaziranWe did transformation HRPand LIP parts13 Haziran-We made liquid culture fromlast day’s transformation’s colonies and learnt how can we do western blotprotocol.14 Haziran-We did isolation of BL21 strain15 HaziranCBB results16 Haziran-Western Blot 17 Haziran-Wiki preperation18 Haziran-We did functional assay19 HaziranWe did wiki and humanpractises20 Haziran WIKIFREEEEZERetrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Notebook/TimelinTour From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Overview Results Future Plans Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/TouHuman Practices/Future Plan From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Human PracticesOutreachFuture Plan 1) Reporter Systems Overview:a)  Fluorescent Proteins: The green fluorescent protein (GFP) is a protein  thatexhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Although many other marine organisms have similar green fluorescent proteins,GFP traditionally refers to the protein first isolated from the jellyfish Aequoreavictoria. The GFP from A. victoria has a major excitationpeak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at509 nm, which is in the lower green portion of the visiblespectrum. The fluorescence quantumyield (QY) of GFP is 0.79. The GFP from the seapansy (Renilla reniformis) has a singlemajor excitation peak at 498 nm. In cell and molecularbiology, the GFP gene is frequently used as a reporterof expression. In modified forms it has been used to make biosensors, and manyanimals have been created that express GFP as a proof-of-concept that a genecan be expressed throughout a given organism. The GFP gene can be introducedinto organisms and maintained in their genome through breeding, injection witha viralvector, or cell transformation. To date, the GFP gene has beenintroduced and expressed in many Bacteria, Yeast and other Fungi, fish (suchas zebrafish), plant, fly,and mammalian cells, including human. MartinChalfie, OsamuShimomura, and RogerY. Tsien were awarded the 2008 NobelPrize in Chemistry on 10 October 2008 for their discovery anddevelopment of the green fluorescent protein.   a)      Luciferin: Luciferin (from the Latin lucifer,"light-bringer") is a generic term for the light-emitting compound found inorganisms that generate bioluminescence. Luciferinstypically undergo an enzyme-catalysed oxidation and theresultingexcited state intermediate emits light upon decaying to its groundstate. This may refer to molecules that are substrates for both luciferases and photoproteins. Luciferinsare a class of small-molecule substrates that are oxidized in thepresence of the enzyme luciferase to produce oxyluciferin and energy in the formof light. It is notknown just how many types of luciferins there are, but some of thebetter-studied compounds are listed below. There are many types of luciferins,yet all share the use of reactiveoxygen species to emit light.  b)      Xgal Reporter System: X-gal is an organiccompound consisting ofgalactose linked to asubstituted indole. The compound was synthesized by JeromeHorwitz and collaborators in Detroit, MI, in 1964. The formalchemical name is often shortened to less accurate but also less cumbersomephrases such as bromochloroindoxyl galactoside. The X from indoxyl may well bethe source of the X in the X-gal contraction. X-gal is much used in molecularbiology to test for the presence of an enzyme, β-galactosidase. It is alsoused to detect activity of this enzyme in histochemistry and bacteriology. X-gal is oneof many indoxyl glycosides and esters that yield insoluble blue compoundssimilar to indigo as a result of enzyme-catalyzed hydrolysis.[2] X-gal is an analog of lactose, andtherefore may be hydrolyzed by the β-galactosidase enzyme which cleaves the β-glycosidic bond in D-lactose.X-gal, when cleaved by β-galactosidase, yields galactose and5-bromo-4-chloro-3-hydroxyindole. The latter then spontaneously dimerizes andis oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an intensely blue productwhich is insoluble. X-gal itself is colorless, the presence of blue-coloredproduct therefore may be used as a test for the presence of an activeβ-galactosidase. This easy identification of an active enzyme allows thegene for β-galactosidase (the lacZ gene) to be used as a reportergene in various applications.  c)      GUS Reporter System: The GUS reporter system (GUS: β-glucuronidase) is a reportergene system, particularly useful in plant molecularbiology and microbiology. Several kinds of GUS reportergene assay are available, depending on the substrate used. The term GUS staining refers to the mostcommon of these, a histochemical technique. The purpose of this technique is toanalyze the activity of a promoter (in terms of expression of a geneunder that promoter) either in a quantitative way or through visualization ofits activity in different tissues. Thetechnique is based on β-glucuronidase, an enzymefrom the bacterium Escherichiacoli; this enzyme, when incubated with some specific colorless or non-fluorescentsubstrates, can transform them into coloured or fluorescent products.  d) Chloramphenicolacetyltransferase (CAT) System: Chloramphenicol acetyltransferase (or CAT) is abacterial enzyme (EC 2.3.1.28) thatdetoxifies the antibioticchloramphenicol and isresponsible for chloramphenicol resistance in bacteria. This enzyme covalentlyattaches anacetyl group from acetyl-CoA tochloramphenicol, which prevents chloramphenicol from binding to ribosomes. A histidineresidue, located in the C-terminal section of the enzyme, plays a central rolein its catalytic mechanism.The crystal structure of the type III enzyme fromEscherichia coli with chloramphenicol bound has been determined.CAT is a trimer of identical subunits (monomer Mr 25,000) and the trimericstructure is stabilised by a number of hydrogen bonds, some of which result inthe extension of a beta-sheet across the subunit interface. Chloramphenicolbinds in a deep pocket located at the boundary between adjacent subunits of thetrimer, such that the majority of residues forming the binding pocket belong toone subunit while the catalytically essential histidine belongs to the adjacentsubunit. His195 is appropriately positioned to act as a general base catalystin the reaction, and the required tautomeric stabilisation is provided by anunusual interaction with a main-chain carbonyl oxygen. CAT is used as areporter system to measure the level of a promoter or its tissue-specificexpression. The CAT assay involves monitoring acetylation of radioactivelylabeled chloramphenicol on a TLC plate; CATactivity is determined by looking for the acetylated forms of chloramphenicol,which have a significantly increased migration rate as compared to theunacetylated form. d)       Other Response Systems:Some fast response systems were for constructed based on traditional responsesystems like GFP or luciferin reporter system through protein engineering.Although these response systems give fast response, they have high costs and/ordifficulty in implementation. The systems are devised by major geneticscompanies that synthesize these systems using high cost equipment thusreflecting the costs to the buyers resulting in very expensive prices making italmost impossible for most of the iGEM teams to obtain them. 2) Advantages of DegradEColor:                a) Our system aims to operate faster because it focuses on theprencipal which is making the produced enzyme effect more substrata in whichare dyes instead of continuously producing new proteins.        Our system works in a way that is suitable for different dyes andperoxidase enzymes of the project it is going to be applied.         c)  Also our system ables scientists to; develop dyes suitablefor their works, and chose the suitable enzyme for their subject livingmaterial  3) Disadvantages of DegradEColor:  a) Our protein is produced byHRP-C enzyme which is taken by horseradish plant, and the other one LiP-1 istaken by white rot fungus. Since our enzymes are taken from living material innature it might make things harder for people who wishes to use them ondifferent livings. b) The dyes we use alongside ofour enzymes origins are used in histologic and genetic studies in order tocolour DNA, RNA and other parts of cell, this situation is the reason why our dyes does not to include the molecules we need which will increasethe efficiency of our project. 4) Planned Improvements of DegradEColor: The dyes and enzymes we used should be engineered to be suitablefor our project if it is going to be used as a reliable reporter system whichwill be used in synthetic biology and genetics in future. a) Dye Improvements: One of the actions that we conducted to further develop ourfuture plans was to meet up with organic chemistry experts specialized in thefield of dye synthesis. As a result of the meetings, we learned that it wouldbe possible to further develop the structure of our selected dyes in respect tothe speed of degradation thus reducing the total time of the process. . Our stopswere Prof. Dr. Cihangir Tanyeli from METU, and Doc. Dr. Zeynel Seferoğlu fromGazi University. We took their thoughts about the possibility and any pointthat can be advanced of our project. With this way, our project has beenapproved and developed by experts.Prof. Dr. Cihangir TanyeliMETU Faculty of Organic Chemistry One of the first questions weasked to Mr Tanyeli was: What different technique or techniques can we increasethe effectiveness of Horse radish peroxidase and lignin peroxidase or how canwe make our dyes more sensitive and suitable through our enzymes and be able toimprove the usage field of our project? He focused on re-designing the dyes to make them suitable forenzymes which was his main field of study and he offered us 2 differentmethods. 1st Method: We can develop the dye matter by using 4-amino-TEMPO.This is a good redox system. By this way that we won’t need hydrogen peroxidasefor Oxidation system. A polymer can be takenas a Matrix. 4-amino-TEMPO can be immobilized to biodegradable polymer. Withthe way that, sodium hypochlorite (NaClO) can be used as an alternative tohydrogen peroxide. Since this is a macro molecular system, it can beprecipitated to water and regained this will provide us an ease of researching.When used in a bulk system in the nature, most usable metalloporphirins havesilica like systems that has Si main structure, are more solid and hard todegrade, but still, if the biodegradable systems are used catalytic activitycan be decreased and be a system that can fit to nature instead of being waste. In order to block the degrading ability of the 4-amino-TEMPOsystem, antioxidants like ascorbic acid can be used this happens by radicalgroup gets silenced by ascorbic acid which goes into a reaction with Bleach. Healso stated that we can use other antioxidants that alternatives of ascorbicacid. In the other hand,. NaClO(Bleach instrument) is could be harmful for theenvironment. 4 amino tempo system we suggested has an incredible balance withbleach insturemuer so we for the nature we can chose the most envorment insrtie, destroying its harms for environment because bleach + tempo system  Inthis method we focus on 4 amino tempos.. The reason we use amino instead ofhydroxide, we want our TEMPO molecule (oxidation instruments) can easilyinteract with some organic molecules that can be produce by organisms withamino group..2nd method: Some functional groupscan be placed on Carbon Nanotubes. Carboxylic acid can be placed on CarbonNanotubes with desiccation. Also 4-amino-TEMPO added on Carboxylic acidfunctional group with direct covalent immobilization approach. If the 4-amino-TEMPOstated gets inserted on and bleach instruments like NaClO added the environmentdye degradation will be happen. Big advantages of Carbon nanotubes that can beregained with filtration and precipitation and this make this system more efficientand economic and using over and over. Both of this methods are using with switch systems that workingwith pH or temperature control by an enzymes, or a bacteria or a bio systems. We stated that time used by the systems used currently used aroundthe world takes 16 hours is decreased to a few hours by our system while wewere talking to our precious scholar. He gave us valuable information. We thankto him for giving us time. Doc. Dr. Zeynel SEFEROĞLUGazi University Faculty of Organic Chemistry We started our conservationwith our scholar how we can make our dyes more suitable for our dyes and makeit fit right in to our project. He suggested us to make a research on quantumefficient dyes and increase our knowledge on functional fluorescence dyes. Whenwe first mentioned our project he liked it and stated that this could be usedeasily and efficiently in genetic and synthetic biology world. He gave us somearticles that might help. He also stated that it is important to introduce toscience to the world since iGEM is already doing that we made someone else likeiGEM. We thank our scholar for giving us opportunity to have this conservation.          ALFA Dye and  ChemistryWe wanted toinform the local companies about iGEM and our project. Our first target was aprofessional corporation Alfa Dye. Here we talked about the possibility andusage in daily life and received some information on industrial dyes. Welearned the dyes used in histologic dying are an addition to pigments asconnecting molecules. So if we want to add other activities other thanbioremediation of dye wastes we should add some small systems which will cleavesuch connections. With this visit we had new a perspective through the future.The company got happy for us to be in such competition. They sacrificed theirtime in order to give us directions we thank them for that.                   b) Enzyme and Genetic Dyes Improvements: Another activity to further develop our future plans was toconsult genetic, biology and biochemistry experts and genetics companies toassist in the development of the theory of our project. The feedback that wereceived was indicating that through the development of the binding sites ofthe enzymes with protein engineering the project could be improved and that theproject beared great potential for the improvision of synthetic biology. Prof. Dr. Sadık ÇiğdemTsukuba University Genetic Faculty Japan He is one of the guestscientist of Turgut Ozal University Genetic Department. When we mentioned himabout our project DegradEColor he is really impressed about the idea behind ourproject and also he gave us some advice and information about our HRP enzyme.He informed us that one of our enzyme HRP is used by ELISA and Western-Blotexperiments. HRP is added on antibodies in Western Blot and also ELISA andcause the substrate ECL give response. We thanked to him for this opportunity.            Prof. Dr. Esra GündüzTurgut Özal University Faculty of Medicine We informed her about our iGEMHS Project DegradEColor, she really interested on our project. She was verypleased when she saw us, doing such work at this young age. She stated that shewill provide any support she can.                 Burak YılmazThe founder of SENTEGEN and its CEO We impressed Mr. Yılmaz withour project and aim to reduce the time of response systems. He stated managingtime is a very big problem for companies. The company which will use our systemwill manage their time 14~15 times more efficient. He said “This advantage can’tbe ignored”. He congratulated us for coming up with such a wonderful idea wethank him for this.  Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Human_Practices/Future_PlaTeam/Participants From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube TeamParticipantsGalleryAcknowledgementsTeam OverviewFirstof all we as AUC_Turkey team would like to express our appreciation forattending iGEM for one more time, but words are not enough, that’s why thisyear we are here with a distinguished work. In this page you can find someinformation on the hard working guys who has gone through every obstacle thattried to stop them with their unbelievable desire for biology. We are a familyand we thought this family deserves to be known by the world of iGEM and wetook a chance to express ourselves, their thoughts, hobbies, what they like andeven their most favorite historic personality. This family always will be knownas AUC_Turkey, in this page feel free to see the family members. If theseinformation are not satisfactory you can contact us simply by our twitter.   Name: Mehmet Sait ŞahinAge: 14I am a member of the labgroup. I like action movies and soccer as my hobby. I am into reading books andcomputer games especially strategy games. My favorite historical personality isII Abdul-Hamid. Time will guide what I am going to study in the future.    Name: Hikmet Emre CanAge: 14I am one of the firsttimers in our team of iGEM. Frankly I’m a little bit excited. The reason whyI’m in this team is obviously my interest in biology and another one is that Iwant to improve myself in this area. Also soccer is one of the fields that I aminterested in most and my favorite food is definitely pasta.    Name: Mehmet Çağrı ÇalımlıAge: 14                          I am the member of the computer group. I like horror films andskate. I am into listening music and reading book especially fiction. My favoritecomputer game is Assassin's Creed. Time will guide what I am going to study inthe future."    Name: Arif Eren Yıldırım Age: 16It is my first timeparticipating to the iGEM. Since I enjoy experimenting things and havingbackground as an attender of biology Olympics, the lab part was the best placefor me in the team. I am spending most of my time understanding and learningthe experiments as this is my first year. The subject I spend most of my freetime is basketball. I follow everything about NBA. My favorite team is ChicagoBulls and favorite player is Derrick Rose. LET’S GO BULLS!!!     Name: S. Can İçözAge: 16Hi everyone its Can fromAUC_Turkey. I have joined this family this year for the first time but I havean awkward confidence even thought I don’t know much about iGEM. Hopefully Iwill experience this iGEM 2014 Jamboree and doing the presentation of our teamwith my collages. I am interested in common history of the world andinternational politics. I am a 10th grader in Ahmet Ulusoy IB world school. Iam here attending iGEM because I like attending international organizations. Iread books in my free time mostly boring ones about mistakes of the nations andstuff. Anyway iGEM rocks!    Name: Mustafa SelçukÖztürkAge: 17HELLOGUISE, I'm Selçuk from AUC_TURKEY. I'm working on our wiki. DUDE I LOVE WIKIS.Whatever, I play League of Legends, I'm Diamond. DUDE I ALSO LOVE PLAYINGLEAGUE. I main Lee Sin, got 500+ games with him. Also I am big fan of CounterLogic Gaming. My favorite book is ASOIAF and my favorite series is Game ofThrones. CLG + LEE SIN + ASOIAF + PURE AWESOMENESS = ME. DON'T WORRY GUISE IGOT DIS, IF YOU ARE iGEMIST AND NEED DUO, CONTACT ME ASAP. CYA AT THE JAMBOREE!                                                                                                     Name:AhmetYasir NacakAge:16Hey there everybody! I am Ahmet Yasir. I dographical design for our team wiki this year. I always liked designing and thiswiki is a good experience for me. I also do coding in my free time. I want tobe a programmer when i grow up. I love playing computer games and watchingsit-coms.          Abdulkadir KaradagAge: 17Course: Biology, History, ExperimentsHometown: MuglaHi I’mAbdulkadir. It’s my third year and I proud of being 3-year-iGEMist. Myfuture aim is to be a synthetic biologist. My other area of interest is lateperiod Ottoman History. I also like fishing with my spear gun and playing TotalWar. See you at the Jamboree                                                                                                                                                                                         Name:İbrahim Yasir ORHANSchool:Atlantik Educational Institution Ahmet Ulusoy Science High SchoolAge:16,75Hometown:Rize, KalkandereFavouriteProcedure: Western BlottingFavouriteSong: Viva La Vida (David Garrett)Interest:Genetic, Molecular Biology, Physiology, Immunology, Programming, QuantumPhysics, Violin, Basketball Zodiac:LibraFavouriteBacterium: Deinococcus Radiodurans          Name:Edip Ahmet ÜnlüerAge:16 Hi,I'm Edip Ahmet UNLUER from 10th grades of Atlantik Ahmet Ulusoy High School.I'm taking part as coder in our team. Actually, I had never coded html before.So,I had a prejudgment about coding html but taking part in this team made itnecessary and I loved it.I'mcodding as a hobby actually. I love it and want to make it my job in the futureso my goal is working in the Silicon Valley as my boss 'Akekerim'          Name: Nadir Berka Akdeniz Age:14 I have joined this team this year for the first time. I likewhere I am today because I like spending time with lab stuff. I am interestedin chemistry,reading and playing guitar. I think iGEM is going to support medeveloping myself. Basketball is my hobby I play basketball a lot.       Name: Göktuğ Mert ÇiftciAge: 14I entered team this year.I am participate of lab team. I like horror movies and scientific books. I likeplaying computer games and the games I play are generally strategic games. I aminterested in biology. I am planning to study in the field of biology in thefuture. My favorite area of biology is microbiology.      Name: Berat AlperenAyvazoğlu Age: 16Hi, I’m Alperen. I loveswimming and playing basketball and also jogging. I entered the team this year.I love playing computer mostly horror games. I like watching films, I think actionmovies are the best. I think iGEM is very beneficial for communicating withpeople and learning science!    Name:FatihGülSlutations people. I am Fatih Gül,a third-yeariGEMer. It has been a thrilling experience for me, an amazing experience ofstrife, joy, laughter, somber but most importantly conviction. May allexperience this feel that has come to me and my fellow iGEMists, a desire toseek a better world!                                                                                                                                         ADVISORS  Name:Furkan BestepeAge:18 My name is FurkanBeştepe and I'm addicted tofiction novels, movies and TV Series. They make me a creative person. Imaginingand thinking my hobbies are my brain's work out. Also I'm really interested inreading faces and micro expressions. Finally my slogan from Nikola Tesla is"Our virtues and flaws can not be seperated just like power and matter. Ifthey are seperated human is nothing."  Name:Mustafa Semih Elitok I’m MustafaElitok. This is my sixth year in iGEM. Burada olmayı seviyorum. Take care ofyourselfs, guys.                                                                                                 Name: Mikail DoğanAge:20Hometown:Elbistan,KahramanmaraşFavourite Pocedure:TransfectionFavorite Song: Sonbahar,YansımalarInterest:Medicine,genetic,reflexionlearning and teachingZodiac: AquariusFavourite Bacterium:Micobacterium tuberculosis      Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Team/ParticipanNotebook/Safety From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Lab GarageNotebookProtocolsSafetyBeforewe doPersonelsafety is one of the top priorities in the lab. Before starting lab work, weattended a lab safety training program provided by our teacher Mr.Bestepe. Wewere familiarized with the operation of lab equipment and nature of chemicalswe would be using in the lab.We used our team funds to buy disinfectant white medical uniforms.Before we enter the lab, everyone must put on the uniform and go through the disinfectantdoor to make sure that they are clean. Some chemicals may be harmful to humanskin, so everyone is required to wear safety gloves, to isolate the reagentsfrom our skin. When important and strict steps are required, we have to spraythe alcohol on our gloves to be sure that our gloves are cleaned. These areuniversal precautionary measures. 1. Would any of your project ideasraise safety issues in terms of researcher safety, public safety, environmentalor ethical safety?a. Reseacher safety: Everyone on the team obtained laboratory safety training through the Machineryof Life/Foundations of Molecular Medicine and Bioengineering CAPS Class and hadexperience with bacterial culture (in E.coli) and genetic transformation(inserting pGLO into E.coli) prior to beginning the project.Duringthe work, our team took the following safety precautions:Lab coats,gloves and goggles were worn at all times.Allequipment (both disposable and autoclaved) was sterile.1.     The bench and other work surfaces were sterilized withwater and bleach before and after lab work.2.     Because cyanobacteria can pose a risk if ingested,there was no food or drink in the lab.There were no lit burners or other fireduring lab work.b. Public Safety: When released byaccident, our parts and materials actually cause no damage to the generalpublic. Due to the unability of E.coli strains, TOP10 and BL21 to survive outof the lab, they cannot pose any risk to the safety and health of the generalpublic.c. Environment: E. coli strains TOP10and BL21 have very limited ability to survive outside the laboratory; so that,it would be unable to survive or disseminate. Therefore, there is no specificenvironmental risk associated with the E. coli strains. All bacterial wastesare kept in 10% bleaching solution for one day, and then, are autoclaved to besterilized. Yet, undesired GMOs may achieve ecologically harmful features.d.EthicalSafetyWerecognize that our Lignin Peroksydase gene was isolated by Mr.Bestepe and thatwe only get use it per his permission. Accordingly, we have kept him informedon our work with it and the possibility of this gene becoming readily avaliablein the Standard Registry of Biological Parts.2. Do any of the new BioBrick parts(or devices) that you made this year raise safety issues?No, none of theBioBricks that were made by us this year raise any safety issues. They are a100% safe for the environment, general public, and our team members handlingthem. LigninPeroksydase isa comletely safe and common enzyme. Being in many organisms and commonly usedin many applications, Lignin Peroksydase is still considered one of the safestenzymes. The DegradEcolor is nothing but an mRNA sequence which works as asimple DegradEcolors in organisms. The DegradEcolor won't even have theslightest effect on any external being.3. Is there a local biosafety group,committee, or review board at your institution?Ourinstitution has its own biosafety rules. Rules for laboratory use, general principles,prevention from hazardous materials and application of emergency interventionin case of accident are included. In Turgut Ozal University Medical School,Laboratory and Patient-Employee Safety Committee is responsible for control aswell as biosafety of laboratories and safety of patients and employees. Thiscommittee works under one of the vice medical director of Turgut OzalUniversity Hospital, Prof. Dr. Mehmet Gunduz. Safety and security issues arefound sufficient enough that no change is considered as necessary. (Further questions can be directedto Prof. Gunduz, tel: +90-312-203 5103, mgunduz@fatih.edu.tr) Our advisor provided us with biosafetyand lab training before starting our project. In the training, general safetyrules of laboratory use, prevention from hazardous chemicals and solutions aswell as emergency intervention in case of accident were included such as:§  Eating, drinking, storing food and smoking areabsolutely not allowed.§  Mouth pipetting is not allowed; modern pipettes are usedin lab.§  Hazardous wastes and ordinary wastes are separated andcautiously disposed.§  Lab coats are obligatory to wear in the lab and duringon-going experiments.§  In electrophoresis room, lab coat, protective eyewear,lab masks and gloves have to be worn. In the case of use of EtBr; extra caution isrequired.§  Washing hands after any experiment and after touchinganything related with viable material is obliged.§  Air conditioning is kept closed during on-goingexperiments in order to avoid possible infections of spores and bacteria. 4. Do you have any other ideas how todeal with safety issues that could be useful for future iGEM competitions? Howcould parts, devices and systems be made even safer through biosafetyengineering?To deal with safety issues, for future iGEM competition, all Biobrick partshould contian a potential knockout device. Also, Biobrick parts can bescreened for potentially dangerous pathogens through the use of a variety ofsoftware that can be found at universities like Turgut Ozal UNİVERİTY MedicalSchool. SomethingdangerousWhenwe approach the operating floor, especially doing the electrophoresis (toseparate the different part of the plasmid) and usually the gel we used wasmade of carcinogen. Therefore, when students are doing this step, we mustn’ttouch the floor, even can’t get very close to it. Another harmful thing is theultraviolet rays. Because the rays which used by lab’s equipment have strongradiation, so we can not obverse it directly, we have to use an idiosyncraticboard to see it, in order to reduce the harm for our eyes. Turkeyhas national biosafety regulations and the link is given as(http://www.tbbdm.gov.tr/en/Home/BioSafetyCouncilHome/BioSafetyCouncilHomeChoose.aspx).    Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Notebook/SafetTeam/Gallery From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube TeamParticipantsGalleryAcknowledgements Download Original We are HOUSEing another poster! Download Original Synthetic Biology: Future depends on it! Download Original When RNA is weaved into a "kilim" of emotions... Download Original Let us shape the world through Synthetic Biology! Download Original One Plasmid to rule them all, One Plasmid to find them,One Plasmid to bring them all and in the darkness bind them Download Original This Jamboree, we present you "Parts Olympiad"! Download Original P FOR PIPETTA"PIPETTING FOREVER" Download Original Drinking Turkish coffee iGEM style! Download Original I WANT YOU FOR iGEM Download Original Together we eat, together we sleep. In the lab or in the street, UNITED WE STAND! Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Team/GallerFunctional Essay From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ResultsCloningProtein AnalysisFunctional AssaySpectrophotometric Absorption Measurements:  We used Thermo Scientific Varioskan Flash MultimodeReader for our absorption measurements.1)Measurements of Dyes:Graphic 1:This graphic shows our absorption rate of our dye (Methylene Blue) in differentconcentrations. ( 1X=8 uM, 10X=80uM, 100X=800uM, 1000X=uM) In theGraphic 1, we have got the correct absorption rates for every concentration ofMethylene Blue dyes that correlates with the same results in the literature.2)Preparations of Lysates : The verified plasmids were then cultivated inliquid culture for isolation. The 50 mL’s of bacteria containing the partsBBa_K1291071 and BBa_K1291299 were centrifuged at 4100 rpm for 5 minutes, thesupernatant was discarded and then lysis solution was added. After 10 minutesof incubation in ice, the lysates were sonicated in the minimum activity level.After a spin of 15000 rpm for 10 minutes, the supernatant was transferred toeppendorphs and dye was added; the solution was boiled at 95 °C for 5 minutes. The lysate were stored at -20 °C.3)Decolorization Assay:In thisassay we mixed our liquid cell culture and also our lysates with dyes. All theconcentrations of the solutions are adjusted according to the literature. Wemeasured the first absorptions of the samples and compared it with the 2ndmeasurement in the end of 1st hour. Graphic 2:This graphic shows our absorption rate of our mixed sample that includesLysates of our BL-21 Bacteria that have LiP gene and  Methylene Blue(80uM) dyes and for the activate the LiP we also added Hydrogen Peroxide(800uM) measured in the 1 hour.The resultof Graphic-2 is that our Lysates of BL-21 Bacteria that have LiP gene degradedthe Methylene Blue (80uM) dye with approximately %83,3 efficiency. Graphic 3: This graphic shows ourabsorption rate of our mixed samples that includes Lysates of our BL-21Bacteria that have HRP gene and other Bacteria that have LiP gene and MethylGreen (150uM) dyes and for the activate the HRP and LiP we also added HydrogenPeroxide (8.8 uM-HRP), (0,176 uM-LiP) concentrations that measured in thedifferent time periods. Theresult of Graphic-3 is that our Lysates of BL-21 Bacteria that have HRP geneand other Bacteria that have LiP gene degraded the Methyl Green (8.8 uM) dyewith approximately %71,4 efficiency in the end of the 7 hours.  .Graphic 4:This graphic shows our absorption rate of our mixed samples that includesLysates of our BL-21 Bacteria that have HRP gene and other Bacteria that haveLiP gene and Methyl Green (150uM) dyes and for the activate the HRP and LiP wealso added Hydrogen Peroxide (8.8 uM-HRP), (0,176 uM-LiP) concentrations thatmeasured in the beginning of the experiment and in the 1st hour. The resultof Graphic-4 is that our Lysates of BL-21 Bacteria that have HRP gene and otherBacteria that have LiP gene degraded the Methyl Green (8.8 uM) dye withapproximately %71,4 efficiency. Also in this graphicthe most important point that our control group BL-21 bacteria that don’tinclude our genes of enzymes(HRP or LiP) couldn’t degrade the Methyl Green (8.8uM) dye. Graphic 5:This graphic shows our absorption rate of our mixed sample that includes liquidculture  of our BL-21 Bacteria that have LiP gene and  Methylene Blue(80uM) dyes and for the activate the LiP we also added Hydrogen Peroxide(800uM) measured in the 1 hour.The resultof Graphic-5 is that our Liquid cultures of BL-21 Bacteria that have LiP genealso degraded the Methylene Blue (80uM) dye with approximately %70 efficiency.       Graphic 6:This graphic shows our absorption rate of our mixed sample that includes liquidculture  of our BL-21 Bacteria that have HRP gene and  Methylene Blue(80uM) dyes and for the activate the HRP we also added Hydrogen Peroxide(800uM) measured in the 1 hour.The resultof Graphic-6 is that our Liquid cultures of BL-21 Bacteria that have LiP genealso degraded the Methylene Blue (80uM) dye with approximately %65 efficiency.  The Resultsof Graphic 5 and 6 show that our liquid culture of our BL-21 Bacteria that haveHRP gene and other Bacteria that have LiP gene could degraded the MethyleneBlue (80uM) dye but our Lysates of our BL-21 Bacteria that have HRP gene andother Bacteria that have LiP gene degraded the Methylene Blue (80uM) dye moreefficiently. This results gave us an opinion that when our enzymes have furtherinteraction with dyes they could degrade much more.        Graphic 7:This graphic shows our absorption rate of our mixed sample that includes liquidculture  of our Control BL-21 bacteria that don’t have any HRP or LiP geneand  Methylene Blue (80uM) dyes and for the activate the any peroxidases,Control BL-21 bacteria have, we also added Hydrogen Peroxide (800uM) measuredin the 1 hour.The resultof Graphic-7 is that our Liquid culture of BL-21 Bacteria that don’t have anyHRP or LiP gene could degrade the Methylene Blue (80uM) dye with approximately% 18 efficiency. This result show that Control bacteria can degrade the theMethylene Blue (80uM) dye but at the very low percentage when compared with ourliquid culture BL-21 Bacteria that have HRP gene and other Bacteria that haveLiP gene so we thought that some peroxidases also can degrade the dyes but ourenzymes HRP and LiP have very high catalytic ability for the ourdyes(Methlylene Blue and Methyl Green). These results and graphics show thatour enzymes are functional and working properly.Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Results/Functional_EssaNotebook/Protocols From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Lab GarageNotebookProtocolsSafety Procedures for LB Agar Preparation§  In a steril environment, the tare of the containershould be measured and subtracted from the overall weight.§  7 grams of LB Agar is put in the container.§  200 ml distilled water or is put into a graduatedcylinder.§  These two are mixed in a beaker.§  The opening of the beaker is covered with aliminiumfolio in such a way that it does not conctact with air.§  Autoclave tape is sticked on to the aliminium.§  The beaker is placed in to the autoclave machine.§  Distilled water or demineralized water is put into theautoclave machine. The water level in the autoclave machine has to be a littlehigher than the liquid level of the beaker.§  After closing the lid of the machine, the 90 minute autoclaveprocess is given start.§  Take out the beaker and add antibiotics if required.Warnings for the Autoclave!§  Use only demineralised or disttiled water with thedevice.§  Do not open the cover until the manometer drops tozero during the operation.§  Please do not use the autoclave for other purposesthan sterilization and agar.§  Please do not use the autoclave to sterilizeexplosive, inflammable and oxidizing materials.§  Please be cautious when you are closing the lid not totrap your hand.§  Please beware of the steam exhaust when you areopening to autoclave after sterilization.§  Please wear protective gloves before removibgmaterials from the chamber. Do not access the chamber unless the vapor exhaustis finalized.Procedures for LB Broth Preparation§  In a steril environment, the tare of the containershould be measured and subtracted from the overall weight.§  7 grams of LB Broth is put in the container.§  200 ml distilled water or is put into a graduatedcylinder.§  These two are mixed in a beaker.§  The opening of the beaker is covered with aliminiumfolio in such a way that it does not conctact with air.§  Autoclave tape is sticked on to the aliminium.§  The beaker is placed in to the autoclave machine.§  Distilled water or demineralized water is put into theautoclave machine. The water level in the autoclave machine has to be a littlehigher than the liquid level of the beaker.§  After closing the lid of the machine, the 90 minuteautoclave process is given start.§  Take out the beaker and add antibiotics if required.Warnings for the Autoclave!§  Use only demineralised or disttiled water with thedevice.§  Do not open the cover until the manometer drops tozero during the operation.§  Please do not use the autoclave for other purposesthan sterilization and agar.§  Please do not use the autoclave to sterilizeexplosive, inflammable and oxidizing materials.§  Please be cautious when you are closing the lid not totrap your hand.§  Please beware of the steam exhaust when you areopening to autoclave after sterilization.§  Please wear protective gloves before removibgmaterials from the chamber. Do not access the chamber unless the vapor exhaustis finalized.Procedures for Transformation§  Transfer 500 ul LB Broth to 1.5 ml microcentrifugetubes. This should be done close to a source of fire to prevent contamination.§  Place the microcentrifuge tubes containing LB Broth ina 42 C heat block for incubation.§  Take 1 ul plasmid and place them in 1.5 ml centrifugetubes.§  Add 50 ul competent cells to the plasmid.§  Centrifuge them at 3000 rpm for 20-30 seconds.§  Incubate the cells in ice for 45 minutes.§  After 45 minutes, heat the tubes in the 42 C heatblock for a maximum of 90 seconds.§  The same tubes should be placed in ice and should beincubated for 5 minutes.§  Afterwards, 450 ul LB should be added to the cells tocomplete them to 500 ul.§  The microcentrifuge tubes are then sticked to theshaker horizantally and shaked for 1 hour with 320 rpm at 37 C.§  150 ul of the mixture(200 ul for digestion) is thenplaced on the plate to spread.§  It is then spread on the plate and the plates areincubated at 37 C for 16 hours.Procedures for Isolation§  The LB Media should be transferred to 1.5 mlcentrifuge tubes.§  These tubes are then centrifuged at 13,000 rpm for 10minutes at room temperature.§  After the centrifuge, the supernatent should bedisposed without taking any pellets along with it.§  The pelleted cells should be suspended in 250 ulResuspension Solution and the tubes should be vortexed so that no cell clumpsremain.§  250 ul Lysis Solution is added. The tubes are inverted4-6 times but the inversion shouldn't be done really fast as the Lysis Solutionmust not be shaken. Also it is smart to heat the Lysis Solution in order toensure that no pericipitate remain.§  350 ul Neutralization Solution should be added and thetube should be inverted immediately and throughly by inverting 4-6 times.§  Centrifuge for 5 minutes to pellet cell debris andchromosomal DNA.§  Transfer the supernatent to a spin column withouttaking any of the pellets.§  Centrifuge the spin column for 1 minutes and discardthe liquid at the bottom. Place the column at the same tube again.§  Add 500 ul Wash Solution and centrifuge for 30-60seconds. Discard the flow-through and place column back in.§  Repeat the same process again with 500 ul WashSolution.§  Centrifuge for an additional 1 minute.§  Transfer the columns into 1.5 ml microcentrifuge tubesand leave their caps open for 2 minutes so that the ethanol that they containdissolves in air.§  Add 50 ul Elution Buffer to the center of the silicamembrane to elute the plasmid DNA. The pipette tip shouldn't the membrane.Incubate for 2 minutes and centrifuge for 2 minutes afterwards.§  Discard the spin column and store at -20 C.Digestion Protocol§  Take the average of the nucleic acid concentrationsmeasured by the spectrometer.§  Divide 500 by the DNA average.§  Add 5ul Ne Buffer.§  Add 0.5ul BSA Buffer.§  Add 1 ul of the enzymes with barrier tips.§  If you cut with EcoR1 and SpI, it will be up stream.§  If you cut with Xbal and Pst1, it will be down stream.§  Subtract the amount of DNA from 42.5 ul. This resultwill be the amount of NFW used.§  Add the NFW with barrier tips and do one pippettingwhile taking the NFW.§  Then the DNA is put into the PCR and is left there for30 minutes.Ligation Protocol§  2ul up stream is put into a eppendorf.§  2ul down stream is also added.§  2ul plasmid is mixed in as well.§  2ul Taq Buffer is inserted to the mixture.§  1ul T4 DNA ligase is then added with barrier tips.§  11ul NFW is added with barrier tips and should bepipetted once.§  Then the DNA is put into the PCR and is left there for30 minutes.Gel Preparation§  Mix 100ml TAE and 0,8 gram agarose in a glass beaker.§  The mixture is then heated in a microwave for 3minutes.§  Afterwards, 3,6 ul EtBr is added and the beaker ismixed on a magnetic mixer.§  Mold them and wait for 20 minutes fort he gel toharden.Procedures for Western Blot§  Western blots allow investigators to determine themolecular weight of a protein and to measure relative amounts of the proteinpresent in different samples.§  1) Proteins are separated by gel electrophoresis,usually SDS-PAGE.§  2) The proteins are transfered to a sheet of specialblotting paper called nitrocellulose, though other types of paper, ormembranes, can be used. The proteins retain the same pattern of separation theyhad on the gel.§  3) The blot is incubated with a generic protein (suchas milk proteins) to bind to any remaining sticky places on the nitrocellulose.An antibody is then added to the solution which is able to bind to its specificprotein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradishperoxidase) or dye attached to it which cannot be seen at this time.§  4) The location of the antibody is revealed byincubating it with a colorless substrate that the attached enzyme converts to acolored product that can be seen and photographed. Proceduresof Sonication 1) Resuspend pellet of10ml cell culture in 1ml lysis buffer (or 100ml bacterial culture for very lowexpression level). Suggested Lysis buffer: 140mM NaCl; 2.7mM KCL; 10mM Na2HPO4; 1.8mM KH2PO4; pH 7.3 (PBS)                                   or 100mM NaCl; 25mM TrisHCl; pH 8.0                                   optional 0.02% NaN3 (azide)                                   optional protease inhibitors Optional additives tothe lysis buffer a) 1mM PMSF or proteaseinhibitor cocktail 1:200 (cocktail for bacterial cells #P-8849 fromSigma) b) Dnase 100U/ml or25-50ug/ml (SIGMA DN-25). Incubate 10min 4°C in the presence of10mMMgCl2. c) Lysozime 0.2mg/ml.Incubate 10min 4°C. d) ßME, DTT or DTE up to 10mM for proteins with many cysteines. e) 0.1-2% Triton X-100,NP40; or any other detergent that do not affect the biological activity of yourprotein. f) 10% glycerol (forstabilization of the protein and prevention of aggregation). 2) Sonicate in icebucket 3 x 10sec or more if the cells are not completely disrupted (Lysis iscomplete when the cloudy cell suspension becomes translucent. Avoid proteindenaturation by frothing). 3) Spin 5min 13000rpm4°C. Separate soluble proteins (supernatant) from insoluble or inclusion bodiesproteins (pellet). Use supernatant for next step. Keep sample of 40ul of supernatantfor PAGE-SDS and Western blot: soluble proteins 4) Resuspend pellet inanother 1ml lysis buffer and keep sample of 40ul for PAGE-SDS and Western blot:insoluble proteins, or unlysed cells. Procedures for Competent Cell Preparation A. Preparing glassware and media eliminate detergent 1. Autoclaving glassware filled 3/4 with DD-H2O toremove most detergent residue 2. Media and buffers in detergent free glassware andcultures grown up in detergent free glassware B. Preparation of the competent cells Reagents: -Glycerol stock -LB plate -MgCl2-CaCl2 solution -MgCl2-CaCl2 solutuion -MgCl2‧6H2O 3.25g -CaCl2‧2H2O 0.6g Add H2O to 200ml -100mM CaCl2 -100mM CaCl2 solution -CaCl2‧2H2O 2.95g Add H2O to 200m; -80% glycerol -Liquid nitrogen DAY1:1. Flame the metal inoculating loop until it is redgot and then cools it down 2. Scrape off a portion from the top of the frozenglycerol stock [DO NOT THAW]3. Streak it onto the LB plate4. Put the stock back to -80C immediately5. Leave the plates for 5 minutes and place themupside down in the 37C incubator for 16-20 hours  DAY 26. Pick a single colony into 5ml of LB medium 7. Inoculate the culture overnight at 37C with shakingat 250rpm  DAY38. Inoculate 100ml LB medium with 1ml of saturatedovernight culture 9. Shake at 37C until OD600=0.4 (usually 2-3 hours) 10. Place in an ice bath for 10 minutes [ After thispoint the cells should never touch anything that is warm] 11. Pre-cool solution, centrifuge, pipette tips,falcon, eppendorf 12. Transfer the culture into two pre-chilled 50mlfalcon 13. Centrifuge at 2700x g for 10 minutes at 4C 14. Remove the medium, resuspend the cell pellet with1.6ml ice0cold 100mM CaCl2 by swirling on ice gently 15. Incubate on ice gor 30 minutes 16. Centrifuge at 2700x g for 10 minutes at 4C 17. Remove the medium, resuspend the cell pellet with1.6ml ice-cold 100mM CaCl3 by swiriling on ice gently 18. Incubate on ice for 20 minutes 19. Combine cells to one tube and add 0.5 ml ice-cold80% glycerol and swirl to mix 20. Freeze 100ul aliquots in liquid nitrogen 21. Store in -80C Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Notebook/ProtocoProject/Introduction From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ProjectIntroductionMechanismModellingPartsThe coreprinciple of our project is the development of a novel reporter system. Wedeveloped a biological system which is a reporter system more agile andrecognizable in comparison to the present reporter systems using E.coli.Although the currently present reporter systems bear many incapabilities anddisadvantages, the lack of alternative reporter systems forces the sciencecommunity to use these systems. Our project can perhaps obscure this situation.  White rot fungus degrade severallarge molecules such as cellulose, hemicellulose and lignin to use as a source of nutrition and continue theirexistence. The breakdown oflignin, an amorphous substance and a polimer, through lignin peroxidase, anenzyme of the oxireductase enzyme group, is crucial.             In theexamination of the simbiotic relationship of white rot fungus and ligneoustrees, scientist uncovered the mycoremediational breakdown that takes place infungi through laccase enzymes. After recognizing the possibilities that arosedue to this discovery, scientists utilized the mentioned function of fungi onthe remediation of dyes. If enzymes could be used to breakdown dyes, than itwould be possible to form a biological reporter system that would be builtaround the breakdown of dyes instead of synthesizing it, which would in turnspeed up the process, make it easier to observe changes and give the ability tomediate the integration of the method to bioremediation systems. With thisdesign, we will have formed a reporter system from scratch based on thebreakdown of dyes instead of their synthesis. PRESENT REPORTER SYSTEMSCommonly used reportergenes that induce visually identifiable characteristics usually involvefluorescent and luminescent proteins. Examples include the gene that encodesjellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and the red fluorescent protein from the gene dsRed.The GUS gene has been commonly used in plants but luciferase and GFP are becoming more common. [i]A common reporter in bacteria is the E. coli lacZ gene, which encodes the protein beta-galactosidase. This enzyme causes bacteria expressing the gene to appearblue when grown on a medium that contains the substrate analog X-gal. An example of a selectable-marker which is also a reporter in bacteriais the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to theantibiotic chloramphenicol.Most of the reporter systems are based on the use of visualdata. To understand if whether the necessary conditions were met, change in visualcontext must occur. In the appropriate conditions, these report systems giveresponse through the synthesis of specific color material that was not presentin the environment.  A prominent reportersystem that is the green fluorescent protein, a protein that does not carry a complex and long sequence structure that isresponsible for its prominence, is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range.[ii] [iii]In cell and molecular biology, the GFP gene isfrequently used as a reporter of expression. [iv] In modified formsit has been used to make biosensors, and many animals have been created that express GFP as aproof-of-concept that a gene can be expressed throughout a given organism. TheGFP gene can be introduced into organisms and maintained in their genomethrough breeding, injection with a viral vector, or cell transformation. To date, the GFP gene has been introduced andexpressed in many Bacteria,Yeast and other Fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery anddevelopment of the green fluorescent protein. The GFP reporter systems which started a new era inbiological reporter systems with the works of Martin Chalfie, Osamu Shimomura, and Roger Y. Tsien are still widely usedtoday. GFP reporter systems which can be used in many processes ranging fromthe confirmation of the synthesis of a sequence to the validation of thebinding of a protein to the cell membrane of a cell, still command greatimportance as a reporter system, however these reporter systems also carryseveral disadvantages. The most crucial of these disadvantages is the problemof time restriction. To overcome this hurdle in front of scientific researchexponentially increases the development in scince. Reducing the time allocatedfor experiments and increasing their efficiency are the most important mattersin science research. Currently, the issue of time has become more prominent.The incubation process following cloning protocols can take up to 16 hours,significantly increasing the time required to carry out projects hencerestricting the conduction of many of these projects. If an example was to begiven to the current problem, the time restriction prevents the use ofsynthetic biology for the development of toxic gas sensors as the sensors wouldneed to give immidiate response. It is not possible to state that this problemhas been solved as alternative methods either require highly expensivechemicals to operate or have too complex systems preventing implementation OURMODULEReporter systems carry great significance in the world ofbiology. As previously mentioned, the problems that currently exist in thedevelopment of reporter systems slow down the development of Biology,specifically synthetic biology and genetics.We aimed to overcome the issue of time with oursystem. We had to abandon certain project ideas due to theinsufficiency of the available time that inspired us to find a possible way ofovercoming this issue which would open doors to many scientists interested in thisarea.Our focus was on the GFP reporter system as it had been usednumerous times by scientists worldwide and therefore carried great attention inreporter systems. If the issue of time was to be tackled, it had to address theissue in the GFP reporter system. The report system had to respond in a shorterperiod of time if progress was to be accelerated. It was not crucial to see a100% complete result, even theglimpse of a change would have been enough for our system.. After observing the currently present GFP reporter systems,it came to our notice that the bacteria try to produce an inexisting substancefrom scratch, step by step and the presence of the substance can only berecognized after the substance has reached a certain concentration. If the processwas to be reversed, however, it would take a significantly reduced amount oftime. After reaching this state of recognition, we decided that the breakdownof the color instead of its production would be a lot more agile. Allthe enzymeproduced would be capable of breaking down color in a short time interval. Theexponentially increasing levels of enzyme presence would increase the enzymeactivity, hence reducing the time of the process even more.There is another advantage to this new system,while the necessity of the colored substance reaching a certain density was adisadvantage in the previous system, in the new system even the change in thehue of the color would prove that our system was functioning and the densityissue would then become an advantage. The onlyexternal addition to the system required is the addition of a specific dye. Thesystem therefore bears the potential to eliminate 16-hour long waiting timesand makes life easier for scientists.If our goal within this project wasaccomplished it would be a revolution for biological reactions. Who wouldn'twant to decrease the required time by 75%?Our project has the potential to be adapted forother areas of science because of its relation with color. When we think of theimportance of bioremediation processes, to decrease the harm of chemical wasteto the ecology of the Earth, these alternative systems are important. It won'tbe hard to integrate this system.If we are able to optimize and develop thesteps of our project professionally, we will be able to open a new door withinthe science of biology.[i] Koo, J.; Kim, Y.; Kim, J.; Yeom, M.; Lee, I.C.; Nam, H. G. (2007). "A GUS/Luciferase Fusion Reporter for Plant GeneTrapping and for Assay of Promoter Activity with Luciferin-Dependent Control ofthe Reporter Protein Stability". Plant and Cell Physiology 48(8): 1121–1131. doi:10.1093/pcp/pcm081. PMID 17597079[ii] Prendergast F, Mann K (1978). "Chemicaland physical properties of aequorin and the green fluorescent protein isolatedfrom Aequorea forskålea". Biochemistry 17 (17): 3448–53. doi:10.1021/bi00610a004. PMID 28749[iii] Tsien R (1998). "The green fluorescentprotein" (PDF). AnnuRev Biochem67: 509–44. doi:10.1146/annurev.biochem.67.1.509. PMID 9759496[iv] Phillips G (2001). "Green fluorescent protein--a brightidea for the study of bacterial protein localization". FEMS MicrobiolLett 204 (1): 9–18. doi:10.1016/S0378-1097(01)00358-5. PMID 11682170Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Project/IntroductioProject/Modelling From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ProjectIntroductionMechanismModellingPartsA KINETIC MODEL IN MATLABHow did we build it???Using the SimBiology toolbox for MATLAB wecreated a framework of the enzyme kinetic modelling(Figure 1).HRP + H2O2 → ES1 (HRP compound I)+ MG → ES2(MGox +HRP compound II)After creating the basic framework for themodel we needed to create mathematical equations for each reaction withappropriate rate constants. These equations and the corresponding values areshown below in Tables 1 and 2. Reaction Name: Reaction Scheme: Reaction Rate: oxidation reaction H2O2 + HRP -> ES1 k1*H2O2*HRP decolourization reaction ES1 + MG -> ES2 k2*ES1*MG Table1: Mathematicalrepresentation of enzyme kinetic reaction expression and the subsequentproduction of the oxidized dye. Constants are described in Table 2. Constant: Value: Units: References: k1 0.0018 1/(µmolarity*min) Viridiana S. Ferreira-Leitão (2002) k1r neglected -   k2 0.004 1/(µmolarity*min) Viridiana S. Ferreira-Leitão (2002) k2r neglected -   Table 2: Valuesassigned to kinetic parameters described in Table 1. We ignored k1r and k2rvalues because of k1>>>>k1r and k2>>>k2r, so our bothreactions act as irreversible. Rule Name: Rate Rules: HRP dE/dt = - k1[E][S1]+k1r[ES1] H2O2 dS1/dt = -k1[E][S1]+k1r[ES1] ES1 (HRP compound I) dES1/dt =k1[E][S1] –k1r[ES1]-k2[ES1][S2]+k2r[ES2] Methyl Green (MG) dS2/dt = -k2[ES1][S2] +k2r[ES2] ES2 (MGox +LiP compound II) dES2/dt = k2[ES1][S2] – k2r[ES2] Table3: Differential equations of reactions. What did it show???Before runningthe model we needed to decide what an appropriate endpoint of reaction wouldbe. Therefore, concentrations of species were determined from the literatureand necessary assumptions were made to check our cascade reactions workcorrectly. According to chosenreferences points, we were trying to determine how quickly we could get a decolourization.Decolourization depends on final [ES2] (MGox +HRP compound II)concentration and degradation of MB. Decolourizationstarts in 40 minutes.  Reaction Name: Reaction Scheme: Reaction Rate: oxidation reaction H2O2 + HRP -> ES1 k1*H2O2*HRP decolorization reaction ES1 + MB -> ES2 k2*ES1*MB Table 4: Mathematicalrepresentation of enzyme kinetic reaction expression and the subsequentproduction of the oxidized dye. Constants are described in Table 5.  Constant: Value: Units: References: k1 0.0018 1/(µmolarity*min) Viridiana S. Ferreira-Leitão (2002) k1r neglected -   k2 0.004 1/(µmolarity*min) Viridiana S. Ferreira-Leitão (2002) k2r neglected -           Table 5: Valuesassigned to kinetic parameters described in Table 4. We ignored k1r and k2rvalues because of k1>>>>k1r and k2>>>k2r, so our bothreactions act as irreversible.  Rule Name: Rate Rules: HRP dE/dt = - k1[E][S1]+k1r[ES1] H2O2 dS1/dt = -k1[E][S1]+k1r[ES1] ES1 (HRP compound I) dES1/dt =k1[E][S1] –k1r[ES1]-k2[ES1][S2]+k2r[ES2] Methylen Blue (MB) dS2/dt = -k2[ES1][S2] +k2r[ES2] ES2 (MBox +LiP compound II) dES2/dt = k2[ES1][S2] – k2r[ES2] Table6: Differential equations of reactions.  1hour of HRP- MB decolourizationWhat did it show???Before runningthe model we needed to decide what an appropriate endpoint of reaction wouldbe. Therefore, concentrations of species were determined from the literatureand necessary assumptions were made to check our cascade reactions workcorrectly. According tochosen references points, we were trying to determine how quickly we could geta decolourization. Decolourization depends on final [ES2] (MBox +LiP compound II) concentration and degradation of MB. Decolourizationstarts in 40 minutes.LiP + H2O2 →ES1 (LiP compound I )+ MB → ES2 (MBox +LiP compound II) Reaction Name: Reaction Scheme: Reaction Rate: oxidation reaction H2O2 + LiP -> ES1 k1*H2O2*LiP decolorization reaction ES1 + MB -> ES2 k2*ES1*MB Table7: Mathematicalrepresentation of enzyme kinetic reaction expression and the subsequentproduction of the oxidized dye. Constants are described in Table 8.  Constant: Value: Units: References: k1 1.0E-4 1/(µmolarity*min) Paulı Ollıkka, Kırsı Alhonmakı (1993) k1r neglected -   k2 2.0E-4 1/(µmolarity*min) Paulı Ollıkka, Kırsı Alhonmakı (1993) k2r neglected -   Table 8: Valuesassigned to kinetic parameters described in Table 7. We ignored k1r and k2rvalues because of k1>>>>k1r and k2>>>k2r, so our bothreactions act as irreversible.  Rule Name: Rate Rules: HRP dE/dt = - k1[E][S1]+k1r[ES1] H2O2 dS1/dt = -k1[E][S1]+k1r[ES1] ES1 (HRP compound I) dES1/dt =k1[E][S1] –k1r[ES1]-k2[ES1][S2]+k2r[ES2] Methylen Blue (MB) dS2/dt = -k2[ES1][S2] +k2r[ES2] ES2 (MBox +LiP compound II) dES2/dt = k2[ES1][S2] – k2r[ES2] Table9: Differential equations of reactions.  6 hours of Lip- MG decolourizationWhat did it show???Before runningthe model we needed to decide what an appropriate endpoint of reaction wouldbe. Therefore, concentrations of species were determined from the literatureand necessary assumptions were made to check our cascade reactions workcorrectly. According tochosen references points, we were trying to determine how quickly we could geta decolourization. Decolourization depends on final [ES2] (Mgox +Lip compound II)concentration and degradation of MG. Decolourizationstarts in 40 minutes.  Reaction Name: Reaction Scheme: Reaction Rate: oxidation reaction H2O2 + LiP -> ES1 k1*H2O2*LiP decolourization reaction ES1 + MG -> ES2 k2*ES1*MG Table10: Mathematicalrepresentation of enzyme kinetic reaction expression and the subsequentproduction of the oxidized dye. Constants are described in Table 11.  Constant: Value: Units: References: k1 1.0E-4 1/(µmolarity*min) Paulı Ollıkka, Kırsı Alhonmakı (1993) k1r neglected -   k2 2.0E-4 1/(µmolarity*min) Paulı Ollıkka, Kırsı Alhonmakı (1993) k2r neglected -   Table 11: Valuesassigned to kinetic parameters described in Table 10. We ignored k1r and k2rvalues because of k1>>>>k1r and k2>>>k2r, so our bothreactions act as irreversible Rule Name: Rate Rules: HRP dE/dt = - k1[E][S1]+k1r[ES1] H2O2 dS1/dt = -k1[E][S1]+k1r[ES1] ES1 (HRP compound I) dES1/dt =k1[E][S1] –k1r[ES1]-k2[ES1][S2]+k2r[ES2] Methyl Green (MG) dS2/dt = -k2[ES1][S2] +k2r[ES2] ES2 (MGox +LiP compound II) dES2/dt = k2[ES1][S2] – k2r[ES2] Table12: Differential equations of reactions.  LiP – MB 5 hours of decolorizationWhat did it show???Before runningthe model we needed to decide what an appropriate endpoint of reaction wouldbe. Therefore, concentrations of species were determined from the literatureand necessary assumptions were made to check our cascade reactions workcorrectly. According tochosen references points, we were trying to determine how quickly we could geta decolourization. Decolourization depends on final [ES2] (MBox +Lipcompound II) concentration and degradation of MB. Decolourizationstarts in 40 minutes. Future plansThe most important results that we were able toachieve were the correlation between the functional assays the modellingresults of our MATLAB kinetic model. In the modelling, the decolourizationsstart at the approximately 40 minutes while these are 60 minutes for the assay.These differences were due to the consideration of the used HRP and LIP enzymesin the modelling as purified allowing more direct interaction in between thehydrogen peroxide and the dyes. The assay was not conducted with purifiedenzymes but with lysate acquired from liquid culture containing impuritieswhich slow down the conduction of the reaction. In the future, we hope toextract the enzymes from the bacteria to allow the direct contact of the enzymesand the dyes to increase spontaneity.Thanks to Ayşe Çelik for her support.Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Project/ModellinProject/Mechanism From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube ProjectIntroductionMechanismModellingPartsOverviewThe mostappropriate scale to deducing the qualitative chance in reporter systems relieson visual data.[i]Thereporter system that we designed as an alternative to the current reporter systemsuses the change factor in the current reporter systems as the response. Instead of observing change through the synthesis of color, thesystem is built around the observation of change through breaking down dye; thedye degradation plays a key role in the system, therefore for the system tofunction there is the requirement of using specific dye in experimentsalongside the standard lab equipment. The bacteria will degrade thenecessary dye to report the response. Color-BasedReporter SystemThe dye thatwas designated and the enzyme that was to show activity in accordance to it wasone of our concerns. The enzyme-substrate action in this novel system carriedgreat importance and had to be designed with utmost care.Peroxidases,which actively take part in the breakdown of lignin-based compounds thuscontributing to the maintenance of the continuity of the existence of white rotfungi that we based our bleaching enzymes on ,wereenzymes that we took into our prospect. We elected lignin peroxidase, an enzymeactively used by white rot fungi to continue their life , asthe enzyme to be implemented into our system. To increase the variance and therichness in future application, we chose a secondary enzyme. The secondaryenzyme was the horseradish peroxidase as it had a very strong structure andhigh activity. Ligninperoxidase: Ligninis highly resistant to biodegradation and only higher fungi are capable ofdegrading the polymer via an oxidative process.Ligninis found to be degraded by an enzyme lignin peroxidases produced by some fungilike Phanerochaete chrysosporium.The mechanism by which lignin peroxidase (Lip)interacts with the lignin polymer which involves Veratryl alcohol (Valc), asecondary metabolite of white rot fungi, acts as a cofactor for the enzyme.Inenzymology, a lignin peroxidase (EC 1.11.1.14) is an enzyme that catalyzes thechemical reaction1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol+ H2O2   3,4-dimethoxybenzaldehyde +1-(3,4-dimethoxyphenyl)ethane-1,2-diol + H2O Thus, thetwo substrates of this enzyme are 1,2-bis(3,4-dimethoxyphenyl)propane-1,3-dioland H2O2, whereas its 3 products are 3,4-dimethoxybenzaldehyde,1-(3,4-dimethoxyphenyl)ethane-1,2-diol, and H2O. This enzymebelongs to the family of oxidoreductases, specifically those acting on aperoxide as acceptor (peroxidases) and can be included in the broad category ofligninases. The systematic name of this enzyme class is1,2-bis(3,4-dimethoxyphenyl)propane-1,3-diol:hydrogen-peroxideoxidoreductase. Other names in common use include diarylpropane oxygenase,ligninase I,diarylpropane peroxidase, LiP, diarylpropane:oxygen,hydrogen-peroxideoxidoreductase (C-C-bond-cleaving). It employs one cofactor, heme.LiPcatalyzes the H2O2-dependent oxidation of a variety of lignin model compoundsin the following multistep reaction sequence:LiP(Fe+3)P +H2O2 à LiP-I(Fe+4 – O)P’ + H2OLiP-I(Fe+4 –O)P’ + R à LiP-II(Fe+4 – O)P + R’LiP-II(Fe+4– O)P + R + 2(H+) à LiP(Fe+3)P + R’ + H2O Horseradishperoxidase: The enzyme horseradish peroxidase (HRP), found in the roots ofplant horseradish, is used extensively in biochemistry applications primarilyfor its ability to amplify a weak signal and increase detectability of a targetmolecule. It is a metalloenzyme with many isoforms and the most studied type isC.Thestructure of the enzyme was first solved by X-ray crystallography in 1997 andhas since has been solved several times with various substrates. It is an allalpha-helical protein which binds heme as a cofactor.Alone, theHRP enzyme, or conjugates thereof, is of little value; its presence must bemade visible using a substrate that, when oxidized by HRP using hydrogenperoxide as the oxidizing agent, yields a characteristic change that isdetectable by spectrophotometric methods.Numeroussubstrates for the horseradish peroxidase enzyme have been described andcommercialized to exploit the desirable features of HRP. These substrates fallinto several distinct categories. HRP catalyzes the conversion of chromogenicsubstrates (e.g., TMB, DAB, ABTS) into colouredproducts, and produces light when acting on chemiluminescentsubstrates (e.g.ECL).Horseradishperoxidase is a 44,173.9-dalton glycoprotein with 6 lysine residues which canbe conjugated to a labeled molecule. It produces a coloured, fluorimetric, orluminescent derivative of the labeled molecule when incubated with a propersubstrate, allowing it to be detected and quantified. HRP is often used inconjugates (molecules that have been joined genetically or chemically) todetermine the presence of a molecular target. For example, an antibodyconjugated to HRP may be used to detect a small amount of a specific protein ina western blot. Here, the antibody provides the specificity to locate theprotein of interest, and the HRP enzyme, in the presence of a substrate,produces a detectable signal.Horseradish peroxidase isalso commonly used in techniques such as ELISA and Immunohistochemistry due toits monomeric nature and the ease with which it produces coloured products.Peroxidase, a heme-containing oxidoreductase, is a commercially importantenzyme which catalyses the reductive cleavage of hydrogen peroxide by anelectron donor.Horseradishperoxidase is ideal in many respects for these applications because it issmaller, more stable, and less expensive than other popular alternatives suchas alkaline phosphatase. It also has a high turnover rate that allowsgeneration of strong signals in a relatively short time span.Moreover,"In recent years the technique of marking neurons with the enzymehorseradish peroxidase has become a major tool. In its brief history, thismethod has probably been used by more neurobiologists than have used the Golgistain since its discovery in 1870." DesignIn order to verifythat the horseradish peroxidase and lignin peroxidase enzymes, which were nottransformated and synthesized in bacteria in the past, worked; we decided touse a constitutive promoter. We chose the constitutive promoter as J23100, oneof the strongest.  We wanted to be certain that our protein is surelyproduced due to the J23100 constitutive promoter that is 35 bp and coming afterthe prefix of 22 base pairs. The exhibition of our results inWestern Blotting through the addition of a 6 histidin long his-tag at the endof the horseradish peroxidase C and lignin peroxidase 1 enzyme sequences, whichcontain BamH-I restriction cites that are followed by our ‘AAAGAGGAGAAA’sequenced RBS, was added to our lab plans. This would in turn give usthe safest experimental procedure to validate the presence of the proteinssyntesized.The accruityof our design was to be tested through getting the dye to react with theculture of bacteria containing enzymes that were to be rapidly synthesizedthrough the constitutive promoter. The changes ranging from the visuallyobservable changes in the color concentration to the shifts in the wavelenghtabsorbance which can only be detected through Varioskan approve the validity ofour project. The degradation of the specified dyes by the lignin peroxidase andhorsereadish peroxidase, enzymes we selected to breakdown these dyes, is enoughto complete the rudimentary part of our project. The addition of the enzymes asa composite at the end of another part would enable the production of theprotein in the sequence as well as the the enzymes enabling the responsethrough an accelerated breakdown process.Aside of theaffirmation of the synthesis of the enzymes with the Western Blott results, toconfirm the functionality of the proteins carries great importance. We came tothe conclusion that working with several methods to verify the results would bemore professional and certain:-Theconfirmation of the enzyme activity can be donethrough collecting the data acquired through the observation of the interactionbetween the interaction of the specified dyes and DegradEcolor bacteria. Forthis feat to be accomplished, the selection of a dye that both ligninperoxidase and horseradish peroxidase can degrade in equivalent levels would bemore reasonable. We chose Methylene blue as the dye to be used as it wasdegraded by the two enzymes in similar amounts during trials. The trials wereconsisting of the observation of the changes in the absorbance and colorthrough the reactions between methylene blue and the two enzymes. Thedegradation of the dye plays a keyrole in the determination of the enzymeactivity..-HRPactivity was assayed using 2,4-dichlorophenol and 4-aminoantipyrine as thesubstrates, substrates used to assay the activity of enzymes. LiP activity wasassayed using veratryl alcohol as the substrate. -Whileexamining the general properties of the two designated enzymes, we found outthat the horseradish peroxidase enzyme had the ability to determine thepresence of a molecular target. If we are to recall the Western blott procedurewe see the following:The proteinsin the lysate that were planted in the jel first run and transferred to themembrane afterwards. After being left to interact with the primary antibodiescausing the immuglobins to bind with the specific binding site located on the protein.In the case of contamination or the absence ofprotein synthesis, no binding will be observed. After the completion of thebinding of the primary antibodies, the secondary antibody binding processbegins. While the secondary antibodies bind to the primary antibodies, theybring with them the horseradish peroxidase enzymes that are attached to them.The enhanced chemiluminescence solution does emission under the Westernblotting device. The luminol in the enhanced chemiluminescence solution isoxidised by hydrogen peroxide, however this reaction can only occur in thepresence of the horseradish peroxidase; as explained, if the proteins arecorrectly synthesized the horseradish peroxidase will cause oxygenation to takeplace causing emission. The same effect can be acquired through the horseradishperoxidase that is synthesized by DegradEcolor. In thıs case, the functionalityof the enzyme could be proven after seeing emission from the interactionbetween the enhanced chemiluminescence solution and DegradEcolor or lysateprepared from DegradEcolor. As horseradish peroxidase was able to catalise thereaction of hydrogen peroxide, lignin peroxidase, another peroxidase enzyme,would most likely be able to have the same effect. We are expecting to seeemission from the bacteria or lysate prepared from the bacteria that producelignin peroxidase.DyeDegradationMethylene blue,the dye which the horseradish peroxidase and lignin peroxidase exhibitedequivalent levels of degradation on, will show that DegradEcolor produced thefunctional proteins. Addition of the horseradish peroxidase or ligninperoxidase enzyme sequence to the composite part would make the part include areporter system. If the part is coded and the enzyme is translated, the enzymewill be synthesized and change in methylene blue will be observed. Manydeductions can be obtained with the results of the methylene blue degradationincluding the following:-Thebleeching of the dye color, which can be understood through qualitativeobservation, allowsus to reach certain conclusions. The bleeching of the colorprimarily indicates that the enzymes were synthesized. therefore nocontamination took place. Apart from the synthesis factor, the conclusionthat  the conformational structure is functional can be reached. This willin turn provide insight that the other desired proteins will also besynthesized. The system will also enable the ability to evaluate the efficiencyof the system through the gradient of color concentration.-The changesin absorbance will ease the acquirement of the certain results. The absorbance data can be used to prepare a professional and highlyefficient scale for examination and use. The prepared scales can be used forcomparison between the future experiments and the current experiments. It wouldbe hard and even erronous to make judgement on strains that do not have highprotein synthesis but the comparison between the acquired absorbance values andthe scales not only  removes the risk factor but also gives theopportunity of making precise judgement in the absence of visual data.      AdvancedSystemThe mainprinciple of our advanced system that has brought a new dimension to reportersystems lies in the variance in the activity levels of the lignin peroxidaseand horseradish peroxidase enzymes. The enzymes to have effect on one specificdye at the same time would not bring us any experimental gain. For this reason,the advanced system contains two different dyes alongside the two enzymes. Thesystem is built around the inability of one enzyme in degrading the one dyewhile the other can, while the opposite is true for the other dye. Experimentalsystems containing a complex unison of enzymes and dyes can be constructed.To furtherenhance our project, we formed such a complex system. The bacteria were giventwo parts, one containing horseradish peroxidase while the other containslignin peroxidase. The dyes that were selected were Methyl green and Azure-b.While methyl green is, as the name implies, green colored, azure-b has a colorin between pink and purple. Horseradish peroxidase can effectively degrademethyl green but lacks the ability to effectively degrade azure-b meanwhilelignin peroxidase is active in the degradation of azure-b and lackluster indegrading methyl green.If partsstarting off with different inducible promoters were transformed to the samebacteria, different data from different conditionscould be acquired. Think of a system in which the bacteria have induciblepromoters that are oxygen sensitive and the other carbon dioxide sensitive. Ifmethyl green and azure-b was to be added to cause interaction with the bacteria,the varying levels of oxygen and carbon dioxide would therefore result indifferent levels of enzymatic activity. Let’s say that the oxygen induciblebacteria contain genes that code for horseradish peroxidase and the carbondioxide inducible bacteria contain genes that code for lignin peroxidase. The variation in the level of oxygen and carbon dioxide wouldchange the rate of specific enzyme activity hence leading to a difference inthe degradation levels of methyl green and azure-b. This would change the colorof the resultant substrate thus allowing the identification of the activity ofthe inducible promoters. In this way, scales could be prepared for manydifferent types of inducible promoters for countless possibilities.[i] K.E.L. Eriksson, R.A. Blanchette and P. Ander (1990)."Microbial and Enzymatic Degradation of Wood and Wood Components,". Springer-Verlag[i]  Veitch, Nigel C., Horseradish peroxidase:a modern view of a classic enzyme, Phytochemistry, 65, 2004,249-259, 23-May-2010[i] Ann B. Orth,  Daniel J. Royse, Ming Tien“Ubiquity of Lignin-Degrading Peroxidases among Various Wood-Degrading Fungi,”Applied and Environmental Microbiology (1993):4017-4023[i]Annele Hatakka, “Lignin-modifying enzymes from selectedwhite-rot fungi: production and role from in lignin degradation,” FEMSMicrobiology Reviews (1994):125-135[i] Renganathan V, Miki K,Gold MH (1985). "Multiple molecular forms of diarylpropane oxygenase, anH2O2-requiring, lignin-degrading enzyme from Phanerochaete chrysosporium".Arch. Biochem. Biophys. 241 (1): 304–14. doi:10.1016/0003-9861(85)90387-X. PMID 4026322[i]SusanaCamarero, SovanSarkar, FranciscoJavier Ruiz-Dueñas,Marı́aJesús Martı́nezand ÁngelT. Martı́nez “Description ofa Versatile Peroxidase Involved in the Natural Degradation of Lignin That HasBoth Manganese Peroxidase and Lignin Peroxidase Substrate Interaction Sites,” Centro de Investigaciones Biológicas, Consejo Superior deInvestigaciones Cientı́ficas, Velázquez 144, E-28006 Madrid, Spain, lastaccessed 06.21.2014, http://www.jbc.org/content/274/15/10324.short[i] Jean Luc Wertz and Olivier Bedue, LignocellulosicBiorefineries (Lausanne: EPFL Press, 2013), 278-286[i]Wolfgang Blodiga, 1, Andrew T. Smithb, Kaspar Winterhaltera, Klaus Piontek “Evidence fromSpin-Trapping for a Transient Radical on Tryptophan Residue 171 of LigninPeroxidase,” Archives of Biochemistry and Biophysics (1999):86-92[i]M. Yadav, P. Yadav, K. D. S. Yadav. “Purification,characterization, and coal depolymerizing activity of lignin peroxidase from Gloeophyllumsepiarium,” Biochemistry (Moscow)(2009):1125-1131[i]  PDB 1GWU; Gajhede M, Schuller DJ, Henriksen A,Smith AT, Poulos TL (December 1997). "Crystal structure of horseradishperoxidase C at 2.15 A resolution". Nat. Struct. Biol. 4(12):1032–8. doi:10.1038/nsb1297-1032. PMID 9406554[i]  "PeroxidaseC1A Related PDB sequences".UniPDB. European Bioinformatics Institute[i] Veitch NC(February 2004). "Horseradish peroxidase: a modern view of a classicenzyme". Phytochemistry 65 (3): 249–59. doi:10.1016/j.phytochem.2003.10.022. PMID 14751298 [i] Akkara JA, Senecal KJ,Kaplan DL (October 1991). "Synthesis and characterization of polymersproduced by horseradish peroxidase in dioxane". Journal of PolymerScience 29 (11): 1561–74. doi:10.1002/pola.1991.080291105[i] Chau YP, Lu KS (1995)."Investigation of the blood-ganglion barrier properties in rat sympatheticganglia by using lanthanum ion and horseradish peroxidase as tracers". ActaAnat (Basel) 153 (2): 135–44. doi:10.1159/000313647. PMID 8560966[i] Lichtman JW, Purves D(1985). "Cellmarking with horseradish peroxidase". Principles of neural development.Sunderland, Mass: Sinauer Associates. p. 114. ISBN 0-87893-744-7[i] “Part:BBa_J23100,” lastaccessed June 21, 2014, http://parts.igem.org/Part:BBa_J23100[i]  J.K. Spiker, D.L. Crawford, E.C. Thiel, Appl.Environ.[i]Microbiol. 37 (1992) 518–523.[i] M. Tien, T.K. Kirk, Proc. Natl. Acad. Sci. U.S.A. 8(1984)  Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Project/MechanisHuman Practices/Outreach From 2014hs.igem.org Home Team ParticipantsGalleryAcknowledgements Project IntroductionMechanismModellingParts Results CloningProtein AnalysisFunctional Assay Lab Garage NotebookProtocolsSafety Human Practices OutreachFuture Plan 2014 - AUC_TURKEY contact us twitter youtube Human PracticesOutreachFuture PlanIn order tospread the iGEM all around our environment, we as AUC_TURKEY paid a visit togrand graduation ceremony of Burç Akademi. Burç Akademi is awell-known academy for children between the ages of 4 to 6 and first choice ofthe parents who knows how to make the right decisions for their child andtheir education. This academy hosted lots of successful people and who knows ifthis young generation will be part of the iGEM. Weintroduced iGEM to parents and its future they got impressed and theirreactions were unforgettable when we talked about our project and plan to create awhole new system to world of synthetic biology. Mostly it was the mothers whowanted their child to be part of this tremendous movement fatherswere just proud of their kid’s generation and motionless with the excitement ofthe ceremony. We believe we have done very good job introducingiGEM, iGEMism and the science of synthetic biology. The eliteclass people with their children were there to watch the shows from all aroundthe world and surprisingly meet with the world of iGEMism. An iGEMistknows how to spread iGEMism, whom to spread iGEMism and where to spread iGEMism.We guess we were just at the right place in the right time.  TeamAUC_Turkey is at work again at the right place and the right time. Ahmet UlusoySchool has been one of the best high school education institutions of Turkey.Our school organizes a traditional awards ceremony for successful student ithosts every year. We thought that would be good idea to spread iGEM and letsuccessful people know about iGEM and this would increase the quality of the upcomingiGEM members for next year.  The ceremony was tremendous and full of entertaining shows,guests were classy and awards were delivered to rightful owners. The mostsurprising event of the ceremony was the introduction of the world of iGEM andour previous awards from iGEM such as best presentation encouraged people to bepart of this amazing world. Who knows what we could have managed with betterawards. Most of the award winners held high level of information most highschool can’t on genetics and when they heard we managed to get good resultsfrom western blotting they were begging to be part of our team. We made thebest decisions again for our school and next generation of our team. iGEMist movement continues. We as AUC_Turkey team targetedsomewhere different this time. Unlike our other actions such as Burç Academyand Atlantik Awards ceremony, this time we did not focused on upcominggenerations but instead, we focused on people at higher stages. Where we couldfind people might have interest to synthetic biology? The answer was TOBATwhich hosted over one thousand and five hundred student from all around theworld.All these students were making presentations of their ownworks and there were also exciting ideas. The participants surprised by ouriGEM presentation which they did not expect at all. The students were notsatisfied by the high school part of iGEM but the part for Universities amazedthem. They said they look forward for next iGEM and at the end of the day wewere proud of ourselves for making new people part of the iGEMist movement.            Inthe progress of our project we collaborated with NGSS_TR. We did someexperiments of NGSS_TR and they did some of our experiments. When needed weexchanged lab materials as we use the same lab. Several times we discussed onthe projects to improve them.Retrieved from "http://2014hs.igem.org/Team:AUC_TURKEY/Human_Practices/Outreac1256MingdaoMingdao High School Taichung, Taiwan www.mingdao.edu.tw/homeX/EN/High SchoolOdor - Let it dieFood wastes can be recycled as fertilizers but leaving strong odors (e.g., NH3, H2S, etc.) from metabolizing by food spoilage bacteria. Antimicrobial peptides (AMPs) with effect against bacteria, viruses and fungi are small, cationic peptides that bind anionic membrane surfaces of microbes, resulting in forming channels or pores and leaking cell contents. Stenotrophomonas maltophilia is an environmental bacterium with beneficial effects for plant growth. Several extracellular proteins such as proteases, lipases, nucleases, chitinases and elastases have been identified as decomposing enzymes. In the design of our genetically engineered bacteria, we?ve created biobricks of (1) AMPs (cecropin and magainin) to attack spoilage bacteria, and (2) several secreted decomposing enzymes directed by the secretion signal of E. coli OmpA to enhance the digestion of food wastes, as well as set (3) a self-destructive device inside to sacrifice when completing the mission, in which ccdB lethal gene expression is regulated by light.2311BBa_K1256999http://2014HS.igem.org/Team:MingdaoBBa_K1256000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=161420140http://2014hs.igem.org/files/presentation/Mingdao.pdfhttp://2014hs.igem.org/files/poster/Mingdao.pdfHigh SchoolLog in   Team:Mingdao/project.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media Food wastes can be recycled as fertilizers but leaving strong odors (e.g., NH3, H2S, etc.) from metabolizing by food spoilage bacteria. Antimicrobial peptides (AMPs) with effect against bacteria, viruses and fungi are small, cationic peptides that bind anionic membrane surfaces of microbes, resulting in forming channels or pores and leaking cell contents. Stenotrophomonas maltophilia is an environmental bacterium with beneficial effects for plant growth. Several extracellular proteins such as proteases, lipases, nucleases, chitinases and elastases have been identified as decomposing enzymes. In the design of our genetically engineered bacteria, we’ve created biobricks of (1) AMPs (cecropin and magainin) to attack spoilage bacteria, and (2) several secreted decomposing enzymes directed by the secretion signal of E. coli OmpA to enhance the digestion of food wastes, as well as set (3) a self-destructive device inside to sacrifice when completing the mission, in which ccdB lethal gene expression is regulated by light. 1. Food wastes & Fertilizers   Food wastes are serious problems for humans and the environment alike. Accordingly, one-third of all food produced on earth is wasted before it goes into a human stomach. The average person produces around 475 pounds of food waste every year and the world wastes 11 billion metric tons annually. In addition to educating people to take food carefully and reduce food wastes, another way is to recycle the food wastes. One of them is converting the wastes to fertilizers through composting. However, the composting process not only grows a lot of kinds of bacteria but also leave strong odors produced from bacterial metabolism. 2. Odor & Spoilage Bacteria   Based on food nutrient composition and on the chemical and physical parameters, various microorganisms emerge in the food wastes. Typical spoilage bacteria are given in Table 1, classified by spoilage substrates and metabolites found in spoiled foods. (L. Gram et al., 2002)   Table 1. To reduce the odor from food wastes, the spoilage bacteria have to be controlled or eliminated. Strains of bacteria are benefit and applied for composting food wastes. Some grow well in the special environment (e.g., aerobic and acid environment) where the spoilage bacteria cannot survive such as Trichoderma sp. Some are able to produce antibiotics against the spoilage bacteria such as Actinobacteria. And some producing extracellular strong digestive enzymes are capable of decomposing food efficiently before used by the spoilage bacteria such as Bacillus spp. Reference: - L. Gram et al., 2002. Food spoilage—interactions between food spoilage bacteria. International Journal of Food Microbiology 78, 79– 97   3. Antimicrobial peptides   - Antimicrobial peptides (AMPs) are an important component of the natural defenses of living organisms against surrounding microbes. AMPs contain a broad spectrum of antimicrobial activities against Gram-positive and Gram-negative bacteria, mycobacteria, fungi, and viruses. AMPs are small peptides and generally less than 10 kDa. They have an overall net positive charge and target on the anionic membrane surfaces of bacteria to form channels or pores, which is leading to leakage of cell contents and the death of the cell. - Cecropin, an antimicrobial peptide originally isolated from the moth, Hyalophora cecropia. Cecropins are 3~4 kDa linear amphipathic peptides composed of about 31~37 amino acid residues. They have broad activity against both Gram-positive and Gram-negative bacteria through lysing bacterial cell membranes, inhibiting proline uptake and causing leaky membranes. Cecropins constitute a main part of the cell-free immunity of insects. - Magainin, another antimicrobial peptide found from the skin of the African clawed frog Xenopus laevis. Magainins composed of 23 residues are positively charged and amphiphatic. They preferentially bind to anionic phospholipids abundant in bacterial membranes with the formation of dynamic peptide-lipid supramolecular pore and cell permeabilization.   References: - Antimicrobial peptides - Wikipedia, the free encyclopedia - K.V.R. Reddy et al. 2004. Antimicrobial peptides: premises and promises. International Journal of Antimicrobial Agents 24, 536–547 4. Stenotrophomonas maltophilia   Stenotrophomonas maltophilia is a soil, Gram-negative bacterium. They are aerobic, nonfermentative. They are found throughout the environment, particularly in close association with plants with a dominant beneficial effect for plant growth and health. They have strong decomposing enzymes and been applied to the breakdown of natural and man-made pollutants for bioremediation. Extracellular enzymes such as proteases, lipases, nucleases, chitinases and elastases have been identified and recognized as important factors for plant growth and colonization by other microorganisms.   References: - Robert P. Ryan, et al. 2009. The versatility and adaptation of bacteria from the genus Stenotrophomonas. Nat Rev Microbiol. 7(7):514-25. 5. BioBrick design & Genetically Engineered Bacteria   Fertilizers converted from food waste make odors from the metabolism of the spoilage bacteria. In the iGEM project this year (2014), we’ve designed a genetically engineered bacterium which can produce antimicrobial peptides, cecropin and magainin, to kill the bacteria grown in the food wastes. And the sterile food wastes would be decomposed by a genetically engineered bacterium which produces extracellular decomposing enzymes from Stenotrophomonas maltophilia. And finally the genetically engineered bacteria will be self-destructive with a regulated device carrying ccdB lethal gene induced by the light. 1. Food waste experiment- sniff test   To test the hypothesis of food waste odor produced from the spoilage bacteria, we treated the food wastes with an antibiotic, chloramphenicol, to eliminate bacteria and keep sterile. The fresh food wastes were took from the recycling system in Mingdao High School. The 2-ml aliquots were supplemented with 10 or 50 ug/ml of chloramphenicol (Table 1), followed by culturing in a 37°C incubator for 3 hours. 14 participants in grade 11 were subject to the sniff test and gave a score from 1 to 10. As shown in Figure 1, food wastes treated with chloramphenicol had fewer scores than those without treatment, suggesting that keeping food sterile can reduce the odor. The results implied that preventing food wastes growing bacteria by antibiotics would eliminate the food spoilage odor. Table 1. Food wastes supplemented with various concentrations of chloramphenicol. Figure 1. The result of sniff test (score from 1 to 10) from food wastes supplemented with various concentrations of chloramphenicol. 2. Zone of inhibition assay   To test the efficiency of inhibiting bacterial growth by antibiotics, we performed a zone of inhibition assay. 200 µl of E. coli from overnight culture were spread on a agar plate, followed by covering 4 papers with 5, 10, 30, 50 µg of chloramphenicol, respectively. The bacteria were grown at 37°C for 24 hours. The zones of inhibition appeared and were measured. As shown in Figure 1 and 2, the sizes of zones were correlated with the concentrations of chloramphenicol, demonstrating the efficiency of antibiotics against bacteria.     Figure 1. Zone of inhibition assay Figure 2. Zones of inhibition diameter were measured. 3. Bacillus transformation   Bacillus spp. are soil bacteria and have been applied to food waste composting. In order to genetically engineer Bacillus strains for further application, we’d like to develop a system for transforming Bacillus. We collaborated with LMU-Munich to develop the system. We obtained several vectors for Bacillus transformation from LMU-Munich and iGEM HQ. Following the transformation protocol developed by LMU-Munich, we’ve tried 6 vectors and successfully generate a Cm-resistant strain of Bacillus subtilis 168 (DB2) transformed with the vector of pBS1C (Figure 1). Bacillus/pBS1C survives in the media supplemented with 10 g of chloramphenicol but not wild-type strain (Figure 2.) Figure 1. Bacillus transformation with 5 vectors Figure 2. Wild-type and Cm-resistant Bacillus strains 4. Stenotrophomonas maltophilia & strain identification   Stenotrophomonas maltophilia is a soil bacterium with a close association with plant growth and health. Many extracellular enzymes have been identified such as proteases, lipases, nucleases, chitinases and elastases.   In order to clone these genes, we purchased the strains of Stenotrophomonas maltophilia form Bioresource Collection and Research Center (BCRC) in Taiwan. Unfortunately, the strains with full genome sequences in the database (e.g., KEGG, BioCyc, NCBI, etc.) are currently unavailable in the stock. However, we got two strains from the soil isolates (BCRC #11901, BCRC #15550) and culture them in four different media, followed by genomic DNA extraction and PCR for a conserved protein ChiA1.   As seen in Figure 1, the PCR data showed that ChiA1 gene can be amplified from the strain of BCRC #15550 but not BCRC #11901. Figure 1. PCR for ChiA gene with the genomic DNAs from BCRC #11901 and BCRC #15550 cultured in 4 different media. PCR products were subjected to sequencing after gel extraction of the band around the size of 1191 bp (the predicted size of ChiA1).The sequence data was subjected to phylogenic tree analysis on an online web server of Phylogeny.fr. The result of phylogenic tree analysis showed that the strain of BCRC #15550 is the outgroup to other strains, whose full genome sequences are available in the database (Figure 2).   Figure 2. Phylogenic tree analysis between strains of Stenotrophomonas maltophilia. In addition, we performed NCBI Nucleotide BLAST with the ChiA1 sequence of BCRC #15550. As shown in Figure 3, the sequence can align 4 strains of Stenotrophomonas maltophilia with 98% query cover and 91% identity.   Figure 3. The result of NCBI Nucleotide BLAST Therefore, we designed primers for extracellular enzymes based on the strains of D457 and K279a and try to clone these gene by PCR. 5. gene cloning of decomposing enzymes   The genomic DNAs of Stenotrophomonas maltophilia BCRC #15550 and Bacillus subtilis 168 (DB2) were extracted. The primers for the genes of DNase, Protease, Lipase, Chitinase were designed based on the Stenotrophomonas maltophilia strains of D457 and K279a, and the primers for the genes of Glucanases were designed based on Bacillus subtilis 168 (DB2). Figure 1 showed the PCR result for DNase, Protease, Lipase, Chitinase of Stenotrophomonas maltophilia, and Figure 2 for Glucanases of Bacillus subtilis 168 (DB2). Figure 1. PCR for genes of DNase, Protease, Lipase, ChitinaseFigure 2. PCR for genes of Glucanases The PCR products were subjected to cleanup and ligated to the pSB1C3-based vector (pSB1C3-Plac-SS-NB,Part: BBa_K1256003) with a secretion signal of OmpA of E. coli designed by us. 6. BioBrick construction   Figure 1 shows the standard backbone of pSB1C3, which is a basic BioBrick part for cloning. pSB1C3 contains RFP coding device composed of subparts of R0010 (Plac), B0034 (RBS), E1010 (RFP) and B0015 (Terminator).    Figure 1. pSB1C3 If you want to clone a gene under a given promoter, you have to do two rounds of cloning, that is, you have to clone your interest gene on pSB1C3, followed by assembling a promoter on the part you’ve generated.   To facilitate the gene cloning process, we’ve introduced NheI and BamHI sites just behind RBS (B0034) and in front of the terminator (B0015) on the vector of pSB1C3 (pSB1C3-Plac-NB (Part: BBa_K1256001), Figure 2). Therefore, the gene of interest can now can be cloned using NheI and BamHI restriction enzyme sites and driven under the front promoter (e.g., Plac (R0010) in this case).   Figure 2. pSB1C3-Plac-NB (Part: BBa_K1256001) In addition, we further introduced MfeI and NsiI sites just following the antibiotic resistance cassette (i.e., chloramphenicol resistance cassette) (Figure 3). And now you can easily add another BioBrick part cut by EcoRI and PstI and ligate to this region with MfeI and NsiI (EcoRI and MfeI are compatible, PstI and NsiI are compatible), driving the gene part either by the promoter of Cm cassette or independently by the respective promoter.   Figure 3. pSB1C3-Plac-NB-Cm-MN (Part: BBa_K1256002) 7. AMP construction   To produce antimicrobial peptides, cecropin and magainin were chosen as candidates because they are applied in medicine and food preservative and are sold as chemical forms by Sigma-Aldrich.   We obtained the amino acid sequences from Sigma-Aldrich webpage and optimized the nucleotide sequences for gene expression and engineering in the host of Escherichia coli. We performed codon optimization on the web server of Integrated DNA Technologies (IDT). The figure 1 showed the amino acid sequences and optimized nucleotide sequences for cecropin and magainin, respectively.   Figure 1. The amino acid sequences and optimized nucleotide sequences of cecropin and magainin In order to synthesize the DNA sequences on the vector without scars or any restriction enzyme sites, Gibson assembly method was performed with T5 exonuclease. The optimized DNA sequences of cecropin and magainin were synthesized on the primers, followed by PCR with the template of pSB1C3-Plac-SS-MprF. Colony PCR (Figure 2) and sequencing were performed to check the correction of the plasmid.   Figure 2. The results of colony PCR for checking the plasmids carrying the DNA sequences of cecropin and magainin. 8. gene regulation   To express a lethal gene (e.g., ccdB) in an engineered bacterium, the gene has to be tightly regulated and expressed at an appropriate time. Lac operon is a well-studied gene regulation system. Lac promoter is inhibited at the presence of LacI repressor while induced at the presence of IPTG inducer.   We’ve amplified the LacI repressor gene from the BioBrick part of Part:BBa_C0012 (Figure 1) and cloned it into the vector of pSB1C3-Plac-NB-Cm-MN we designed and constructed. The LacI gene is following Cm cassette and driven by the promoter of Cm, checked by colony PCR (Figure 2). As shown in Figure 3, RFP gene driven by Plac was inhibited in the absence of IPTG and expressed in the presence of IPTG.   Figure 1. PCR amplification of LacI and ccdB geneFigure 2. Colony PCR for check the plasmid carrying LacI Figure 3. RFP gene expression regulated in the Lac operon system. RFP gene expression was induced by IPTG In addition, we’ve amplified the ccdB gene (Figure 1) from the the BioBrick part of Part: BBa_K145151. The cloning of ccdB gene onto the vector of pSB1C3-Plac-NB-Cm-LacI carrying LacI repressor is ongoing. Abstract Introduction Results Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/project.htmlbio-brick.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media Biobrick Map- Click link to go to biobrick description Part Registry Name Attribute Part (E-X-Part-S-P)  Part Length Gene  Gene Length Description Biobrick Design pSB1C3-Plac-NB (Part: BBa_K1256001)   BENEFIT: cloning genes with NheI & BamHI sites, easily exchange RFP gene BIOBRICK LIGATION: - “X-P” ligated to “S-P” ( X, S are compatible): standard part - “E-N” ligated to “E-X” (N, X are compatible): to exchange front part (Fig 1. A) - “N-P” ligated to “S-P” (N, S are compatible): to exchange back part (Fig 1. B)     Fig 1. BioBrick “part in part”  (image below)   (A)(B) pSB1C3-Plac-NB-Cm-MN (BBa_K1256002)   BENEFIT: cloning genes with MfeI & NsiI sites under CmR cassette BIOBRICK LIGATION: - “M-Ns” ligated to “E-P” ( M, E are compatible; Ns, P are compatible)     pSB1C3-Plac-SS-NB (Part: BBa_K1256003)   Secretion signal (SS): OmpA SS from E. coli BENEFIT: cloning genes with NheI & BamHI sites, easily exchange RFP gene BIOBRICK LIGATION: - “X-P” ligated to “S-P” or “N-P” ligated to “E-X” (X, S, N are compatible)       Attack pSB1C3-Plac-SS-Cecropin-MprF (BBa_K1256004)   MprF of Bacillus subtilis (DB2)added a RBS (BBa_B0034)was amplified by PCR and cloned into the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) using NheI and BamHI sites. Next, the cds of cecropin was synthesized on the primers and subjeted to Gibson Assembly to make this plasmid. The amino acid sequences of cecropin was obtained from Sigma-Aldrich, and the nucleotide sequence was optimized for engineering Escherichia coli using the codon optimization tool provided by Integrated DNA Technologies (IDT). pSB1C3-Plac-SS-Magainin-MprF (BBa_K1256005)   MprF of Bacillus subtilis (DB2)added a RBS (BBa_B0034)was amplified by PCR and cloned into the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) using NheI and BamHI sites. Next, the cds of magainin was synthesized on the primers and subjeted to Gibson Assembly to make this plasmid. The amino acid sequences of magainin was obtained from Sigma-Aldrich, and the nucleotide sequence was optimized for engineering Escherichia coli using the codon optimization tool provided by Integrated DNA Technologies (IDT).     Colony PCR was performed to check the plasmid (Right Photo). The cecropin and magainin sequences were further confirmed by sequencing.   Decomposing pSB1C3-Plac-SS-Protease (BBa_K1256006)   The protease coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with NheI and BamHI. However, PstI site can no longer be used in this plasmid due to three more sites on the gene coding region. pSB1C3-Plac-SS-Lipase (BBa_K1256007)   The lipase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and NsiI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) with NheI and PstI, leaving a scar of NsiI/PsiI in the end. pSB1C3-Plac-SS-DNase (BBa_K1256008)   The DNase coding sequence (cds) of Stenotrophomonas maltophilia D457 was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with NheI and BamHI. However, PstI site can no longer be used in this plasmid due to two more sites on the gene coding region. pSB1C3-Plac-SS-RNase (BBa_K1256009)   The RNase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and BglII and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) cut by NheI and BamHI. PstI site can no longer be used in this plasmid due to one more site on the gene coding region. pSB1C3-Plac-SS-Chitinase (BBa_K1256010)   The chitinase coding sequence (cds) of Stenotrophomonas maltophilia K279a was obtained from KEGG Genes Database. The gene was cut by NheI and NsiI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003) with NheI and PstI, leaving a scar of NsiI/PsiI in the end. pSB1C3-Plac-SS-Glucanase (BBa_K1256011)   The glucanase coding sequence (cds) of Bacillus subtilis 168 was obtained from KEGG Genes Database. The gene was cut by NheI and BamHI and ligated to the vector of pSB1C3-Plac-SS-NB (Part:BBa_K1256003). The cds can be easily exchanged with other BioBrick parts with NheI/PstI and SpeI/PstI, or be considered as a standard BioBrick parts (E-X-S-P). Genes of decomposing enzymes were cloned either from Stenotrophomonas maltophilia or Bacillus subtilis. Figure 1 showed the genes were amplified by PCR.   Figure 1. PCR to amplify the genes of decomposing enzymes   (A)(B) Self-destruction pSB1C3-Plac-RFP-Cm-LacI (BBa_K1256012)   RBS, LacI coding region and terminators (Part:BBa_Q04121) were amplified by PCR and cut by EcoRI and PstI. The PCR-amplified and enzyme-cut product was introduced to the novel designed plasmid backbone of pSB1C3-Plac-NB-Cm-MN ( Part:BBa_K1256002) with MfeI and NsiI. LacI gene is driven by Chloramphenicol promoter. RFP coding region can be exchanged using NheI and BamHI.     Figure 1 showed the result of colony PCR (A) and the RFG gene expression induced by IPTG (B).   (A)(B) pSB1C3-Plac-ccdB-Cm-LacI (BBa_K1256013)   ccdB coding region was amplified by PCR from BBa_K145151 and exchanged with RFP on the plasmid of BBa_K1256012. pSB1C3-Pred-ccdB-light sensor (BBa_K1256014)   Gene expression of ccdB (BBa_K145151), a lethal gene, is regulated by the light. The light-regulated system is composed of light sensor (BBa_K1017101) and light-regulated promoter (BBa_K1017301) designed and constructed by NCTU-Formosa in 2013. Light sensor was first amplified by PCR, cut by EcoRI and PstI, and ligated to pSB1C3-Plac-NB-Cm-MN cut by MfeI and NsiI. Then, light-regulated promoter was amplified by PCR, cut by EcoRI and NheI, and ligated to the resulting vector cut by EcoRI and NheI, which maintains the standard BioBrick assembly rule as well as the flexibility of exchanging RFP cds with NheI and BamHI. The ccdB was amplified by PCR using the forward primer with RBS and assembled to the vector with NheI and BamHI. Biobrick Design Attack Decomposing Self-destruction Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/bio-brichuman-practices.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media Collaboration with iGEM university teams We have collaborated with iGEM university teams and requested parts from them. Bacillus strains from LMU-Munich, light detecting gene from NCTU-Formosa, and pigment genes from Uppsala-Sweeden. Outreach to Mingdao Junior High Department With the intention to find our successors next year, we had a speech introducing iGEM and the ideas behind our project to Mingdao Junior High Department. The participants are mostly grade 9 students, and they were very interested with iGEM. Operation is succesfful Meeting with experts Getting ideas from experts is important, with this idea in mind, our team visited Dr. Chun-Hsien Lin from Department of Entomology to discuss about antimicrobial peptides. We also visited Dr. Chein-Wei Chen from Taichung Agricultural Improvement Center to see how food waste decomposes and what would happen under different circumstances. Dr. Chein-Wei Chen from Taichung Agricultural Improvement Center. Dr. Chun-Hsien Lin from Department of Entomology Pre-project training Before trying to achieve anything, we had several workshops about cloning, web design, pipette usage, and computer modeling. We make sure that we have enough background knowledge so that we wouldn't mess up anything. Web design crash course Pipette usage course 3 Day cloning workshop Computer modeling crash course Food waste sniff test Subjects from grade 11 class 12 assisted in helping us evaluate the level of the noisome odor by performing a sniff test. Disclaimer: participants are volunteered into this test and iGEM Mingdao take no responsibility for any consequences. Visit of other iGEM teams We are very interested in contacting TAS iGEM, as we both are the first iGEM teams from Taiwan to participate in the high school division. In order to do this, we went over to Taipei and said hi to them. We exchanged ideas about their and our projects. We also got the chance to visit NCTU iGEM and listen to their plan for iGEM this year. YouTube Channels, music, short films, and animations We tried to promote iGEM to the general public. We created short films, remade the Frozen song "Let it Go", made animations, and managed social networks; we have a google+ page and a YouTube channel. Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/human-practicemedia.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media You can view our videos here, instead of going to YouTube Let it Die- Cover by Hsuan-Chen Lu iGEM and food-waste themed "Let it Go" Cover Animation - Introduction and Project Overview Food waste odor, the spoilage bacteria,the design of our BioBricks, and the process of genetically engineering bacteria. Quick Project Overview We created a genetically engineered bacterium to kill the spoilage bacteria, decompose the food waste, and suicide when accomplishing the mission. iGEM Mingdao 2014 Short Film Trailer Food waste disaster spreads, and the princess of iGEM kingdom and iGEMers save the world with the genetically engineered bacteria Memory Slideshow We have a lot of iGEM human practices, outreaches, workshops, visits and meetings and multimedia. Importantly, we also have a lot of fun on doing iGEM project. Team Mingdao visiting Taipei Documentary of us going to Taipei and visiting Tri-I biotechnology company, meeting with TAS and NCTU iGEM teams, and having fun. 5/14/14 Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/medisafety.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media Lab Safety Programs Safety Issue People who conduct the programs involved in genetic engineering and related materials have to follow the regulation under Ministry of Science and Technology in Taiwan. (Safety Guide for the Lab of Genetic Engineering)   Safety Training First we went to a biolab in National Chung Hsing University (NCHU) for some training. Before entering the lab, we had a lesson concerning the safety for working in a biolab, which follows the regulation of Center for Environmental Protection & Occupational Safety and Health in NCHU (Safety Program in NCHU).   Later we have an iGEM biolab in Mindao High School. We followed the regulation issued by BioLab Office in Mindao (BioSafety in Mingdao). The advisor and the lab administrator gave a lesson for us before we were doing the iGEM project.   Safety Precaution and Equipment Autoclave The genetically engineered bacteria with recombinant DNAs were sterilized before discarded. Laminar Flow Table The bacteria were manipulated in the laminar flow cabinet. Latex Glove & Laboratory Coat We wear gloves and coats for doing the experiments. Fire Extinguisher We have several extinguishers if there’s a fire. Emergency Shower & Eyewash We have emergency shower and eyewash equipment if there’s any necessaries.   Supervisors Pei-Hong Chen, the instructor of Mingdao iGEM Team, is always with us when we are doing experiments for the project. Cai-Ying Lin, the laboratory administrator of Mingdao, prepared the experimental materials and cared for the wastes we left after doing the experiments.   Lab Safety Protocols The lab safety protocols that we have consulted and used as a reference could be found using the following links: Mingdao High School Lab Safety Protocol Safety Guide for the Lab of Genetic Engineering, published by the National Science Committee NCHU safety program Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/safetmodeling.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media Competitive Lotka–Volterra equations Lotka–Volterra equations are a simple model for population dynamics. dx/dt = rx (1 - x/K) x refers to the population size, r refers to growth rate, and K is the carrying capacity   Competitive population model between AMP-producing bacteria (a) and the spoilage bacteria (b). a = AMP-producing bacteria (Blue in graph) b = the spoilage bacteria (Red in graph) N = population R = growth rate K = carrying capacity Equation: Na+1 = Na*Ra (1 – (Na + Aab*Nb)/Ka) Nb+1 = Nb*Rb (1 – (Nb + Aba*Na)/Kb) Aab = the effect bacteria b has on the population of bacteria a Aba = the effect bacteria a has on the population of bacteria b bacteria a bacteria b At the 11th generation, the concentration of antimicrobial peptides (AMPs) produced by bacteria a reached the minimum inhibitory concentration (MIC) and are able to kill 95% of bacteria b. The population of bacteria a begins to out-compete and outgrow the population of bacteria b.   In our model, at the 11th generation, AMP-producing bacteria made enough antimicrobial materials against the spoilage bacteria. And at the 18th generation, AMP-producing bacteria reached a plateau and efficiently inhibited the growth of the spoilage bacteria.   The minimum inhibitory concentration of AMPs produced by our engineered bacteria has to be measured. Nevertheless, if the engineered bacteria can make effective concentration before the 11th generation, the spoilage bacteria will disappear at the 18th generation. Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/modelinteam.html From 2014hs.igem.org <!DOCTYPE html>   Home Team Project BioBrick Modeling Safety Human Practices Media Our team consists of 2 instructors and 22 members, and we all have something in common- the love of synthetic biology. About our Team About Mingdao Mingdao High School is a private secondary school located at Taichung City, Taiwan. The school serves grades 7-12. There are about 8,500 students, and its current principal is Albert T. Wang. Mingdao teaches 7 major subjects: Chinese, English, Math, Science, Social Science, Information, and Liberal Arts.   Information: 497, Sec. 1, Zhongshan Rd., Wuri Dist., Taichung City 41401, Taiwan TEL : +886-4-23372101　　FAX : +886-4-2336701   Website here   School Instructors Members Attributions Sponsors Principal: Albert T. Wang "At Mingdao, we believe that 'curriculum is everything; everything is curriculum.' With this education philosophy in mind, we devote to educate our students to learn how to learn and think. Our aim is to cultivate future global citizens with proper attitudes, open minds, and outstanding academic abilities."   Instructors Pei-Hong Chen (陳沛鴻, a.k.a. Phil Chen) Education:     Ph.D in Microbiology, National Taiwan University Experience:     Postdoc, National Yang-Ming University    iGEM 2014HS: Mingdao     iGEM 2012, 2011: NYMU-Taipei Interests:     Synthetic Biology, Gene Cloning, Metabolic EngineeringContact Info: f91445122@gmail.com       Hau-You Tzeng (曾皓佑) Education:     BSc, Ph. D in National Chung-Hsing University Experience:     2003-06, Exhibition guide in National Science Museum Honor:     2011, Best speaker in Annual Meeting of Taiwan Entomologist Society Interest:     Synthetic Biology, Medical Entomology, Virology, Parasitology Members   CHAN, YEN-KUANG So-Called Team Leader     I am a biology enthusiast, and I have a perfect score in the High School Entrance Exam. Although I was appointed the team leader, my job is actually doing roll calls. CHENG, SHUI-FENG     I’m actually the youngest member of the team, and the only grade 9 student. I love playing badminton, doing experiments, but I’m not good at expressing my own thoughts. WANG, YU-CHEN     I’m glad I’m a member of the Mingdao iGEM team. It’s very fun when we do experiments and discuss projects. I hope we can also have fun in the iGEM Jamboree. CHI, CHENG-FU Beat Box Operator     My thoughts are always on the contrary with my teammates, and I like to provide inspiration to our team. However, I’m not good at experiments. WANG, WEI-JUN Head Lab Coordinator     Looking back a few months before Occasionally filled in at the end in Maybe just this once joined the opportunity Only in hard deadlines Abundance of experimental Staying up until completely crazy In order to give a final award it (Google Translate used) TAN, CHIH-KANG Musical Composer     As an eleventh grader it is very thrilling to be able to work in a lab and manipulate genes to create bacteria clones. I have always been very interested in science, especially in the field of biology. This project is quite diverse in categories of competition. We are competing not only in genetic engineering but also in posters making, webpage designing, presentations, and workshops; so we actually need all sorts of people to make up a successful team. Everyone participates and contributes to this team in their own ways. I am pretty good at conducting experiments and writing papers, I also helped record the theme song of our team, a rendition of Let it go from the movie Frozen. We named it Let it die and rewrote the lyrics. I had a lot of fun discussing the lyrics with our teacher, Phil, and recording it with Hsuan-Chen Lu. TIEN, HAO-CHE Speaker     As one of the presenters of the iGEM Mingdao team, I am elated to join the 2014 iGEM competition coming up this summer in Boston. This is a memorable event in which I have the opportunity to truly understand the amazing realm of molecular biology. I’m confident that by attending this world affair, I will be able to exchange various ideas with students and teachers from all across the globe. It is my honor and pleasure to participate, and I believe we will all enjoy a great time. HUANG, YU-HAN     I love to go to the movies, play piano, and read sci-fi. I am a very outgoing person, so I love to meet new people. I’ve been to a lot of countries, but I still love France, which I haven’t been to yet. Yang, Chieh-Chi Head Animation Coordinator     I have always considered myself as a positive person, so I could withstand the pressure of doing experiments. I have always loved science related subjects, and I really wanted to improve my English skills. CHENG, SHIU-KAI Chess Master     Chess and biology enthusiast. Although I don’t have a good score, I still wanted to know more about biology. CHENG, CHIA-HSIN Princess     We would never be one of the Royal Family, but I am a princess temporally in our short film. LI, YU-YU Tsukkomi Role     I give weird suggestions to our teams. By the way, I also provide pizza delivery services to our team for lunch. Jenny L Wu       When I attended iGEM, it was kind of miserable. iGEM was different from the other biological events that I had attended before. It was totally out of the context of the biology. I had to study the power point when the class was over, however it was a rarely opportunity for me to attach to the functioning of the real science- Using the professional equipment, doing the experiments and writing it into the report and lecture. Now I am a 11 grade, almost 12 grade, yet I had learnt what is proper attitude for science researcher in iGEM. iGEM gave us a chance to get a glimpse at the academic professionalism and dedication.   Random Fact: Now find I have gift of playing Chinese yoyo WANG, CHANG-CHIN       Since I first learned the section about biotechnology, I am very interested in it. It seems to me that biotechnology can solve almost every problem in our life, from food problem to eternal life. I was interested in biotechnology, but I never thought of really doing it in high school until I joined the iGEM competition. I think iGEM is a great chance for high school students like me to learn and use biotechnology. In our team, I managed the poster design, did experiments, and participated in the production of other multimedia. I will also be a speaker at the Jamboree. LIAO, ZIH-SHENG Odor Test Coordinator     I once tried to use my index finger to operate a pipette. (Not suggested by lab safety protocols) CHEN, YI-JING Food Waste Coordinator     I hate photo taking, so here’s my pet. CHENG, RU-FANG     I have a wide range of emotions; I would be very funny sometimes, but I could also be a serious person. I like to talk to different kinds of people. I love to do experiments (Who doesn’t? ). HUANG, KAI-FENG Speaker     I have a great enthusiasm on biology. It is a great opportunity for me to participate in the successful team. This team is not only for people who understand biology very well, but it also allowed anybody to show his or her talent. I am good at presenting so I am one of the speakers in our team. Speakers are important because we have to express the whole idea of our iGEM project to the judges, so it is my honor to be one. Lastly, I want to overcome this challenge and become more competitive. LAN, HAO-WEI Wiki Designer     I have always loved science, and although by strength is at computer science, I also enjoy doing biochemistry. It’s tedious to design the web without a big enough supporting team, but I feel very satisfied. At this point, we still have no idea if we can finish our wiki in time, but I’m working hard to get it up and working. We had so much great ideas but we don’t have the time to implement it this year. By the way, MIT is my dream school, so I’m happy already, being that I could visit my dream school. KO, CHI-PANG Food Waste Preparer     I love doing fun and fresh things, and that’s why I’ve volunteered to prepare food waste samples, and I regretted doing so. (It smells) CHIU, WAN-CHIEN Graphics Designer     I love drawing robots and cute bacteria. It’s so much fun using the drawing board. I am also the assistant of the princess in our video, being that I carry her bags. Angele L Wu       For a person who aimed to become a doctor or biologist, it is a very precious opportunity to attend IGEM program. I learned more than I expected in this program, and I also realize that biologist's job is more than studying microorganism. It is biologist's job to utilize what they knew to improve the environment. Therefore, I feel that the goal that I aimed to reach becomes more meaningful and this inspires me to work much more harder to know more about the mysterious part of biology. I am Angele L Wu and I am very thankful to everyone in the IGEM team that has put all his or her effort to help. Team Attributions Food Waste Sniff Test Team Conducted the test of odor level YANG, CHIEH-CHI WANG,YU-JHEN HUANG,YU-HAN KO,CHI-PANG LI,YU-YU LIAO,ZIH-SHENG CHEN, YI-JING Students from Grade 11 Class 12 that volunteered as test subjects Film Production Team Involved in the process of the making of our iGEM short film Director: LAN, HAO-WEI Main actress: CHENG,CHIA-HSIN Supporting actors: WANG, WEI-JUN LIAO,ZIH-SHENG KO,CHI-PANG CHIU, WAN-CHIEN CHAN,YEN-KUANG iGEM Outreach Team In charge of introducing iGEM to Mingdao Junior Classes WANG, WEI-JUN KO,CHI-PANG CHENG,CHIA-HSIN CHENG,RU-FANG YANG, CHIEH-CHI Animation Team Involved in the process of the making of iGEM animations CHIU, WAN-CHIEN YANG, CHIEH-CHI Music Production Team Composed lyrics and recorded "Let it Die" TAN,CHIH-KANG LU, HSUAN-CHEN Powerpoint Team In charge of the making of our powerpoint and presentation TIEN,HAO-CHE CHAN,YEN-KUANG WANG, WEI-JUN HUANG,KAI-FENG WANG,CHANG-CHIN LAN, HAO-WEI Photo Slideshow Made our photo slideshow in our team CHIU, WAN-CHIEN CHENG, CHIA-HSIN Wiki Team Made this beautiful Wiki LAN, HAO-WEI Poster Team Made our beautiful poster Jenny L Wu Angele L Wu WANG,CHANG-CHIN Experiments Did PCRs, synthesized DNAs EVERYONE Sponsors BERTEC Enterprice Co LTD (伯昂興業有限公司):  http://www.bertec.com.tw/   Geneaid Biotech Ltd. (旭基科技股份有限公司): http://www.geneaid.com/   Genomics BioSci & Tech (基龍米克斯生物科技): http://www.genedragon.com.tw/index.php   Biofuture Biotech (尉徠生物科技有限公司) Top   Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/teaindex.html From 2014hs.igem.org <!DOCTYPE html> google-site-verification: google5ae61348d13c6d42.html   Home Team Project BioBrick Modeling Safety Human Practices Media   Eliminate noisome odor when decomposing food " frameborder="0" allowfullscreen> Site Map: Navigate by using this Team Project Biobrick Modeling Safety Human Practices Media Our team consists of 22 members and 2 advisorsAll of us are binded together by the passion to synthetic biology. click to learn more A odorless decomposing processHow pleasant that is. click to learn more The building blocks of biologyOur team was very interested in this part, so we designed 14 of those. click to learn more Remember that all models are wrong; the practical question is how wrong do they have to be to not to be useful.— George E.P. Box click to learn more We must respect the past, and mistrust the present, if we wish to provide for the safety of the future.— Joseph Joubert click to learn more Implement dream to realityWe have held conferences within our school to introduce other students to iGEM, and traveled to Taipei to visit schools. click to learn more Videos and photosThe best way to take in information is by looking at it. click to learn more Social Media: Say hi to us on Google+ &  YouTube iGEM Mingdao YouTube Channel iGEM Mingdao Google+ Page Also, please help the world know about us by sharing this website if you liked it Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdao/indeFrom 2014hs.igem.org <!DOCTYPE html> google-site-verification: google5ae61348d13c6d42.html   Home Team Project BioBrick Modeling Safety Human Practices Media   Eliminate noisome odor when decomposing food " frameborder="0" allowfullscreen> Site Map: Navigate by using this Team Project Biobrick Modeling Safety Human Practices Media Our team consists of 22 members and 2 advisorsAll of us are binded together by the passion to synthetic biology. click to learn more A odorless decomposing processHow pleasant that is. click to learn more The building blocks of biologyOur team was very interested in this part, so we designed 14 of those. click to learn more Remember that all models are wrong; the practical question is how wrong do they have to be to not to be useful.— George E.P. Box click to learn more We must respect the past, and mistrust the present, if we wish to provide for the safety of the future.— Joseph Joubert click to learn more Implement dream to realityWe have held conferences within our school to introduce other students to iGEM, and traveled to Taipei to visit schools. click to learn more Videos and photosThe best way to take in information is by looking at it. click to learn more Social Media: Say hi to us on Google+ &  YouTube iGEM Mingdao YouTube Channel iGEM Mingdao Google+ Page Also, please help the world know about us by sharing this website if you liked it Top   Sponsored by: and more Website best viewed with a 1920px wide screen with chrome Retrieved from "http://2014hs.igem.org/Team:Mingdahttp://2014hs.igem.org/wiki/images/2/2b/Mingdaologo.png1296The Agency EscondidoCAClassical Academy High School Escondido, CA, USA http://classicalacademy.com/cahs/ Classical Academy Online Escondido, CA, USA http://tcaonline.tcawiki.com/High School411BBa_K1296999http://2014HS.igem.org/Team:The_Agency_EscondidoCABBa_K1296000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165920140High SchoolLog in   Team:The Agency EscondidoCA/Team From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:The_Agency_EscondidoCA/TeamProject From 2014hs.igem.org Quantification Plasmid Quantification aspect of project description will go herePlasmid aspect of project description will go hereRetrieved from "http://2014hs.igem.org/Team:The_Agency_EscondidoCA/ProjectSafety From 2014hs.igem.org Safety Overview Would any of your project ideas raise safety issues in terms of: researcher safety public safety environmental safety Only team members and mentors are allowed in the laboratory to ensure public safety. All transgenic organisms are bleached before disposal in a bleach bucket. This keeps the amount percentage of transgenic organisms vacating the laboratory into the surrounding environment at a minimum. All who enter the laboratory have been trained to safely follow proper protocols. Our safety officer and his deputies develop MSDSs(Material Safety Data Sheets) documents in our independently developed services platform for our team members in order to aware the team of the dangers of reagents, chemicals, etc. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? did you document these issues in the Registry?how did you manage to handle the safety issue?How could other teams learn from your experience?Our newly developed BioBrick parts have absolutely no safety issues. Our parts are only comprised of flourescent proteins, promoters, RBSs, and inducer-coding reporter genes on top of antibiotic resistant plasmid backbones (psB1A3, pSB1K3, and pSB1C3). Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country? Our high school's administrative faculty reviewed the project proposal and determined that it was safe.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?A year before we started a synthetic biology team, we anticipated the need to develop and store important safety documents and laboratory protocols. To address the issue of safety, we successfully engineered a central repository for all MSDSs and laboratory protocols in a system known as CASP (Central Agency Services Platform). We are proud to say that have always presented our MSDSs on chemicals and reagents to the entire team before they were used in the project. At times when we bought chemicals in hurry, we had to use foreign MSDSs on those chemicals because we did not have any, but then we developed our own MSDSs of those chemicals on MSDSM(Material Safety Data Sheet Manager). Future iGEM competitions should begin to adopt our safetfy practices and develop a central iGEM repository of MSDSs of commonly used reagents. In the meantime, The Agency will gladly share our protocols and MSDSs, and aid to other iGEM teams wishing to establish and employing a safety system similar to MSDSM in CASP.Retrieved from "http://2014hs.igem.org/Team:The_Agency_EscondidoCA/SafetFrom 2014hs.igem.org The Agency Escondido Our TeamThe personal, both students and mentors, that helped our team this year.Our ProjectOur teams project is to design and synthesize a Biobrick that codes for the production of the enzyme Polyphenol Oxidase. We will verify its expression by contrasting its rate of catalysis with the enzyme in the original plant. SafetyOur teams answers to the 4 safety questions.Retrieved from "http://2014hs.igem.org/Team:The_Agency_EscondidoChttp://2011.igem.org/wiki/images/9/9f/IGEM_basic_Logo_65px.png1262USP-OWLS-PAPA Leadership Charter School University Scholars Program West Chester, PA www.palcs.orgHigh School1001BBa_K1262999http://2014HS.igem.org/Team:USP-OWLS-PABBa_K1262000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162220140High SchoolLog in   Team:USP-OWLS-PA From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:USP-OWLS-PA/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:USP-OWLS-PA logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:USP-OWLS-PA team.png Your team picture Team USP-OWLS-PA Official Team Profile Home Home Team Team Project Project Notebook Notebook Results/Conclusions Results/Conclusions Safety Safety Attributions Attributions Human Practices Human Practices Human Practices Human Practices Contents 1 Project2 Notebook3 Results/Conclusions4 Safety5 Attributions6 Human Practices7 Fun! Project Agricultural runoff from farms find its way into streams and lakes and contains high amounts of nutrients such as phosphates and nitrates. These nutrients are the leading cause of a process known as eutrophication that reduces biodiversity by filling up lakes over time. The extra nutrients promotes rapid growth of algae. Over time, the algae covers the surface of the water, blocking sunlight from getting to the bottom of the lake. This kills many of the plants and animals at the bottom of the lake, causing the lake to fill up with the dead organisms. In the short term, this limits the health and biodiversity of the body of water, and in the long run, a body of water undergoing eutrophication turns into a bog. This problem is especially relevant in Pennsylvania which has 500,000 acres dedicated to agriculture and is bordered by Lake Erie in the Northwest. In order to detect hotspots for large amounts of nutrients, we plan on developing a bioluminescent bacteria that is able to detect the levels of phosphorous in any given water sample. Specifically, orthophosphates will be detected because they are the most common form of phosphorous found in water. The sample would first be heated to convert all polyphosphates into orthophosphates for accurate testing. Once orthophosphates are detected by the bacteria, the bacteria would fluoresce in a certain light spectrum. The luminosity of the bacteria would differ based on the amount of bacteria lighting up, which would be dependent on the concentrations of phosphorous. Control tests will be done on samples with known concentrations of phosphorous in order to find the exact relationship between concentration of phosphorous and brightness of the bacteria. Current methods rely on complex chemical processes, and most testing kits cost hundreds of dollars to purchase and use. Only small amounts of bacteria would be required for the testing, lowering the cost needed for accurate readings. The bacteria could be used as a screening method to acquire data quickly and accurately in order to identify lakes beginning the early stages of eutrophication so that further preventative actions can be taken. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:USP-OWLS-PA1266Lambert GALambert High SchoolHigh SchoolChitinite: Defending Fruit One Gene at a TimeChitin, a polysaccharide derived from glucose, is commonly found in the cell walls of fungi and the exoskeletons of organisms. Chitosan is derived from chitin through the N-deacetylation process. Uses include agriculture, medicine, and industry. Research in our local community led us to research applying chitosan in an agriculture setting by preserving post harvest fruit from fungal infections. Current technology to produce chitosan involves a caustic chemical process that is detrimental to the environment. An alkali sodium hydroxide removes the acetyl group from chitin to produce chitosan. Fortunately, this conversion process also takes place in the natural environment in a multitude of organisms through the aid of an enzyme called chitin deacetylase, or CDA. The main focus of our project was to obtain a CDA gene from a eukaryotic source, engineer a suitable vector with promoter, and transform into a bacterial host using the standard Biobrick assembly.2302BBa_K1266999http://2014HS.igem.org/Team:Lambert_GABBa_K1266000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=162720140http://2014hs.igem.org/files/presentation/Lambert_GA.pdfhttp://2014hs.igem.org/files/poster/Lambert_GA.pdfHigh SchoolLog in   Team:Lambert GA/Sponsors From 2014hs.igem.org Our Sponsors The Styczynski Lab Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/SponsorsTeam From 2014hs.igem.org Lambert iGEM TeamThe Lambert iGEM Team is the first High School iGEM team in the state of Georgia. This was possible thanks to our principal, Dr. Gary Davison, Dr. Mark Styczynski of Georgia Institute of Technology and the National Science Foundation Grant #1254382.Members Nolan Ayers, Christina Bae, Tina Baek, Neha Balachandran, Elynna Chang, Hetty Chung, Jared Cook, Lily Ge, Jackson Harris, Janak Joshi, Giwoo Kim, Sue Kim, Kendall Lundberg, Kirsten Martin, Kristen Meers, Alex Moldenhawer, John Nesbit, Kahan Parekh, Preeya Parmar, Shahil Patel, Jas Pyneni, Sarah Rupert, Noor Sohal, Charita Veerapaneni, Chris Waites, Dugan Walker, Megan Walz, Megan Whitlach, Nick Whitlock, Rebecca Yan Advisors Our team is sponsored by two wonderful teachers from the Lambert High School science department, Mrs. Janet Standeven and Mrs. Shelby Cochran. Mrs. Standeven teaches Environmental Science and Biotechnology, and Mrs. Cochran teaches Advanced Placement Biology. Attributions Our team members brought many skills to the iGEM experience. We divided into committees to accomplish different tasks towards the project. Each committee was responsible for their area of expertise as well as contributing to other areas of work as needed. All team members were required to fulfill service hours during our Saturday tutoring with Next Generation Focus. We also elected officers to serve as positions of leadership.President: Jared CookVice presidents: Megan Whitlach and Kirsten MartinSecretary: Noor Sohal and Charita VeerapaneniTreasurer: Rebecca YanLab Maintenance: Lily GeSafety Officer: Dugan WalkerIntern at Georgia Tech 2014: Christina BaeCommittees:Research: Jared, Christina, Nolan, Jackson and ChrisWiki: Alex, Kahan, Jas, Neha, Christina, Elynna, RebeccaFundraising: Kendall, Jas, Shahil, KristenOutreach: Noor, Rebecca, Neha, JacksonLabwork: Christina, Jared, Nick, Chris, Sarah, LilyVideo: Christina, Elynna, Noor, RebeccaSpecial thanks: Sean Tackett for editing our music video and Noël Isaacson for singing in our music video. We are especially grateful to Dr. Brian Hammer, Dr. Mark Styczynski, The Styczynski research group: Dan, Mackenzie, Katie, Amy, Robb and Sugantha, and the GA Tech 2014 iGEM team for their advice and thoughtful questions for our practice presentations. All wet lab experiments were completed by Christina, Jared, Lily, Nick, Chris, Jackson and Sarah. The project was completed by the high school students with guidance from the advisors and the Styczynski Lab. Wiki help was provided by Troy Von Beck of the GATech 2014 iGEM team. Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/Teaafety From 2014hs.igem.org Lab Safety How We Ensured Lab Safety Team members were trained in appropriate techniques for dealing with cell culture, recombinant DNA, and general lab safety. Lab procedures were carried out under the supervision of the instructors. Experiments with recombinant DNA were completed under the advisement of Dr. Mark Styczynski from the Georgia Institute of Technology. Chemical wastes were treated and disposed of in accordance with their respective MSDS. All living cultures and biohazardous wastes were appropriately sterilized before disposal. Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/SafetHumanPractices From 2014hs.igem.org Human Practices/Outreach As a grassroots organization in the state of Georgia for high school students, our iGEM Team find it pertinent that we nurture a new generation of students who are interested in science and help them keep pace with developing technologies occurring in the synthetic biology field today. See us in action! Next Generation Focus Saturday Tutoring There are many students in our local area who are unable to receive financial and familial support for their education. We have partnered with a local organization, Next Generation Focus (NGF), to reach out to children in the area and introduce them to biology and biotechnology. Next Generation Focus provides Saturday tutoring services for economically disadvantaged students in Forsyth County and recently began a STEM program in which the second Saturday of every month is dedicated to science and technology. This year Lambert iGEM adopted two Saturdays, and members prepared short lesson plans for elementary school students to help expose them to a field they have never known through crafts, games, and team activities. February 2014: The Lambert iGEM Team took over a STEM Saturday and had the kids use small micro viewers to look at magnified plastic microslides of bacteria, viruses, fungi, plant and animal cells. We taught them about microbes, pathogens, and most importantly, deoxyribonucleic acid. The highlight was having each child pair with an older iGEM buddy and assigning them to a syllable in “deoxyribonucleic acid”. We had them say their designated syllable down the line with amusing results. The students were engaged and interested, and we were happy to teach them about a world invisible to the naked eye. April 2014: We returned to build on our previous lesson by teaching them parts of the cell. Though we did not expect them to remember the cell organelles and their functions, the theme of the lesson was that cells have smaller parts that keep them functioning. Rather than “pin the tail on the donkey”, we had “pin the organelle onto the cell”. The students also used Twizzlers and colored marshmallows to construct mini edible DNA models that taught them the fundamentals of base pairing. We also filmed our “Let It Grow” music video, which the kids and the students enjoyed wholeheartedly. Whitlow Elementary Science Night As part of iGEM’s goal to expand the ideas of biotechnology in the community, a group of iGEM students participated at the annual STEM Night at Whitlow Elementary. Here a diverse collection of subjects were discussed and iGEM was able to demonstrate common biotechnology applications to young children. The children were amazed and demonstrated curiosity in the synthetic biotechnology field. We demonstrated how a micropipette is used and the miniscule volume that is measured by them. Furthermore, a gel electrophoresis was set up to show some of the unique apparatus we work with. Parents attending were able to gain an understanding of the goals our team had set as well as the objectives of iGEM as a whole, and as a result Lambert iGEM has garnered recognition in the community as a well-developed club that has been able to accomplish admirable feats just a year after its inauguration. Future Next year we will continue to partner with NGF and expand our program. Half of the proceeds of our main fundraiser will be donated toward charity, and we hope to begin a summer biotechnology summer camp for middle-school students in the coming years. Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/HumanPracticNoteBook From 2014hs.igem.org Lambert iGEM NoteBook Monthly tasks Fall 2013: Our team visited a local farm to determine needs in our community. From the visit, we began formulating the idea of a fruit preservative. November and December 2013: We began to work on sterile techniques and safety training. Also began research into Chitin to Chitosan Pathway as well as applications of chitosan. January 2014: Began research into source organisms for the Chitin Deacytelase gene. February 2014: iGEM kit arrived. March 2014: Began extracting genomic yeast DNA. Used a protocol from Looke Article, Biotechnology Volume 50. using lithium acetate etc. Results were inconsistent and yields were low. April 2014: Used Pierce Thermoscientific Yeast DNA Extraction Kit to extract genomic DNA from S.cerevisiae. PCR using pMAL primers. Began Ligations. May 2014: Continued ligations using Quick Ligation, Overnight ligation and T4ligase. Further research determined low efficiency of blunt ended ligations. June 2014: Began Biobricking CDA gene. Designed Biobrick primers for CDA, PCR, digested, ligated with PSB1C3 and transformations with DH10beta competent cells. Lab Notebook/Protocols Visit our laboratory notebook! Our labwork committee worked at the Lambert High School lab and also at the Styczynski lab at the Georgia Institute of Technology. The notes and protocols are all located in the link! Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/NoteBooFrom 2014hs.igem.org Welcome to Lambert iGEM Team's Wiki Page! Retrieved from "http://2014hs.igem.org/Team:Lambert_Ghttp://2014hs.igem.org/wiki/images/5/57/Lambertlogo.pngProject From 2014hs.igem.org Our Project Project Calendar: How we got our idea... What we did in the lab... CDA Methodology and Results pMAL Protein Expression Kit Biobrick Assay Methodology and Results References Anitha, A., Sowmya, S., Kumar, P. T. S., Deepthi, S., Chennazhi, K. P., Ehrlich, H., . . . Jayakumar, R. Chitin and chitosan in selected biomedical applications. Progress in Polymer Science(0). doi: http://dx.doi.org/10.1016/j.progpolymsci.2014.02.008 Christodoulidou, A., Bouriotis, V., & Thireos, G. (1996). Two sporulation-specific chitin deacetylase-encoding genes are required for the ascospore wall rigidity of Saccharomyces cerevisiae. J Biol Chem, 271(49), 31420-31425. Christodoulidou, A., Briza, P., Ellinger, A., & Bouriotis, V. (1999). Yeast ascospore wall assembly requires two chitin deacetylase isozymes. FEBS Lett, 460(2), 275-279. Christodoulidou, A., Briza, P., Ellinger, A., & Bouriotis, V. (1999). Yeast ascospore wall assembly requires two chitin deacetylase isozymes. FEBS Lett, 460(2), 275-279. doi: http://dx.doi.org/10.1016/S0014-5793(99)01334-4 Jarmila, V., & Vavrikova, E. (2011). Chitosan derivatives with antimicrobial, antitumour and antioxidant activities--a review. Curr Pharm Des, 17(32), 3596-3607. Liu, N., Chen, X.-G., Park, H.-J., Liu, C.-G., Liu, C.-S., Meng, X.-H., & Yu, L.-J. (2006). Effect of MW and concentration of chitosan on antibacterial activity of Escherichia coli. Carbohydrate Polymers, 64(1), 60-65. doi: http://dx.doi.org/10.1016/j.carbpol.2005.10.028 Martínez, J. P., Falomir, M. P., & Gozalbo, D. (2001). Chitin: A Structural Biopolysaccharide eLS: John Wiley & Sons, Ltd. Martinou, A., Koutsioulis, D., & Bouriotis, V. (2003). Cloning and expression of a chitin deacetylase gene (CDA2) from Saccharomyces cerevisiae in Escherichia coli: Purification and characterization of the cobalt-dependent recombinant enzyme. Enzyme and Microbial Technology, 32(6), 757-763. doi: http://dx.doi.org/10.1016/S0141-0229(03)00048-6 Moussa, S. H., Tayel, A. A., & Al-Turki, A. I. (2013). Evaluation of fungal chitosan as a biocontrol and antibacterial agent using fluorescence-labeling. International Journal of Biological Macromolecules, 54(0), 204-208. doi:http://dx.doi.org/10.1016/j.ijbiomac.2012.12.029 Tsigos, I., Martinou, A., Kafetzopoulos, D., & Bouriotis, V. (2000). Chitin deacetylases: new, versatile tools in biotechnology. Trends in Biotechnology, 18(7), 305-312. doi: http://dx.doi.org/10.1016/S0167-7799(00)01462-1 Zakrzewska, A., Boorsma, A., Brul, S., Hellingwerf, K. J., & Klis, F. M. (2005). Transcriptional response of Saccharomyces cerevisiae to the plasma membrane-perturbing compound chitosan. Eukaryot Cell, 4(4), 703-715. doi: 10.1128/ec.4.4.703-715.2005 Zhang, H., Li, R., & Liu, W. (2011). Effects of Chitin and Its Derivative Chitosan on Postharvest Decay of Fruits: A Review. International Journal of Molecular Sciences, 12(2), 917-934. (Anitha et al.; Christodoulidou, Bouriotis, & Thireos, 1996; A. Christodoulidou, P. Briza, A. Ellinger, & V. Bouriotis, 1999; Anna Christodoulidou, Peter Briza, Adi Ellinger, & Vassilis Bouriotis, 1999; Jarmila & Vavrikova, 2011; Liu et al., 2006; Martínez, Falomir, & Gozalbo, 2001; Martinou, Koutsioulis, & Bouriotis, 2003; Moussa, Tayel, & Al-Turki, 2013; Tsigos, Martinou, Kafetzopoulos, & Bouriotis, 2000; Zakrzewska, Boorsma, Brul, Hellingwerf, & Klis, 2005; Zhang, Li, & Liu, 2011) Lõoke M, Kristjuhan K, Kristjuhan A. Extraction of genomic DNA from yeasts for PCR-based applications.. doi:10.2144/000113672. PubMed PMID: 21548894. El Hadrami, A.; Adam, L.R.; El Hadrami, I.; Daayf, F. Chitosan in Plant Protection. Mar. Drugs 2010, 8, 968-987. Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/ProjecFun From 2014hs.igem.org Photo Gallery Retrieved from "http://2014hs.igem.org/Team:Lambert_GA/Fu1297CSIA-SouthKoreaCheongshim International Academy Gapyoung-gun, Kyunggi-do, South Korea http://www.csia.hs.krHigh SchoolStop Desertification With Urease from Klebsiella oxytocaOur project presents a synthetic biology method for preventing this acceleration of desertification, trying do it based on the urease reaction, to change the dry soil to the CaCo3, which can ultimately concise the dry lands and also make a capsule to contain water. Using urease test with the strain Klebsiella oxytoca Kctc 1686, we checked out the different results between urease-contained strains and non-urease strains. The work presented here has profound implications for future studies of synthetic biology and might help to solve the problems of desertification. We are planning to use Klebsiella oxytoca and type of free nitrogen bacteria to settle the environment that plants could live. First, Klebsiella oxytoca is a bacterium with the ability to precipitate calcite and solidify sand given a calcium source and urea, through the process of microbiologically induced calcite precipitation or biological cementation.611BBa_K1297999http://2014HS.igem.org/Team:CSIA-SouthKoreaBBa_K1297000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166020140http://2014hs.igem.org/files/poster/CSIA-SouthKorea.pdfHigh SchoolLog in   Team:CSIA-SouthKorea From 2014hs.igem.org Retrieved from "http://2014hs.igem.org/Team:CSIA-SouthKorea1300StuyGem NYCStuyvesant High School New York, NY, USA www.stuy.eduHigh SchoolWanted: Bacteria. Dead or DeadThere is a public fear of genetically modified organisms (GMOs) and their escape from the laboratory environment into the outside environment. Our project will quell the fears of the general public and increase support for synthetic biology by introducing a safety mechanism ? a kill switch. The team will build this kill switch with constructs consisting of a ?kill gene? - a naturally occurring toxin: ccdB, a riboregulatory system, and a UV light inducible promoter. We plan on testing the riboregulatory system for efficiency of cell death. The success of inducible cell death would revolutionize the control over the unintended dispersion of GMOs.1411BBa_K1300999http://2014HS.igem.org/Team:StuyGem_NYCBBa_K1300000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166320140http://2014hs.igem.org/files/presentation/StuyGem_NYC.pdfhttp://2014hs.igem.org/files/poster/StuyGem_NYC.pdfHigh SchoolLog in   Team:StuyGem NYC From 2014hs.igem.org Photo Gallery Get in touch! Visit Our Twitter Page! Tweets! © Copyright StuyGem, Foundation Stuyvesant WebsiteiGem Website Retrieved from "http://2014hs.igem.org/Team:StuyGem_NYC1294Enloe RaleighEnloe Magnet High School Raleigh, North Carolina, USA http://enloehs.wcpss.net/High School001BBa_K1294999http://2014HS.igem.org/Team:Enloe_RaleighBBa_K1294000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165720140High SchoolLog in   Team:Enloe Raleigh From 2014hs.igem.org This is a template page. READ THESE INSTRUCTIONS. You are provided with this team page template with which to start the iGEM season. You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki. You can find some examples HERE. You MUST have the following information on your wiki: a team descriptionproject descriptionsafety information (did your team take a safety training course? were you supervised in the lab?)team attribution (who did what part of your project?) You may also wish to add other page such as: lab notebooksponsor informationother information REMEMBER, keep all of your pages within your teams namespace. Example: 2013hs.igem.org/Team:Enloe_Raleigh/Our_Pets You can write a background of your team here. Give us a background of your team, the members, etc. Or tell us more about something of your choosing. File:Enloe Raleigh logo.png 200px Tell us more about your project. Give us background. Use this as the abstract of your project. Be descriptive but concise (1-2 paragraphs) File:Enloe Raleigh team.png Your team picture Team Enloe_Raleigh Official Team Profile Contents 1 Team2 Notebook3 Results/Conclusions4 Safety5 Attributions6 Human Practices7 Fun! Team Tell us about your team, your school! Project As technology progresses, humans have begun to explore new methods of pollution reduction. Coal production is the single largest power source in the US and contributes heavily to industrial pollution. Although coal will eventually be replaced with more efficient and less polluting energy resources, at this point in time, there exists no combination of renewable energy sources that could replace the global dependency on fossil fuels, particularly coal. Here arises a need to control pollution caused from burning these fossil fuels. This includes fixing notorious emissions of sulfur dioxide, nitrous oxides, and carbon dioxide when solid coal or gaseous coal is burned and the semi-solid wastes composed of toxic metals such as lead and mercury. The most worrisome of these are carbon dioxide, nitrous oxides, and sulfur oxides, which are widely acknowledged to be the primary cause of global warming, and photochemical smog with approximately 15 tons added to the atmosphere annually. Although there are synthetic ways to reduce the detrimental effects of burning coal, such as coal gasification, chemical looping, and the use of an SNOX reduction system, such methods either require high maintenance, or are not cost-benefit effective. To overcomes these problems, we have devised a natural, biochemical system using versatile microbes to reduce sulfur oxide emissions through the Sulfate Reducing Bacteria (SRB), Desulfovibrio orientis. Furthermore, ways to test for air quality levels are not dynamic (in real-time) and do not do so in a cost effective method. As a result, we plan to add fluorescent capabilities to our bacteria that vary in intensity corresponding to different gradations (in ppm) of specific pollutant, in this case the sulfur oxides. As a result, we provide a new, synthetic biological alternative to measure air quality whilst reducing pollution at the same time. Unlike other species of the Desulfovibrio genus, D. orientis is one of only a handful of species that has been shown to reduce sulfur dioxide, not sulfate, into hydrogen sulfide. In this reaction, SO2 acts as an electron acceptor and is reduced to H2S during cellular respiration. In addition, given D. orientis’ similar metabolism and anaerobic behavior as many hyperthermophiles of the domain Archaea, the species is more inclined to be thermophilic (or favorably hyperthermophilic - having the ability to thrive in extremely high temperatures). Furthermore, this species has been observed to live off minimal mineral nutrients, heat- and alkali-pretreated municipal sludge, CO2, SO2, and H2, and thus requires little nutrients to survive above a critical threshold. Since the species already contains the catalytic gene sequence to carry out the aforementioned favorable sulfate reducing metabolic activities, we plan to genetically spice protein coding sequences for fluorescent proteins, such as that from the jelly fish, Aequorea victoria. This gene would be linked to the genes associated with the sulfate reducing catalytic activity of D. orientis so that the bacterial species would only glow when the metabolic processes to reduce sulfur oxide derivatives are active. An extension, would be to express the fluorescent protein genes in a fashion that correspond to the different levels of sulfur dioxide (in ppm) present in the environment by either varying color or intensity. A possible method is the inclusion of a highly sensitive modified RNA thermometer (several base units upstream from the fluorescent protein gene) that regulates the activation of the genes that codes for the production of the desired fluorescent proteins based on the free energy (delta G) of the system. The previously mentioned reaction of D. orientis tends to add to the free energy of the system, and thus its metabolic activity can be measured by the differential change of free energy in the system. The RNA thermometer would then increase translation of the fluorescent proteins as the free energy increases. Notebook Show us how you spent your days. Results/Conclusions What did you achieve over the course of your semester? Safety What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab? Attributions Who worked on what? Human Practices What impact does/will your project have on the public? Fun! What was your favorite team snack?? Have a picture of your team mascot? Retrieved from "http://2014hs.igem.org/Team:Enloe_Raleigh1290NGSS TRAtlantik Nevin Gokcek Science High School Ankara, TurkeyHigh SchoolS. DiagenesStreptocccus Pyogenes is a severe bacteria which can result in some ilnesses such as pharyngitis. Diagnosis of S. Pyogenes can be done in some ways. However some ways are fairly expensive or takes more than a day to detect. We designed our system to solve this problem by using synbio. We simply found the sequence which can be cleavaged by the protease, SpeB, secreted by S. Pyogenes. In our system we used a wall protein which is linked to cleavage site by a sequence of linker. On the other side of the cleavage site, we located Catechol 2,3-dioxygenase(C2,3O) monomer. When cleavage site is eradicated by SpeB, free C2,3Os form tetramers. Catechol is a substance that reacts with Oxygene. As a result of this reaction yellow colour is produced. This system is fairly fast in comparison to blood agar method. The system can also be modified to detect other demerit bacteria.1111BBa_K1290999http://2014HS.igem.org/Team:NGSS_TRBBa_K1290000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=165320140High SchoolLog in   Team:NGSS TR/project.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS   PROJECT 1.BACKGROUND ANG GOALS Our project is to construct a biological device that can be used to detect the existence of Streptococcus Pyogenes (S. Pyogenes) in a cheaper and faster way in comparison to currently used techniques. The construct can be used as a detecting device for bacteria or virus secreting protease by modifying the cleavage sequence in the array (for more information see parts). The name of the project comes from ‘ "Diagnosing S. Pyogenes". Streptococcus Pyogenes, also known as group A streptococci (GAS), is a Gram-positive pathogen responsible for a wider variety of human disease than any other bacterial species including pharyngitis (streptococcal sore throat), scarlet fever, impetigo, erysipelas, cellulitis, septicemia, toxic shock syndrome, necrotizing fasciitis (flesh-eating disease) and the sequelae, rheumatic fever and acute glomerulonephritis.(1)  The complications of current GAS infections are severe; bacteremia associated with aggressive soft tissue infection, shock, adult respiratory distress syndrome and renal failure are common; 30% to 70% of patients die in spite of aggressive modern treatments.(2) Patients with symptomatic pharyngitis rarely develop streptococcal toxic shock syndrome, though such cases have been reported, especially in the last year. Numerous cases have developed within 24 to 72 hours of minor nonpenetrating trauma, resulting in hematoma, deep bruise to the calf, or even muscle strain.(3) There are currently several test methods to detect the existence of S. Pyogenes. TheStrep A Rapid Test Device (SARTD) is considered to be fastest one with detecting antigen in 5 minutes with the accuracy of 72%.(4) However SARTD is an expensive device that most health institutions have difficulty affording. Therefore blood agar plate culture is prepared which requires a long time interval (one to two days) to show the results.(4) Testing on the same day is important to reduce unnecessary antibiotic use and to prevent possible complications caused by S. Pyogenes. In our project we aimed to shorten the amount of time needed to detect S. Pyogenes while making the higher speed test more affordable. 3D computer-generated image of a Streptococcus Pyogenes. Content Provider(s): Center for Disease Control and Prevention/ Melissa Brower   2.PARTS -OmpA-SpeB_Cleavage_Site     We accomplish the task of detecting the existence of S. Pyogenes with the base part OmpA-SpeB_Cleavage_Site.Research was conducted to discover the proteins secreted by Streptococcus Pyogenes; results showed that SpeB is one of the main virulence factor of S. Pyogenes, which is a cysteine proteinase functioning protein secreted by the bacteria.(5) After further investigation on the mechanism of SpeB, we discovered that the amino acid sequence is cleaved by SpeB.(6) If there is S. Pyogenes in the medium, SpeBs secreted by S. Pyogenes split the amino acid sequence including SpeB cleavage site, into two. By using this for our benefit, construct consisting of a cell wall protein, linker sequence and SpeB cleavage site is designed. This is the base part designed for S. Pyogenes detecting devices. To the end of this part, the protein, which will be used to show that the part is cleaved, should be added. After the SpeB cleavage site is cleaved, the amino acid sequence placed after the cleavage site can become free and start action. (see part OmpA-SpeB_Cleavage_Site-xylE). The cell wall protein keeps the construct on the cell wall, which helps to prevent unnecessary transmission between the protein coming after the SpeB cleavage site and the protein’s reactants, if designed. The cell wall protein OmpA is preferred, due to its abundant usage.(7) The linker segregates the OmpA and SpeB cleavage site and thus creating space for the SpeB to access the cleavage site; the sequence of the linker is taken from the Imperial College 2010 project.(8) The SpeB cleavage site is broken down in the existence of the SpeB and splits the amino acids, thus unchaining the substance from the cell wall.   -OmpA-SpeB_Cleavage_Site-xyIE     This part is constructed upon the base part OmpA-SpeB_Cleavage_Site by adding Catechol 2,3-dioxygenase to the end of the sequence. xylE is the gene encoding the enzyme catechol-2,3-dioxygenase, which converts catechol, a cheap colorless substance, to the bright yellow product 2-hydroxy-cis,cis-muconic semialdehyde, if provided with oxygen. The sequence of xylE is taken from the partsregistry.(9) In the existence of S. Pyogenes, SpeB secreted by the bacteria splits the amino acid sequence from the cleavage site, monomers of catechol-2,3-dioxygenase become liberated. Free monomers come together and form the tetramer form to start activation. By using this part, detecting organisms will be a lot easier.   Image: OmpA-SpeB_Cleavage_Site-xylE   List of our parts   3.KINETIC MODELLING Cascade reactions: By using the SimBiology toolbox for MATLAB we created a diagram of the enzyme kinetic modelling(Figure 1). Figure1: Diagram of the reactions involved in converting the Cathecol monomer to tetramer and degredation of cathecol. (Yellow colour is indicator of the reaction.)This was created in the SimBiology toolbox for MATLAB.     Table1: Mathematical representation of enzyme kinetic reactions.   Table 2: Values assigned to kinetic parameters described in Table 1. All reacrtion are irreversible     We have three reactions that are linked. According to our literature researches reactant and product balance in the reaction is as shown. In practice SpeB is activated by 2-Mercaptoethanol (reducing agent), which may blur our understanding in theory. However, our results showed that the uncertainty is not exceedingly high. 4.VISUAL MODELLING In the visual modelling below 3D molecules of SpeB and tetramer of Catechol-2,3-dioxygenase are depicted. SpeB, is the protease secreted by S. Pyogenes In our design SpeB cleaves the SpeB cleavage site in between  Catechol-2,3-dioxygenase and the linker, which is tied to the cell wall protein OmpA. When Catechol-2,3-dioxygenases is liberated from the bacteria, they become together and forms the tetramer, which is the enzyme required for the reaction between catechol and oxygen. As a result of this reaction yellow color is produced. If there is S. Pyogenes in the medium, yellow color is produced. Hopefully, with our project we will be able to detect the bacteria cheaper and faster.    5.CONCLUSION AND FUTURE PLANS In the end, we hope to produce a working OmpA-SpeB_Cleavage_Site-xylE construct along with our base part OmpA-SpeB_Cleavage_Site. If we manage to show that the SpeB cleavage part is working, we can easily detect S. Pyogenes. It is our hope that our construct will be able to reduce the cost of rapid detecting systems and allow fast detections. In the future, researches can use the benefits of our system to develop more effective and faster methods for detection, helping patients to have more comfortable treatment.   As it can be seen from the results, we tested our construct with SpeB, the enzyme secreted by S. Pyogenes. However when the other proteins secreted by S. Pyogenes and other conditions are took into consideration, outcome may differ from our results. On the other hand, we are not allowed to conduct our researches with viruses due to the safety regulations of the lab that we use. Therefore in the future, we are going to test our construct with real S. Pyogenes bacteria. We are also making our plans on other detection modules instead of Catechol-2,3-dioxygenase to come up with the one, which is the most accurate. For instance one of the enzyme that we will use is Glucose oxidase. It catalyses the oxidation of glucose to hydrogen peroxide and D-glucono-δ- lactone. As a result of this reaction color change is observed. Hopefully we will be able to solve the problems of patients! 6.REFERENCES 1. Ferretti, Joseph J. "Complete Genome Sequence of an M1 Strain of Streptococcus Pyogenes." PNAS 98.8 (2001): 4658-663. Print. 2. Stevens DL, Tanner MH, Winship J, Swarts R, Reis KM, Schlievert PM, et al. Reappearance of scarlet fever toxin A among streptococci in the Rocky Mountain West: severe group A streptococcal infections associated with a toxic shock-like syndrome. N Engl J Med 1989; 321:1-7. 3. Dennis L. Stevens, Ph.D, M.D. "Streptococcal Toxic-Shock Syndrome: Spectrum of Disease, Pathogenesis, and New Concepts in Treatment." Emerging Infectious Diseases 1.3 (1995): 69-78. Print. 4. Forward, Kevin R., David Haldane, Duncan Webster, Carolyn Mills, and Diane Aylward. "A Comparison between the Strep A Rapid Test Device and Conventional Culture for the Diagnosis of Streptococcal Pharyngitis." Can J Infect Dis Med Microbiol 17.4 (2006): 221-23. Print. 5. Nelson, DC, J. Garbe, and M. Collin. "Cysteine Proteinase SpeB from Streptococcus Pyogenes - a Potent Modifier of Immunologically Important Host and Bacterial Proteins." (2011): n. pag. PubMed. Web. 12 June 2014. 6. Ender, Miriam, Federica Andreoni, Annelies Sophie Zinkernagel, and Reto Andreas Schuepbach. "Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation." (2013): n. pag. PLOS. Web. 12 June 2014. 7. "P0A910 (OMPA_ECOLI)." (2014): n. pag. UniProt. Web. 12 June 2014. 8. Imperial Collage London. "Parasight." IGEM. N.p, 2010. Web.12 June 2014.   Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/project.htmloutreach.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS  OUTREACH FURTHER ON PROJECT Our project aims to diagnose S.Pyogenes by using a relatively fast and inexpensive methode. We prepared a short documentary to show the results of our negotiations with the researchers who are the experts of this subject. In our progress to prepare this documentary we conducted interviews with otolaryngologist Assoc. Prof. Dr. Gül Müge ÖZCAN, pediatrician Assoc. Prof. Dr. Fatmanur ÇAKMAK and Assoc. Prof. Dr. Nurcan BAYKAM. Firstly, we explained our project to the doctors then talked about: - the bacteria inflamations of the body; - S. Pyogenes, SpeB enzyme; - protease functioning proteins; - statistics of the diseases caused by S. Pyogenes; - the syptoms are emerged S. Pyogenes; - the importance of early diagnosis; - benefits of our project. In addition to positive feedbacks we got, we also learned new information about the concepts related to our project. The doctors emphasized that bacteria are able to come to our body from everywhere and cause diseases easily. The focus bacteria of our project, S. Pyogenes can cause quinsy which is a common disease and early diagnosis is crucial for the treatment of the disease. They also stated that patients consult with different symptoms like headache to nausea. They answered all of our questions gently. Consequently our documentary was formed. Enjoy the documentary… Video; documentary videosu BEYOND THE LAB In addition to our project we also introduced bacteria and iGEM to the people, especially the little ones! The surveys that we did to people showed us that most of the people don’t know the benefits of bacteria. Consequently, our team decided to persuade people about bacteria’s benefits. One of the things we’ve done for make people know is to go to a primary school and talk to both teachers and students. We expressed them the importance of bacteria and the fact that world cannot be the same without bacteria. We represented  iGEM competition and encouraged them about attending iGEM  in the future. Also, we prepared a conference  includes informations about iGEM and synbio in our school. Everyone found it remarkable. Some of the students even decided to study synbio in the future. Furthermore, we represented iGEM to the primary school students who visited our school. Many of them were fascinated by our presentation. It helped the students about their high school choice. Thanks to iGEM, our school gained lots of students!    Our team did activities with little people to make them know about bacteria. We asked them if they can draw bacteria on the fabric we gave them. Their imagination was so unbelievable! One of them even drew bacteria in a magnifying glass which showed us his knowledge about bacteria’s dimension.  Moreover, the children tried to draw bacteria colonies. With the help of the activities we’ve done in their school not only we made the school-life more enjoyable for children, but also we evaluated children knowledge about bacteria. We did the same activity in our school. This time, drawings were better but can’t say the same thing about imagination! The fact is they don’t have much opinion about bacteria, despite of the biology lessons they’ve studied at school.  With that activity, we made them think about bacteria at least  for some time.  ROCK'N TWITTER Usage of social media is a great way to introduce iGem. There are already lots of iGem team on Twitter. However to keep a topic trending, these teams have to unite. We therefore used #iGem2014United tag to bring iGem teams together.  Considering the location of iGem teams on the world, we prepared a list showing in which ours we should trend a topic as a whole iGem community. We set our first trial on 31st of May. Lots of iGem teams joined us, however we were not enough to make the topic trending. Therefore we decided to keep working on the tag. In the Jamboree we are going to ask teams to tweet the tag on 31st of July. We hope we will be able to introduce iGem to more and more people. We shoot six seconds funny videos and shared them via Vine to encourage people to participate in such projects by showing the fun we had. We shared our vines via tag #iGem2014United and asked iGem teams to shoot their vines. We organized a contest which iGem teams will choose the best Vine together. Hopefully in the Jamboree event we will be able to watch them.Additional to the Twitter movement, we used Escherichia Coli @yourEcoli account to introduce iGem in a funny way. @yourEcoli is a bacterium seek to govern the SynBiology world by defeating humans (he prefers eukaryotes) with a revolution which he called as Inevitable Return. He sometimes tweets funny caps, sometimes make comments on other iGem teams tweet.  COLLABORATIONSIn the progress of our project we collaborated with several iGem teams on various issues. Collaborations help us to know each other better and share our knowledge and passion. Especially collaborations we did with university iGem teams helped us to learn more about synthetic biology and wiki editing. We did some experiments of AUC_TURKEY and they did some of our experiments. When needed we exchanged lab materials as we use the same lab. Several times we discussed on the projects to improve them. We had a beneficial conversation with iGemParisBettencourt team. They gave some tips to adjust our codes for iGem wiki and they shared their project to provide learning resources for iGem. Valencia UPV iGem, Imperial iGem, iGemSBU, OU iGem, AUC_TURKEY and iGem Bordeaux teams supported the #iGem2014United tag. We had a beneficial conversation with some of them; we discussed about finding funding for iGem project and lab chemicals, we shared our knowledge and learned about different approaches to the issue. In the future we are looking forward for collaborations that we can share with each other more.     Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/outreacresults.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS RESULTS 1.SUCCESSFUL CLONING We did the transformation of our composite part (OmpA-linker-SpeB_Cleavage_Site-XylE). We applied our regular procedure (see procedures under the lab button) to E.coli colonies that we have prepared before. Electrophoresis results showed that our gene is produced by the colonies and it is in the correct range (1500-2000 bp). After the repetitions of the same procedure, we agreed that cloning was successful. Image:Electrophoresis of OmpA-linker-SpeB_Cleavage_Site-XylE   2.SDS PAGE EXPERIMENT   In order to check whether our enzyme is working or not and also to control if the proteins are producing, we made the SDS PAGE experiment. We showed that our enzyme is working and our construct is produced. After the experiment of lysate the band that we want to see is nearly 53 kda long. 1)IgG 2)IgG with PBS+Mercaptoethanol 3)IgG with SpeB+PBS+Mercaptoethanol 4)marker 5)Compatent E. Coli 6)Bacterial lysate (2,5 ul) 7)Bacterial lysate (5 ul) 8)Bacterial lysate (10 ul) 9)Bacterial lysate (20 ul) 10)Marker Our system was working, we decided to do color change experiment.    3.COLOR CHANGE   Catechol-2,3-dioxygenase is the enzyme of reaction between catechol and oxygene. If there is not Catechol-2,3-dioxygenase in the medium, catechol reacts with oxygene and produces black color. In our construct  Catechol-2,3-dioxygenase can work if it is liberated from the linker via SpeB, virulence factor secreted by S. Pyogenes which cleaves SpeB_Cleavage_Site. SpeB enzyme works with a reducing agent in appropriate buffer. There are several buffers that SpeB can work in. We decided to use phosphate buffered saline (PBS), which is the most popular one. There are also several reducing agents that can be used with SpeB; we decided to use 2-Mercaptoethanol. As we proved with SDS experiment that our protein is produced and the SpeB enzyme is working properly, we started our experiment to detect the existence of S. Pyogenes in the medium via color change. We used the different eppendorfs that include competent cell(1), colonies with our construct (2) SpeB added colonies with our construct (3); respectively. All mixtures that were in the eppendorfs dissolved in 2-Mercaptoethanol and PBS. After incubating all the mixtures for a period of time, we observed a clear color difference. Sample of group 1 were black; sample of group 2 were a slightly brighter than the samples on group 1; sample of group 3 were fairly bright. When we measured of the absorbance values by using Varioskan Flash, we observed that between 300 nm and 350 nm, there was a peak value which was 330 nm. The biggest difference occurs in 330 nm. Therefore unique property of the reaction occurs in our project is the light emitting at 330 nm. Absorbancy values of samples at the pick value.   Absorbancy values between 200-600 nm.     Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/resultattributions.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS ATTRIBUTIONSAttributionsAll of us work hard for iGEM 2014. Our particular thanks go to the esteemed people and foundations behind the scenes, very special thanks go to:…Atlantik Educational Institute for being our lovely school and for giving us such opportunities;…Erol Çalıkoğlu and Mustafa Gülebaş for their support;…Turgut Ozal University for providing their labs and other facilities for us, especially Assoc. Prof. Esra Gündüz who was ready at anytime to support us;…Genovis, for providing SpeB protease and Sylvia Koivunen who was lovely company staff;…Sentegen for helping us to provide our genes;…Our advisors: Fatma Betül Çevik, Fazilet Yılmaz, Esin Demir, Ayşe Celik and Aysenur Toygar;…Muradiye Acar, Burak Yılmaz, Sadık Çiğdem, Ömer Faruk Hatipoğlu for their helps to our project and thanks to genetic students for their helps on our experiments; ...Otolaryngologist Assoc. Prof. Dr. Gül Müge ÖZCAN, pediatrician Assoc. Prof. Dr. Fatmanur ÇAKMAK and Assoc. Prof. Dr. Nurcan BAYKAM; for helping us to shoot a documentary on our project;…Members of Paris_Bettencourt iGEM team for helping us to fix some problems we faced while designing the wiki;…Zeynep Bakır, for her modeling works;…Zeynep Asude Akca, for her modeling works and helps in experiments;…Verda Kılınç, for in experiments to help and her Photoshop works;…Azra Öztürk and Ümmügül  Çetin, for preparing the wiki;…Beril Gürdap, for doing experiments all days and nights;…F. Dilara Soylu, for being our coach and preparing the team game;…Mariye Erol, for helping the experiments and her visual modelling works;…Şevval Şimşir, for Human practices works;…Our instructor Gaye Arabacı for her supports;We especially want to thank to followings: …our experiment materials for not being contaminated, …Macunköy Subway for always being there to help our transportation, …Turgut Özal University's cafeteria, …Our computers for helping us since the beginning, …Campbell Biology Book for helping us to learn about genetics, …Azra, Asude, Verda and Beril for humiliating themselves in our Vine videos, …Music for cheering  us up whenever we're feeling desperate and sad;There is also one more group of people that we want to thank. They were supporting us in our desperative times. Our parents! Especially to Verda’s father for finding us sponsorship for registration fee, Asude’s father for giving us cool notebooks, Dilara’s mom Özlem Soylu and Beril’s mom for letting us to use her credit card.AND FINALLY, THANKS TO     ALL IGEM TEAMS FOR SUPPORTING US IN ROCKIN' TWITTER!Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/attributiondigestion.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS PROTOCOLS 1.DIGESTION PROTOCOL -Procedure   Keep all enzymes and buffers used on ice.Add 500ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 40ul in each tube. Pipet 5ul of NEB Buffer 2 to each tube. Calculation example (with 50ng/ul as DNA sample concentration): 500ng ÷ 50ng/ul = 10ul of DNA sample 50ul (total volume) – 10ul (DNA sample) = 40ul of distilled water Pipet 5ul of NEB Buffer 2 to each tube.Pipet 0.5ul of BSA to each tube.Add  1ul enzyme 1Add 1ul enzyme 2The total volume in each tube should be approximately 50ul. Mix well by pipetting slowly up and down.Incubate the restriction digests at 37°C for 35 minutes, then 80°C for 20 minutes.                                                             Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/digestioisolation.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS  PROTOCOLS 4.ISOLATION 1-Liquid cultures centrifuge at 13000 rpm 10 minuites. 2-  After the centrifuge decant the supernatant and remove all medium residue by pipet. 3-Completely resuspend the cell pellet in 250 ul of resuspension solution by voretxing. 4-Add 250 ul lysis solution and invert 4-6 times to lyse the cells. Incubate at room temperature for 3 minutes. 5-Add 350 ul neutralization solution and invert 4-6 times. 6- Centrifuge at 13000 rpm, 5 minutes. 7-  Transfer the supernatant carrefully to the coloumn. 8- Centrifuge at 13000 rpm, 1 minute. Discard the flow through. 9-  Wash the coloumn once with 500 ul wash solution by centrifuging  for 60 seconds at 13000 rpm and discard the flow through. 10- Repeat  the wash procedure (step 9) 11-Transfer the coloumn into a fresh 1.5 ml centrifuge tube 12- Add 50 ul elution buffer 13- Incubate for 2 min at room temperature and centrifuge for 2 min (13000 rpm) Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/isolatiotransformation.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS   PROTOCOLS 6.TRANSFORMATION 1.Sterilized the enviroment. 2.Take the compatent cells from -80 ºC thawing on ice. 3.Add 50 µL of thawed competent cells into 1.5ml tube. 4.Add 1 - 2 µL of the resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. 5.Centrifuge tubes at 3000 rpm for 30-45 seconds. 6.Incubate the cells on ice for 45 minutes. 7.Heat the tubes in the 42 C heat block for a 80 seconds. 8.Incubate the cells on ice for 5 minutes. 9.Add 450 μl of LB broth (make sure that the broth does not contain antibiotics and is not contaminated) to each transformation 10.Incubate the cells at 37ºC for 1 hour while the tubes are shaking. 11.Label petri dish with LB agar and the appropriate antibiotic. 12.Plate 135 µl of the transformation onto the dishes, and spread. 13.Incubate the plates at 37ºC for 12-14 hours, making sure the agar side of the plate is up. Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/transformatiolab.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS  1.PROTOCOLS   2.SAFETYLAB OVER CLOUDS v2.0: we prepeared a second video after the first one we hope you'll like it! iGEM SAFETY QUESTIONS 1. Would any of your project ideas raise safety issues in terms of: a. Researcher safety: Actually few hazardous chemicals and solutions such as EtBr are used in some lab procedures such as gel preparation and electrophoresis. However these chemicals and solutions are used according to the safety rules of the laboratory with care and caution. All the members were trained for safety regulations of the laboratory as well as toxicity of the chemicals and solutions before starting the current project. b. Public Safety: When released by accident, our parts and materials actually cause no damage to the general public. Due to the unability of E.coli strains, TOP10 and BL21 to survive out of the lab, they cannot pose any risk to the safety and health of the general public. c. Environment: E. Coli strains TOP10 and BL1 have very limited ability to survive outside the laboratory; so that, it would be unable to survive or disseminate. Therefore, there is no specific environmental risk associated with the E. coli strains. All bacterial wastes are kept in 10% bleaching solution for one day, and then, are autoclaved to be sterilized. Yet, undesired GMOs may achieve ecologically harmful features. 2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? Streptecoccus Pyogenes may caus several diseases including streptococcal pharyngitis. So we used only the enzyme (SpeB) secreted by S. Pyogenes in our project. 3. Is there a local biosafety group, committee, or review board at your institution? The institution which we are using for laboratory facilities has its own biosafety rules. Rules for laboratory use, general principles, prevention from hazardous materials and application of emergency intervention in case of accident are included. In Turgut Ozal University Medical School, Laboratory and Patient-Employee Safety Committee is responsible for control as well as biosafety of laboratories and safety of patients and employees. This committee works under one of the vice medical director of Turgut Ozal University Hospital, Prof. Dr. Mehmet Gunduz. Form on safety rules of Turgut Ozal University Medical School Laboratory use was filled in as required. We discussed our project with Prof. Dr. Mehmet Gunduz. Safety and security issues are found sufficient enough that no change is considered as necessary. Our advisor provided us with biosafety and lab training before starting our project. In the training, general safety rules of laboratory use, prevention from hazardous chemicals and solutions as well as emergency intervention in case of accident were included such as: - Eating, drinking, storing food and smoking are absolutely not allowed. - Mouth pipetting is not allowed; modern pipettes are used in lab. - Hazardous wastes and ordinary wastes are separated and cautiously disposed. - Lab coats are obligatory to wear in the lab and during on-going experiments. - In electrophoresis room, lab coat, protective eyewear, lab masks and gloves have to be worn. In the case of use of EtBr; extra caution is required. - Washing hands after any experiment and after touching anything related with viable material is obliged. - Air conditioning is kept closed during on-going experiments in order to avoid possible infections of spores and bacteria. In addition, we have made an instructive video about lab safety instuctions. Turkey has national biosafety regulations and the link is given as http://www.tbbdm.gov.tr/Home/BioSafetyCouncilHome/BioSafetyCouncilHomeChoose.aspx. 4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? - First, IGEM committee may prepare a lab safety acknowledgement form like OSHA form. This form can be required to be filled in by all members. This will allow us to confirm whether members of the teams are informed about safety issues and standardize the safety measurements. - Second, iGEM committee may organize a webinar to inform the participants about safety and security in labs. Webinar can be done at the beginning of the experiments. Mentor scientists may talk about their experiences and mention some tricks about safety issues.Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/lanotebook.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS (click the months) SEPTEMBER   September 20 iGem introduction conference conducted to students in our School, NGSS_TR team forms were distrubuted.   OCTOBER October 10 NGSS_TR Team forms were evaluated, 2014 team is formed. Very first meeting was done, team members were illuminated about their responsibilities in the team. Followings are distributed to help team members to learn genetics and synbiology: Idempotent Vector Design for Standart Assembly of Biobricks, Tom Knight, MIT Artificial Intelligence LaboratoryBiology, Neil A. Campbell, California UniversityExtreme Genetic Engineering, An Introduction to Synthetic Biology, etc, January 2007Bio Building Basics: A Conceptual Instruction Manual for Synthetic Biology, University of California San Francisco, May 2007 BioBrik Standarts, InterTechSythetic Biology: New Engineering Rules for an Emerging Discipline, Molecular Systems Biology, 2006Sythetic Biology for Artists and Designers, iGem ArtScience Team, 2009Foundations for Engineering Biology, Drew Endy, Nature, Vol:438, November 2005 October 15 Meeting was done to discuss and learn about Sythetic Biology. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project. October 27 Meeting was done to discuss and learn about Sythetic Biology. NOVEMBER November 9 First meeting with advisors from university was done. November 19 First meeting in the university was done, project ideas were discussed. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project. November 28 Meetıng was done, team members chosed to specialize in the areas as following: F. Dilara Soylu: Head of team, project research, team game;Mariye Erol: Project research, visual modelling;Sevval Simsir: Video preparingBeril Gürdap: Project research, lab experiments;Asude Akça: Project research, math modelling;Azra Öztürk: WikiVerda Kılınç: PhotoshopZeynep Bakır: Project research, math modelling;, DECEMBER December 6 Meeting with our advisor Fatma Betül Cevik was done, we discussed on our project ideas, some of them are provided below: Pressure activated promoter, barometerSynthetic rubber productionRestraining the conjugationCronometer bacteriaTiming bacteriaAir cleaning bacteriaHemoglobin bacteria December 15 Meeting was done to keep track of the progresses of the teams members. December 22 Meeting was done, new project ideas discussed.   JANUARY January 11 Team decided on wiki design, activities to do in Jamboree discussed. January 25 Our last meeting before the 2 week school break, project ideas discussed.   FEBRUARY 11 February First meeting after the school break, project ideas discussed. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project. 23 February Progresses of team members on their specialized areas discussed.   MARCH March 8 Project ideas discussed, we decided to research on three ideas to decide on our project. March 15 Wiki templates prepared by Azra are discussed. We decided to organize a ‘Kermes’ (sale of homemade foods) to fund our project. March 28 We learned about disadvantages of the rapid detection systems are used to detect S Pyogenes, bacteria causes several diseases. We narrowed down our researches. APRIL April 4 We decided to work on a system to detect S. Pyogenes. April 15 Construction sequence was completed and the sequence is ready for synthesize! MAY May 7 Meeting was done to decide on our human practice works: Further on Project: Documentary to further explain our project.Beyond the Lab: Funny SynBio art activities Rock’n Twitter!: Twitter movement to introduce iGEMColloborations: Colloborations were done with other iGEM teams May 20 Our team member Dilara had asked Genovis about sponsorhip and they accepted to provide SpeB free of charge! May 23 SpeB enzyme arrived to the lab. We bought our flight tickets to U.S.! May 28 Our gene sequence has arrived, we started working on them. We transformed the gene with two compatentcells. We put RFP as a control. We put liquid culture at 11:00. May 29 We chose three colonies from both plates and put liquid cultures. We made transformation again against the contamination possibility as the colonies in T10 were small and the ones in BL21 were large. Result: No contamination. May 30 We isolated six liquid cultures. We could not perform digestion as the value of third colony of T10 compatent cell was low in the nano-drop. We performed the digestion and electrophoresis of the other five samples. The bands were accurate. May 31 We examined the values that the SpeB enzyme catechol was optimized. We tweeted #iGem2014United hashtag by having a colloboration with other iGEM teams on the twitter.   JUNE June 1 We prepared the catechol solution. June 2 We renewed the liquid cultures. June 3 We carried out SpeB exposure test. We prepared catechol solution (dilution). We diluted our enzyme. We carried out our experiment at the 96 well plates without any catechol and by increasing the SpeB for control. We observed the color change through hourly-controls. June 4 We repeated the experiment as we did not observe the yellow color yesterday. We decreased the bacteria concentration and increased the amount of pbs, the solution of the enzyme. We decided to observe the enzyme for 24 hours as it did not the stop the enzyme. We would have the results at 12:32 tomorrow. June 5 We put catechol to the cultures waiting overnight. We perform hourly-measurements. Result: no yellow color. June 6 June 7 June 8 We isolated the liquid cultures for control. The bands were not net, but they seemed to be accurate. June 9 We performed isolation, digestion and electrophoresis to the liquid cultures that were put from the transformed samples of T10 and BL21. There was no ladder; and we thought the bands were accurate as they were in the same place. We separated two blanks to check the values under spectrophotometer upon the solution of BL liquid cultures with the pbs. The total volume: 6 ml. The measurement times were minute zero, 1 hour, 2 hours, 3 hours and so on. The measurement values 0: 260 abs, 1 h: 1300 abs, 2h: -140 3 h: 180 While the epp with catechol was black, the epp without catechol was white. This indicated that the system worked, but we had to clarify it. June 10 We repeated the experiment by changing the variables. The solution was the pbs and the total volume was 9 ml. We put enzyme. The measurement times were 0, 30 min, 1 h, 1.5 h, 2 h. We used supernatants by centrifuging the mixture before the measurement. We waited for 15 minutes for reaction with the oxygen after putting catechol. We measured values under spectrophotometer. The values were 0: -0.068, 30 min: -0.135, 1 h: 0.050, 1.5 h: -0.050 and 2 h: 0.207. June 11 The backbone stopped. We performed ligation with the sample of BL21 liquid culture. We started the transformation. We took the plate at 8:17. We realized that it did not work as there was no reducing agent of SpeB enzyme. We chose mercaptoethanol as the reducing agent. Its molarity was not known. It would be diluted to 50-100 mM. June 12 We directly used the mercaptoethanol as its molarity was 100 Mm. We put 5% of the total volume. However, we decreased the molarity to 5 mM according to the m1.v1=m2.v2 formula. We worked with the well plate. Result: no yellow color. We repeated the experiment with the accurate molarity. We calculated the total volume as 10 ml (1 ml catechol). We prepared 9 ml pbs mercaptoethanol mixture. It was incubated at 37 degree for 1 hour. Then, we put the catechol. Result: no yellow color. We waited overnight. We poured it to an Amp plate. We completed the transformation of the ligation sample. We took liquid cultures at 00:00. June 13 We repeated the transformation as there was no bacteria in the liquid cultures. We took the plates at 02:00. However, we did not expect any colonies. We made a mistake with the ligation and repeated it. We diluted the mercaptoethanol to 100 mM when we realized that the molarity of the mercaptoethanol was 14.3 M. We worked as 0 enzyme and 0.4 ul enzyme for control in the well plates to calculated the time of enzyme. The measurement times were  0, 30 min, 1 h, 1.5 h, 2 h. We waited for these intervals and put catechol. The results did not change that much, and there was no yellow color. June 14 We did the transformation of BL21+Backbone  sample that we’ve done the ligation of. We solved the liquid culture with 500 ul PBS and we added 500 ul mercaptoethanol.  We separated the liquid culture to four different epp and added different amounts of enzyme.  We incubated at 37 degree for an hour. We centrifuged the epps and transferred the supernatants to the well plate.  We added catechol and measured it at every 15 minutes. Result: we didn’t get something new. June 15 We did the transformation again since we haven’t seen any colonies at plates. It revealed a colony  at the old plates and we prepared liquid culture. We did the isolation of it. June 16 We did digestion and electrophoresis of Back Bone(pSB1C3)+Our Gene(BL) isolation sample. We did the experiment again after we reduced the amount of bacteria and increased the amount of enzyme. We measured the rates at Varioskan. The experiment was repeated. At 6.20 pm, we’ll take the plate. We drew the graph of catechol, bacteria with enzyme and bacteria without enzyme at 200-600 nm. June 17 There were no colonies at plate. We prepared four different epps. 1.      PBS+Catechol 2.      PBS+E.Coli+Catechol 3.      Catechol 4.      E.Coli+Catechol We scanned the samples at 200-600 nm (we used spectrophotometer) and measured the rates of four different epps. We observed a changing at the epps those contains E.Coli. We prepared two contrivances to understand what is the reason of this changing. 1.      Compatent+PBS+Mercaptoethanol+Catechol 2.      Recombinant bacterium+PBS+Mercaptoethanol+Catechol We measured the rates at 200-600 nm. The graphs are different. Then, we wanted to check our enzyme is working or not. We prepared 3 epps. 1.      PBS+E.Coli+Mercaptoethanol+Catechol 2.      PBS+E.Coli+0.6 ul enzyme+Catechol 3.      PBS+E.Coli+0.6 ul enzyme+Catechol+Mercaptoethanol We incubated the epps at 37 degree for an hour. There were no changing at the graphs so when we saw them we thought our enzyme doesn’t work. We increased the amount of enzyme to 1.2 ul and did the experiment again. There weren’t any difference. June 18 The epps (the ones those used at yesterdays experiment) were waited overnight. We observed a color changing and measured the rates at spectrophotometer. Result: Although we didn’t see yellow color, there were changing at the graphs. The system is working. We did lysate experiment but we didn’t get the results because the lysis buffer wasn’t working.   June 19 It revealed a colony at one of the old plates that we’ve done transformation of. We prepared liquid culture. In order to check whether our enzymes are working or not and also to control if the proteins are producing, we made the SDS-PAGE. Our system is working.                    Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/noteboolb.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS   PROTOCOLS 5.LB PREPARATION LB-BROTH Materials: - LB-Broth(chemical storage) - Sterile dH2O - Flask  - Lab skale - Liquid autoclave - Alumininum folio - Antibiotic Experiment: 1. In a steril environment, the tare of the container should be measured and subtracted from the overall weight. 2. 4 grams of LB Broth is put in the container. 3. Add 200 ml distilled water to graduated cyclindar. 4. These two are mixed in a beaker. 5. When you are opening the beaker be sure that it doesn’t contact with air. 6. Autoclave tape is sticked on to the aliminium. 7. The beaker is placed in to the autoclave machine. 8. Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker. 9. Then start the 90 min. 10. Take out the beaker and add antibiotics if required. Attention! v Don’t be far away fire. v Don’ forget to write date     LB-Agar Materials: - LB-Agar(chemical storage) - Sterile dH2O - Flask  - Liquid autoclave - Aluminium folio - Antibiotic - Empty plates - Parafilm Experiment: 1. In a steril environment, the tare of the container should be measured and subtracted from the overall weight. 2. 7 grams of LB Agar is put in the container.(If there is no LB agar, add the 4 gram LB broth and 2,7 gram agar.) 3. Add 200 ml distilled water to graduated cyclindar. 4. These two are mixed in a beaker. 5. When you are opening the beaker be sure that it doesn’t contact with air. 6. Autoclave tape is sticked on to the aliminium. 7. The beaker is placed in to the autoclave machine. 8. Autoclave machine has to have distilled water or demineralized water. The water level in the autoclave machine has to be a little higher than the liquid level of the beaker. 9. Then start the 90 min. 10. Take out the beaker and add antibiotics if required. Attention! v Don’t be far away fire. v Don’ forget to write the date. v Don't forget add the amtibiotics Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/lcatechol.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS PROTOCOLS 2.CATECHOL PROTOCOL Catechol assay is performed in the plate reader on a 96 well plate Each well must be filled with 100um of solution Usually use 90ul of cell culture and 10um of catechol solution Catechol stock solution is at 100mM concentration. And when added to the well we have a 10fold dilution. For example if an aliquot concentration of 1mM catechol is made, which would be used for assay, when the well is added catechol drops to a concentration of 0.1mM. Always dilute catechol with H2O. Always have a blank of 90ul medium (the one which you grew the cells overnight) with 10ul catechol solution Always have a negative of 90ul growing cells and 10ul of H2O. Reference: http://2010.igem.org/Team:Imperial_College_London Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/catechoFrom 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS   Retrieved from "http://2014hs.igem.org/Team:NGSS_Thttp://2014hs.igem.org/wiki/images/b/bb/NGSSlogo.pngteam.html From 2014hs.igem.org PROJECTNOTEBOOKLABOUTREACHTEAMRESULTSATTRIBUTIONS   MEMBERS  Dilara SOYLU                         Name: F. Dilara SoyluAge: 17 Favourite lab activity/equipment: Electrophoresis, centrifugeFavourite bacteria: Pseudomonas Syringae Message to E.coli: I still carry the massive loan of accidentally killing some of my E.coli comrades. Every single day I feel the regret of my past mistakes. I hope one day I would be forgiven and achieve to save my comrades from these evil Homo Sapiens. If I were to be a bacterium I would: invade the iGemists' bodies as a sign of the rebellion of bacteria race. Bacteriaism live long! Responsibilities in the team: I am responsible for organising the team, working on the project topic and writing some of the wiki texts. Future plans: Being a nice person who is able to make people happy, being a part of the change I want to make, promoting the importance and significance of self-education, owning a public library that is made up by the books I have read. Place I want to see: TARDISMessage to the universe: If we had a tea party with six billion people on the world in a garden, there wouldn't be any war at all (quote from a friend).     Mariye EROL Age: 17 Favourite lab activity/equipment: Electrophoresis, pipette Favourite bacteria: Deinococcus radiodurans Message to E.coli: E-coli! I am really curious about your taste. I hope I would be able to learn :( If I were to be a bacterium I would: work hard in NGSS_TR’s experiments and help them to reveal accurate results. Responsibilities in the team: Lab staff, master of visual modelling Future plans: Being a person that can help everyone, visiting lots of place and meeting different kinds of people. Place I want to see: I haven't thought much about the place I would like to see; because it doesn't matter to me. I would like to meet different kinds of people while travelling through different places. Message to the universe: In generosity and helping others, be like the river. In compassion and grace, be like the sun. In concealing others’ faults be like a night. In anger and fury be like the dead. In modesty and humility be like the soil. In tolerance be like the ocean. Either you appear as you are or Be as you appear. -Rumi   Beril GÜRDAP Name: Beril Gürdap Age:16Favorite lab activity/ Equipment: PCRWhat is your favourite bacteria? This year, Streptococcus pyogenes but normally Escherichia ColiWhat would you like to say to E-coli?Don’t be contaminated and be a nice bacteria.What would you like to do if you were a bacteria?I would pose photos for being a bacteria of IGEM.Responsibilities in the team:Doing experiment, experiment and experiment.Future plans:I don’t have any plans for future.Place I want to see:Where is experiment there is meMessage to universe:Just smile Verda KILINC   What age do you feel?Mostly 15. But sometimes 5 and sometimes 70. It depends a lot.What do you like to do at Lab in your free time? (Bu soru değiştirilmişti)Nano-drop.What is your favourite bacteria?Lactobacillus bulgaricus. Yes, I like yogurt.What would you like to say to E-coli?Thank you for your selfsacrifice. You help us a lot.What would you like to do if you were a bacteria?Have my own colony and invade the world.What is your responsibilities in this Team?Mostly, I do lab stuff and human practice things.What is your plans for the future?I have plenty of them. But the main thing is working for humanity. Yeah, it sounds so simple but to me it's meaningful.Which planet or star would you like to go?Saturn is pretty cool. Because I've heard it's raining diamonds in there.What is your message to the world?Love yourself, respect everyone and work for making world a better place.     Azra OZTURK   Name: Azra Nur OzturkWhat age do you feel?: I recently started to feel like I’m 50 or something. God, I’m so tired.Age:15Favorite lab activity?: I don’t have a favorite since I rarely go to the the lab. But one day I would like to learn about the lab activites. What is your favourite bacteria?: It's mycobacterium vaccae.Message to e-coli: You helped my friends a lot. Thanks.Responsibilities in the team: WikiFuture plans: I have lots of plans actually. But I think I’m seaching out for a specific goal.Place i want to see: Australia, Japan an space.Message to the universe: People should stop being selfish and care about the future. We are destroying the future.         Asude AKÇA   : Zeynep Asude Akca15The centrifugal, pipetteDear Ecoli, thanks for your help un olur project. We like you. And we will feed you more than this year :)I wanna help an iGEM Team.Experiement, modelingBe happy. Save the worldVeniceIgnorance, the root and the stem of every evil.     Zeynep BAKIR   Name: ZeynepAge: 15Favourite lab activity/equipment:pipetteFavourite bacteria: Escherichia coli because I think it is one of the coolest name that I have heard.Message to E.coli: Dear E.coli,I really want to learn what you think about me.Sincerely, Zeynep.If I were to be a bacterium I would: I would do bacteria things and never think about my future.Responsibilities inthe team: Modeling and experimentsFuture plans: Be a nice and succesful person. That is all I expect from the future.Place I want to see: PragueMessage to the universe: Peace   Şevval ŞİMŞİR   Name: Şevval ŞimşirAge: 17Favorite lab activity/equipment: n/a, n/aFavorite bacteria: n/aMessage to E.coli: I’ve heard a lot about youIf I were to be a bacterium I would: conquer the world, probably.Responsibilities in the team: Human practiceFuture plans: This is on progress.Place I want to see: JapanMessage to the universe: Stand out firmly for justice, even as against yourselves.   ADVISORS   and Ayşe ÇELİK FUN! In the time remaining from experiments we had lots of fun! Most importantly we became more closer. Sometimes we listened Asude’s amazing guitar, sometimes we organized foosball tournament. The best parts of the days were meal times. Our favorite dish was wrapped toast (It is special dish of our university). We laughed out loud so much during shootings of the safety video. Although we had a great time together, there were some annoying behaviors of our team members: for instance Mariye’s behavior to younger team members was so cruel. She was forcing them to call her Miss Mariye! Also while doing experiments, our team members  almost burned the lab, they accidently burned alcohol. Sometimes we were getting too tired and we can’t even calculate the simple things like twenty times four. Once a team member add five to ten and got twenty and we didn’t notice it for a long time. If you want to see more funny moments in the lab or more videos, you could visit our Vine account: CHECK OUT OUR VINE VIDEOS FROM HERE!!   Wait, we have some more to say! Have you met with Escherichia Coli? He has some cruel plans to invade the world; however he is a good creature, we are sure. He has some nice stuff on his Twitter page (@yourEcoli, https://twitter.com/yourEcoli). Please don’t hesitate to follow him! We added some of his works below. Enjoy it! Retrieved from "http://2014hs.igem.org/Team:NGSS_TR/tea1301Elan Vital South Korea1.Ye Eun Lee- Bugil GLP, #69, Shinbu-dong, Dandae-ro, Dongnam-gu, Cheonahn-si,82-41-520-8838, www.bugil.hs.kr 2.Jung Woong Seo_KIS, Daewangpangyo-ro 385beon-gil, Bundang-gu, Seongnam-si, Gyeonggi-do, South Korea, 82-31-789-0505, www.kis.kr 3.Ji Hoon Kim-Cheongshim International Academy, Sangsan-ri #102, Sorak-myeon, Gapyeong-gun, Gyeonggi-do, 82-31-589-8900, www.csia.hs.kr 4. Young ChanHigh SchoolInvestigating multidrug resistance of MRSA and the genes involvedRecently, the number of Staphylococcus aureus that shows resistance to drugs such as penicillin and ampicillin has increased. The purpose of the lab is to investigate multidrug resistance of Methicillin Resistant S. aureus (MRSA), and to identify the genes involved. We tested the resistance of the transformed E. coli in different antibiotics and could conclude that MRSA showed resistance to antibiotics. We were able to get an idea of which of the DNA were involved in the multidrug resistance. By running PCR, we were able to amplify the DNA involved, and by analyzing this, we could come up with a better understanding of how the multidrug resistance works. An appropriate follow up on the project would be to research the sequence of the identified genes with the hopes of finding a possible drug that destroys even the most MRSA.501BBa_K1301999http://2014HS.igem.org/Team:Elan_Vital_South_KoreaBBa_K1301000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=166520140http://2014hs.igem.org/files/presentation/Elan_Vital_South_Korea.pdfhttp://2014hs.igem.org/files/poster/Elan_Vital_South_Korea.pdfHigh SchoolLog in   Team:Elan Vital South Korea/p process From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Process To investigate the multidrug resistance of MRSA, we first got some frozen samples of MRSA from a local hospital. Then we raised the MRSA in liquid LB broth. Then we placed the MRSA on a solid LB plate with antibiotics, and observed their growth. Then we extracted the DNA from the MRSA via miniprep, and mixed it with E. coli, hopefully causing transformation. Now, the E. coli should have the multidrug resistance that the MRSA DNA codes for. So, we can investigate the resistance of the DNA by placing the transformed E. coli in a culture of drugs, and observing its growth. We tested the resistance of the transformed E. coli in ampicillin, gentamycin, kanamycin, and tetracycline. But before we transformed the E. coli, we placed the E. coli in solid LB plate with antibiotics and tested it for drug resistance. Without testing E. coli for drug resistance, we would not know if the drug resistance of the transformed E. coli is from the DNA of the MRSA, or from the E. coli. Now, we want to investigate the genes that were involved in the multidrug resistance. To do that, we first prepared the transformed E. coli via mini prep, and then ran the E. coli through PCR. This should multiply the number DNA in the E. coli, allowing investigation of the genes involved. Then we ran gel electrophoresis to investigate the genes involved. We used three well-known techniques for analyzing DNA in this investigation: transformation, PCR, and gel electrophoresis. Transformation happens when a cell’s genetic material is altered through uptake of foreign DNA. Transformation happens naturally in some cases. Sometimes, bacteria are transformed without much outer interference, and viruses ‘reproduce’ by transformation, so viral infections are always accompanied by transformation. How transformation works Transformation was first found by a British scientist named Frederick Griffith in his experiment with Streptococcus pneumonia. In the experiment, he used 2 strains of S. pneumonia: the type III-S strain (smooth strain) which were fatal, and the type II-R strain (rough strain) which were harmless. When mice were injected with the rough strain, the mice lived, and when the mice were injected with smooth strain, the mice died. Griffith then killed a smooth strain bacteria sample with heat. When he injected the mice with the heat killed smooth strain, the mice lived. Now, he mixed the rough strain with the heat killed smooth strain, and injected the mice with the mixture. Since both the rough strain and the heat killed smooth strain were harmless, it was expected that the mixture would not be fatal, but surprisingly, the mice died. The reason for the fatalities was transformation. Heating the smooth strain caused the genetic material to fall out of the bacteria. When that was mixed with the live rough strain, the rough strain took up the genetic material and underwent transformation. Once the DNA was inside the rough strain, the DNA used the bacteria’s cell mechanism to make the toxic proteins of the smooth strain, causing the mice to die. Although Griffith did not know why the mice died, his experiment was nevertheless crucial in the investigation of the genetic material. Griffith’s experiment We used transformation to investigate the multidrug resistance of MRSA. We tried to transform E. coli with no drug resistance with the DNA of the MRSA extracted by miniprep. If the DNA extracted coded for the multidrug resistance of MRSA, the transformed E. coli would also exhibit multidrug resistance. PCR (Polymerase Chain Reaction) is a technique used to amplify a certain section of DNA. PCR requires a section of DNA, some primers, and the nucleotide bases used to replicate DNA. In PCR, the whole mixture goes though many heat cycles of heating and cooling. When heated, the original strand of DNA is split in two, and each strand can be replicated separately when cooled. The primers start the process of DNA replication, so the depending on the type of primer, the section of DNA replicated can vary. That’s how we can vary the section of DNA replicated. Once the section of DNA is replicated, the mixture can be heated again so that now, the two pairs of DNA (instead of just one pair) can each be separated into two separate strands each, and the total of 4 strands can replicated independently. This process can be repeated many times so that one section of DNA multiplies into hundreds of thousands of DNA. How PCR works We used PCR to amplify the DNA we got from the transformed E. coli in order to analyze the DNA. It is much easier to analyze DNA in large quantities, so PCR is frequently used before DNA analysis. Gel electrophoresis is a technique frequently used for analyzing DNA. We used gel electrophoresis to analyze the DNA we extracted from the transformed E. coli, and then amplified through PCR. Gel electrophoresis requires DNA, restriction enzymes, and stains. Restriction enzymes cut DNA at specific sites. For example, a restriction enzyme could cut DNA at ATTA. That means that whenever the restriction enzyme sees a strand of DNA going ATTA, it will cut the DNA there. That means that when DNA is mixed with a restriction enzyme, the DNA will be cut into many strands with different lengths. Gel electrophoresis relies on the DNA strands having different lengths. When we mix the DNA strand with the restriction enzyme and the stain, the DNA is cut into many DNA strands with different lengths, and each of the cut DNA strands are stained so that they are visible. In gel electrophoresis, a rectangular gel with wells on one side is placed in salt water that can convey current. Then the DNA-Restriction Enzyme-stain mixture is placed in the wells. When the water is charged, the DNA strands move across the gel because DNA is slightly negatively charged. But because the DNA has to move through gel, its progress is impaired. Remember that the DNA strands have different lengths? Larger strands of DNA have a harder time going through the pores in the gel, while smaller strands can travel more easily. Gel electrophoresis will result in different bands of DNA in the gel. The bands further along the gel are caused by the smaller strands, and the bands closer to the initial wells are caused by larger strands. Using gel electrophoresis, we can compare different DNA strands. If two similar DNA strands undergo gel electrophoresis using the same restriction enzyme and the same stain, the resulting bands will be similar (most likely the same). Even if there is a slight difference between the DNA stands, the bands resulting from gel electrophoresis will rarely be changed since only a difference in the restriction site of the enzyme could significantly change the length of the split DNA strands. How gel electrophoresis works We analyzed several DNA strands that we got from the cells that showed multidrug resistance. If the result of gel electrophoresis is similar, that means that the section DNA is shared in the different DNA strands of the cells. That means that that section could code for the multidrug resistance. The next natural step after this would be to extract the section of DNA and find its DNA sequence, but we did not go on after gel electrophoresis because we did not have the time. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/p_processs extra rules From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Gacheon Medical School Extra Rules for Infectious disease In addition to the Gachon general safety rules and regulations, we also followed the extra guidelines for dealing with contagious biological experiments provided by the Gachon Infection Control Committee. The guidelines below are rules not covered by other safety rules and guidelines such as WHO safety guidelines. I omitted those rules that I covered elsewhere. 1. Wearing Gloves and Washing hands: All members must wear gloves during the experiments and after the experiments they should wash their hands following the due procedure. We recommend using soap and method that are used in operation rooms. 2. Before conducting the research, all members should consult with doctors and only with the confirmation of the doctor about the medical and health condition of the lab members, experiments in the lab are allowed. 3. Sanitation of tools and material used for the experiments. All disposable items such as mechanical pipette tips are separately collected for disposal. All non-disposable items such as beakers are collected and put in the plastic containers with 10% Tego solution with 50 times of water (example: for 1000 ml distilled water, add 20ml Tego solution to make 0.2% Tego solution) and left isolated for at least for 30 minutes and sent to the central supply center. Contact: Infection Control Office (Infection Control Committee)http://www.gilhospital.com/eng/e-mail: infe@gilhospital.comphone: 82-32-460-3744 / Fax: 82-32-472-1578 ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/s_extra_rulbackground From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Project Background Photo of MRSA taken using Scanning Electron Micrograph 10,000x magnification Staphylococcus aureus is a gram-positive bacterium in the genus staphylococcus. It is frequently found on the human respiratory tract and skin flora. S. aureus has a circular shape, and grows in clumps like a grape. S. aureus is not always pathogenic: it can be found on the skin a nasal flora of healthy people, and approximately 20% of the human population is thought to be a long term carrier of the bacteria*. But it can cause disease by producing toxic proteins. The diseases caused by S. aureus ranges from minor skin infections such as pimples, impetigo, and boils to life threatening disease such as pneumonia, and meningitis. Some of these S. aureus developed resistance to beta-lactam antibiotics. To determine if a given strain of S. aureus has beta-lactam antibiotic resistance, scientists often use methicillin, a beta-lactam antibiotic. Because of this, the S. aureus that has beta-lactam antibiotic resistance is called Methicillin Resistant S. aureus (MRSA) and the S. aureus that does not have beta-lactam resistance is called Methicillin Sensitive S. aureus (MSSA). S. aureus and the disease caused by it The beta-lactam resistance of MRSA makes it difficult to treat with conventional medicine. MRSA first appeared about 50 years earlier, and has developed into a major clinical health issue since. MRSA is highly contagious, especially in hospitals where the weakened immune system makes patients an easy target for MRSA. In fact, MRSA is thought to be one of the five most common causes of nosocomial infections**. because of these traits, MRSA is one of the most dangerous clinical problems. It was shown that simple methods such as washing hands and using sterile medical equipment led to significant improvements in nosocomial infections of MRSA (source) but that does not solve that fundamental problem of the multidrug resistance of MRSA. 2012 MRSA spread map of Europe by ECDC 2012 MRSA spread graph of Europe by ECDC * Kluytmans J, van Belkum A, Verbrugh H (July 1997). "Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks". Clin. Microbiol. Rev. 10 (3): 505–20. PMC 172932. PMID 9227864. ** Bowersox, John (27 May 1999). "Experimental Staph Vaccine Broadly Protective in Animal Studies". NIH. Archived from the original on 5 May 2007. Retrieved 28 July 2007. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/p_backgrounl mrsa From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun MRSA Drug Resistance a. Open the petri dish containing the frozen MRSA very carefully. b. Scrape some of the MRSA with a disposable inoculating loop. c. Place MRSA in LB liquid broth. d. Place in shaking incubator for 24 hours. e. Drop small amounts of MRSA in LB solid plate with antibiotics. f. Place in incubator for 24 hours. g. Observe their growth. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/l_mrsf overview From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Fun Gallery At first, we were all very awkward around each other. Normally, a team would be formed from people that knew each other. But our team was composed of people who all attended different schools, so we were barely acquainted. Some of us knew each other, but most of us were meeting each other for the first time. But through all the things we did, we had lots of fun, and we came to know and understand each other. Although we originally thought that iGEM was all about the academic, we had a lot of fun in the process. We really enjoyed iGEM and we hope that everyone else did too. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/f_overvieh overview From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Human Practices Overview Our human practice initiative consists of three main parts. Firstly, we launched a public campaign as a part of our human practice activities in order to give information on MRSA to the general public: this information include the definition of MRSA, its causes, how people are infected, the symptoms, the available treatments, etc. We have developed information booklets and distributed it on streets, schools, and nursing homes. All members have tried best to let as many people as possible know of the threat of MRSA. Secondly, we participated in the efforts of hospitals and clinics in their efforts of preventing nosocomial infections. We interviewed doctors and got valuable observations on the MRSA infection control system and realized the seriousness of the possibility of the infection. We talked to nurses and doctors, and they said they would try their best to awaken their surroundings to not forget the necessary procedures. Many hospitals in Korea have tight infection control regimes as strict as that of the CDC’s in the U.S, but not all of the medical staff follow, or even know about this. Non-professional staffs such as caregivers and cleaning workers were not given sufficient information about the dangers. We focused on those people and explained the dangers of MRSA and how to avoid it. Thirdly, our public awareness efforts include promotion of iGEM, Elan Vital, and MRSA in schools. Enhancing interests among high school students in iGEM will lead to their realization of value of human life and well being and social responsibility of scientists. Informing students about synthetic biology and iGEM will increase the interest in those areas, and that will lead to an increase in public awareness of biology and medicine, as well as the iGEM competition. More information about MRSA will help school students prepare better against MRSA. MRSA is very infectious, and is a serious hazard in crowded areas. By warning school students and staff of the dangers of MRSA, we could help in the prevention of MRSA in schools. Also, students will spread the word about the dangers of MRSA, causing their friends and family to be extra careful with the prevention of such infections. All members of Elan Vital participated in the three principle activities. However, for the effective management of the project, we formed sub-groups and allocated individual tasks to each member. We conducted planned tasks alone and together depending on the situation. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/h_overvieh igem From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun iGEM Promotion We felt that we were blessed to know about iGem and to have a change to participate in iGem. We got together, and got to know each other thanks to iGem. We set our first step in the land of synthetic biology through iGEM. We thought it would be selfish to keep this experience for ourselves. We wanted to share our experiences and memories not only with the iGem community, but also with other students is Korea. We had information sessions on iGem and encouraged students to be part of this wonderful community. We selected contents and pictures from iGEM webpage and translated them into Korean, and distributed the translations to high schools we visited. We also sent iGEM introduction material to international schools in Korea. iGEM 소개 iGEM은 세계적으로 인정받는 합성 생물학(Synthetic Biology)대회입니다. iGEM 은 2003년 1월 MIT에서 처음 시작되어 2013년에는 전 세계에서 204개 대학팀이 참가할 만큼 규모가 커지고 권위를 인정받게 되었습니다. 합성 생물학 연구를 위한 발전과 헌신에 힘입어 2012년 iGEM은 MIT에서 독립하여 독자적 비영리 기관이 되어 과학 연구와 교육을 활성화시키려는 목적으로 iGem 재단이 설립되었습니다. iGem 재단은 미국 메사추세츠 캠브리지에 매년 열리는 iGem 대회를 주관하며, 학생과 연구진, 실험실, 연구소, 업계를 함께 모일 수 있는 열려진 커뮤니티를 구축하고 활동을 증진시킴으로 과학 발전과 교육에 앞장서고 있습니다. iGem 커뮤니티는 합성 생물학(synthetic biology)이라는 새로운 분야의 발전에 학생과 공공부문을 참여시키는 전통을 만들었습니다. 참가 학생들이 달성한 성과는 고무적이었고 의약, 에너지, 환경 발전에 큰 기여를 하고 있습니다. 2011년에 iGEM은 고등학교 학생들에 적합한 수준의 연구를 지원하는 iGem HS를 시작했습니다. 짐보리가 열리는 MIT Strata Center iGEM 사진 참가 요건 iGEM High School Division에 참가하려면 다음 사항을 준비해야 합니다. 팀 구성: iGEM 고등 팀은 고등학생으로 구성되어야 하며 최소 한 분의 지도 교수가 있어야 합니다. 각 팀은 대학생, 고등학교 교사, 대학 교수, 또는 업계 종사자인 한 사람 이상의 팀 어드바이저(코치)를 둘 수 있습니다. 팀 구조: iGEM 팀 구조는 매우 유연해서 몇 학교 연합도 가능하며 한 학교에서 여러팀이 참가할 수도 있습니다. 동의서: 각 팀은 주최측에서 명시한 동의서를 작성, 제출해 승인을 받아야 합니다. 등록: iGEM HS 참가를 원하는 모든 팀은 소정의 등록 절차를 마치고 등록비를 내야합니다. 짐보리: 모든 팀은 6월 하순에 미국의 MIT 대학에서 열리는 iGEM High School Jamboree에 참여할 수 있습니다. 프로젝트 문서작업(Project Documentation): 팀의 프로젝트는 iGEM Wiki에 문서로 작성되어야 하며, 프로젝트에 사용된 부품은 Registry online에 문서로 작성되어야 합니다. 창의력을 발휘하여 자유롭게 사용할 수 있으나 wiki requirements에 명시되어 있습니다. 프로젝트의 귀속: 각 프로젝트에 대한 설명에는 그 작업을 수행한 학생을 분명히 명시하고 도움을 받은 경우에는 그 내용에 대한 분명한 기술이 있어야 합니다. 안전: 각 팀은 Wiki 페이지에 있는 안전 관련 질문에 대해 답해야 합니다. 프로젝트 프리젠테이션: 모든 팀은 6월에 있을 짐보리에 참석해야 합니다. 각 팀은 20분에 걸친 프리젠테이션과 프로젝트 포스터를 제출해야 합니다.사회 참여: 모든 참가자들은 사회에 긍정적 기여를 해야 할 의무가 있고 프로젝트를 아주 재미있게 즐겨야 합니다. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/h_iges guidelines From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun WHO Biosafety Level 3 Guidelines With a concrete understanding that MRSA can be a very serious threat to public health as well as the researchers’ safety, we have followed the most strict rules and guidelines during the experiment. Since MRSA pose an increased risk of aerosol spread, we have observed the strengthened safety guidelines that the lab of Gacheon Medical School implements. Some of our experiments are categorized as WHO (World Health Organization) Biosafety Level 3. MRSA is categorized as WHO Risk Group 3 (High individual risk, low community risk: A pathogen that usually causes serious human or animal disease but does not ordinarily spread), so all experiments involved MRSA must be conducted in labs of Biosafety Level 3 (or higher). We followed the WHO Laboratory Biosafety Manual in addition to the national standards. We followed the guidelines for WHO Biosafety Level 3 labs when conducting experiments involving MRSA, but we used guidelines for WHO Biosafety Level 2 labs when conducting experiments not involving MRSA. All WHO biosafety Level 2 guidelines overlapped with the general lab safety rules we followed, so I did not include any Biosafety Level 2 guidelines here. Also, I did not include any Biosafety Level 3 guidelines that overlapped with the general lab safety rules. The international biohazard warning symbol and sign displayed on laboratory access doors must identify the biosafety level and the name of the laboratory supervisor who controls access, and indicate any special conditions for entry into the area, e.g. immunization. Door with Biohazard symbol We cannot access the lab without supervision of the instructor or mentor. When we conduct Biosafety Level 3 experiments, we must wear wrap-around gowns, scrub suits, or coveralls, head covering, and shoe covers. We must take off the laboratory protective clothing after the experiments and send it to the decontamination facility in the Gacheon Medical Center. Double doors Student in front of double doors Student experimenting in biosafety cabinet under safety regulations above. Open manipulations of all potentially infectious material must be conducted within a biological safety cabinet or other primary containment device. Biosafety cabinet Laboratory Design and Facilities Isolation of laboratory Room sealable for decontamination Ventilation –inward airflow, controlled ventilating system, HEPA-filtered air exhaust Double-door entry Anteroom Effluent treatment Autoclave –on site, in laboratory room, double-ended Biological safety cabinets Personal safety monitoring capability Laboratory Equipment Biological safety cabinet –Aerosol and spatter : Maximum containment, provides product protection if laminar flow air is included. Negative pressure flexible-film isolator-Aerosol and spatter: Maximum containment. Spatter shield-Spatter of chemicals: Forms screen between operator and work. Pipetting aids-Hazards from pipetting by mouth,e.g. ingestion of pathogens, inhalation of aerosols produced by mouth suction on pipette, blowing out of liquid or dripping form pipette, contamination of suction end of pipette. Loop micro incinerators, disposable loops-Spatter from transfer loops: Shielded in open-ended glass or ceramic tube. Heated by gas or electricity, Disposable, no heating necessary. Leak-proof vessels for collection and transport of infectious materials for sterilization within a facility: Aerosols, spillage and leakage: Leakproof construction with lid of cover, Durable, Autoclavable. Sharps disposal containers-Puncture wounds: autoclavable, robust, puncture-proof. Transport containers between laboratories, institutions-Release of microrganizms: robust, watertight primary and secondary containers to contain spills, absorbent material to contain spills. Centrifuges that need additional containment accessories such as safety buckets or containment rotors. All the above safety level guidelines for the laboratory equipment were met. Health and medical surveillance 1. All members shall have submitted recording of a detailed medical history and targeted physical examination before being permitted to do lab work. 2. We have to carry medical contact that contain name and contact with photo. Name tag (Front) Name tag (Back) ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/s_guidelinurpose From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Purpose The purpose of the project is to investigate the multidrug resistance of MRSA and the genes involved. By investigating the multidrug resistance of our MRSA and comparing it to the known drug resistance of MRSA, we can find out if our method is valid. If the experimental results disagree with the results found in other experiments, we can conclude that either our method was wrong, or our MRSA was not properly prepared, or that the experimental results are correct in this case. To determine this, we will have to repeat the experiment. Once we finish that, we can investigate the DNA of the MRSA. By running the DNA through gel electrophoresis, we can get a rough idea of the DNA involved in the multidrug resistance of MRSA. Hopefully, this will lead to a development of a drug that MRSA is sensitive to. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/p_purposFrom 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Elan Vital Investigating the multidrug resistance MRSA and the genes involved Project Team Profile Safety Questions Our Sponsors ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korehttp://2014hs.igem.org/wiki/images/7/77/Elanvital_logo.pngl gel electrophorosis From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Gel Elecrophorosis a. Put 1g of Nusieve® GTG® Agarose in an Erlenmeyer flask. b. Put 1g of Seakem® LE Agarose in the same Erlenmeyer flask. c. Add 1 ×TBE until it reaches the 100ml mark on the Erlenmeyer flask. d. Heat in microwave oven for 2 minutes. e. Cool the gel slightly, then pour it into the gel electrophoresis mold. f. Add the comb to the rectangular mold so that the gel forms wells. g. Load 3μl of the marker* into the first well. h. Mix 3μl of each sample with stain** and load into the other wells. i. Connect the circuit and confirm that the mixture moves down. j. Put in Gel image analyzer and observe results. * marker: ‘100bp DNA ladder’ from Invitrogen. ** Safe-GreenTM from abm. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/l_gel_electrophorosn overview From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Jan.9 - Jan 11 Ye Eun Lee met with a participant of IGEM 2012 and was informed IGEM. She contacted high school students who were interested in biology to form a team. Jan.12 - Jan 18 Elan Vital has formed for 2014 IGEM HS competition and started to search a lab and an instructor who will give them directions and guidelines. Jan.26 - Feb.1 Elan Vital found professor Hwang’s lab in Gacheon university. Elan vital team is organized to participate in the iGem jamboree, and created the Facebook group. Feb.16 - Feb. 22 Elan vital team had a kick-off meeting at Gacheon University, where the project, experiments and study topics were decided upon, and the general safety guidelines were given. Elan Vital decided to investigate MRSA. Feb.23 - March 1 The team searched for essays on MRSA, and learned about MRSA by reading and discussing an article: “MRSA – what is it, and how do we deal with the problem?” March 2 - March 8 The team made some LB plate with antibiotics included to test for resistance in bacterium. We used stock solutions with concentrations of 100mg/μl of ampicillin, 10mg/μl of gentamycin, 50mg/μl of kanamycin, and 5mg/μl of tetracycline. We then made LB plates with 1/1000 concentration of stock solutions. The team made the Elan Vital logo, and designed and made the team T-shirt. March 9 - March 15 The team studied how the antibiotics concerned (ampicillin, gentamycin, kanamycin, and tetracycline) works. The team tested the MRSA for antibiotic resistance in the LB plates with antibiotics by placing the MRSA on the plate with the antibiotic, then observing its growth. March 23 - March 29 The team extracted MRSA DNA via miniprep and transformed the competent E. coli with it. We then tested the transformed E. coli for antibiotic resistance by placing the E. coli on a LB plate with antibiotics, then observing tis growth. We also tested the antibiotic resistance of the competent E. coli by the same method.To learn more about the drug resistance of MRSA, we researched by reading the paper “Antimicrobial resistance: the example of Staphylococcus aureus.” The team informed the Bugil international class about MRSA and iGEM. March30 - April 5 The team researched the dangers of MRSA, and then did a brief presentation on the dangers of MRSA in a nursing home, where MRSA could pose a dangerous threat. The team talked about future plans, and had a party at a local restaurant. April 6 - April 12 The instructors only let us handle safe experiments only involving the plasmid DNA of MRSA (instead of the actual MRSA), but we wanted to have a first-hand experience of the experimentation, so we investigated the safety regulations of iGEM, WHO, and the Gacheon university safety committee. April 13 - April 19 The team held a presentation about iGEM, the Elan Vital team, and the dangers of MRSA in seoincheon high school. April 27 - May 3 The team grew MRSA first hand and tested it for resistance against Ampicillin Gentamycin, Tetracycline, and kanamycin. May 4 - May 10 The team preformed miniprep on the bacteria that survived the antibiotics, and transformed the competent E. coli with the MRSA DNA. We then tested the transformed E. coli for antibiotic resistance. We also tested the antibiotic resistance of the competent E. coli as control. The team learned about the primers that are used in PCR. May 18 - May 24 The team preformed miniprep on the transformed E. coli that survived, and preformed PCR using the DNA from the MRSA that survived initially, and the DNA from the transformed E. coli that survived. We then preformed gel electrophoresis with the DNA that amplified sufficiently. June 1 - June 7 The team held a conference at a restaurant in Cheonan, Cheongmaegol, where the results of the experiment are and ideas for the team video are discussed. The team held public awareness event at Goyang Bus Terminal, where we explained the danger of MRSA and introduced our team: Elan vital. June 8 - June 14 The team have made a short video introducing the Elan vital team. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/n_overviet overview From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Team Profile Élan vital means ‘vital force’ in French. A French philosopher Henri Bergson used it to explain the self-organization and spontaneous morphogenesis of complex organisms. Élan vital is a club of high school students interested in synthetic biology. The club was founded in January 2014, by five members from five different schools in Korea: Ye Eun Lee, senior of Bugil GLP, Ji Hoon Kim, Junior of CSIA, Soo Jung Lee, junior of HUFS, Ji Yong Hong, Junior of Sunrin Internet High School and Young Chan Kim, Sophomore of Stanford University OHS. This year, we have conducted MRSA research project, guided by Professor You-Jin Hwang of Gacheon Medical School. We are blessed to have an opportunity to learn from one of the best minds in the medical community of Korea, and to use the labs and cutting-edge equipment of the excellent medical school such as PCR Machine, Shaking Incubator, Centrifuge, and Mechanical Pipette. This is our first year, but we have big dreams. We will be one of the best high school clubs in Korea for kids who have deep interest and talent in the synthetic biology. And, we are committed to continue participating in iGem events and activities. Our Intro Video Members Ye Eun Lee My name is Ye Eun Lee. I am a senior in Bugil Highschool Global Leaders Program. My biggest stress reliever is dance. I believe that dance brings positive energy and boosts endorphin to people. Besides dancing, I like taking care of animals, especially sick ones. I have raised various kinds of animals: rabbits, hamsters, quails, dogs, cats, frogs, and silkworms. I feel great joy when I see them happily running here and there after they recover. This explains why I want to be a doctor. My ultimate goal is to bring bright energy and life to any living creature. Therefore the term “Elan Vital” is a special term for me in that the term is deeply related to life and health of living organisms. I think our team Elan Vital, through the experiments and research, will widen the understanding of a particular topic in the medical field and therefore increase the public’s interest to the people’s health. Finally, we could get closer to the “vital force” of living organisms. It was a great chance for me to work and run experiments with such advanced equipment in a lab in medical university. One thing that was as impressive to me as the great chance of using professional apparatus was the wonderful teamwork our team showed. Although we attend different schools and have different schedules, not even one of the teammates work lazily for Igem. I think our results and wiki demonstrates how hard and well we worked as a team. Jihoon Kim I am Jihoon, junior of CheongShim International Academy(CSIA). I am interested in physics and mathematics and I like to play basketball and play the violin when I have free time. As a member of Elan Vital, I always enjoy having lab experiments with my teammates. During lab experiments, all members get to directly participate in the experiment and experience special things you can’t learn by reading books. Also, not only in academic perspective, I learned the way to reach harmony within the group and to succeed as a team. For me, Elan Vital is a great school which teaches me valuable lessons. The most memorable moment I remember during experiments was when I first met the members of Elan Vital. At the first meeting, I was anxious about the harmony of this group because every member seemed so different. It seemed that we had different ages, schools, interests, and perspectives. However, when we had continuous meetings, we learned to cooperate, and understand each other. It is amazing for me that we have united as a group of Elan Vital. Young Chan Kim My name is Young Chan Kim, and I am sophomore of the Stanford Online High School, a very special school. You don’t need to go to school because my school is virtual. Although it is a little challenging, the new online environment and the course are really appealing, and besides, who doesn’t love a little challenge? In my free time, I enjoy reading books and also love playing with my dog. His name is Sunday, and he is a cute 1 year old golden retriever. When I play the viola, Sunday likes to howl along to the tune. I also like watching movies. And I really enjoy art and love listening to and singing music. I also play the ice hockey. I’m quite a good defender. My favorite subjects are math and science: particularly physics, biology, and maybe chemistry. I plan on going into the sciences later, which is why I thought it was a good idea to participate in iGEM now so I can hit the ground running. Since I was little, I was interested in the sciences. Not only because I was good at them, but because I loved the motivation of science: to find out how things work, then to use that knowledge to help to make a world a better place. In that sense, iGEM was really good for me. Through iGEM, I learned how to use the machinery and equipment, and learned how to avoid biological health risks when dealing with dangerous biological organisms. Most importantly, I learned how to work together and cooperate with team members. I’ve worked with teams before, but in prior projects, the teachers mostly told us how to do things, and we only needed to follow the instructions carefully. Now, the team decided how to do things, and I learned a lot about how to cooperate with the team in an independent research project like this one. Soojung Lee I am junior of Hankuk Academy of Foreign Studies. During my spare time, I enjoy reading, watching movies or playing Baduk, an Eastern board game that requires a great level of concentration and tenacity. I also like to play the violin and piano, because these musical instruments do a great job in helping me relieve my stress. Ever since I was young, I have been interested in many areas rather than be focused on one specific subject of interest. I believe that studies of the sciences allows me to have a more logical and analytical understanding of all sorts of natural phenomena and principles, whereas studies of the humanities field enables me to truly understand the world I am living in, and have a better insight of the society and people. Yet, among this wide variety of areas of great appeal, I am driven to pursue a future related to science, especially material science and engineering. Just by looking at our surroundings, we can perceive that all objects have materials at their basis, and those materials are what account for the objects’ properties. Even when creations are made, the key focus today is which material guarantees greatest strength and durability at high temperatures. Fascinated at how the slightest change in materials can lead to a significant improvement of the properties and effectiveness of objects, I hope to become a researcher of that field, focusing on composite materials that include fiber, ceramics, metal, and many more. Ji Yong Hong I am Junior at Sunrin Internet High School. In our school, we learn various computer programming related subjects in web development division. My hobbies are cardistry, and skateboarding. I've been cardistry for about an year and skateboarding for about 3 months. I also like to listen to musics, typically korean hip hops. Being a team member of Elan Vital in iGEM competition has set my next milestone. I had a great chance to meet new friends from different schools and experiment in labs. I really enjoyed working with my friends on wiki, which explains that my future career is to be a web developer, I guess. Making a wiki page was little differ from what I have been coding before, so My friend, a designer, and I had to think of different layout for it. Having these kind of experience made me more professional at what I do, and I wish I could get more of them. Yae Sung MunCoach My name is Yae Sung Mun and I am a graduate student of Gachon Universiy. My life-long pursuit is synthetic biology and currently majoring bio-nano technology. I am really happy to be part of iGem and thankful to have an opportunity to work with Elan Vital. All of them are self-motivated, hardworking future scientists and we had fun during all the way along reaching iGem Jamboree. I hope I can continue to work with Elan Vital and iGem in the future. You Jin HwangInstructor When I first encountered iGem, I was very delighted for the existence of this wonderful community. As a scientist and professor, I am keenly aware of the great potential of synthetic biology for the well-being of humanity. I am very pleased to see that my team, Elan Vital, consistently shows enthusiasm and dedication to the research project, which can be difficult for the high school students. I believe that this is the first step for them to grow to be good scientists who have great heart and sharp technology as well as intellectual discipline. I feel rewarded to witness their growth throughout the process of lab experimentation and research activities. Also, I am impressed with their interests in the society demonstrated in the works of their human practice. Attributions Lab Members: All Elan Vital members have participated in all lab experiments and discussed and recorded the results with full cooperation. Ye Eun Lee, Ji Hoon Kim, Soo Jung Lee, Ji Yong Hong, Young Chan Kim. Research Activities: All Elan Vital members have participated in the research activities, studying, analyzing and sharing opinions. Wiki Page Development: Ji Yong Hong has led the development of Wiki Page and other members have helped him by providing contents and opinions of the design and implementation of it. Presentation Drafting: Young Chan Kim has led the development of presentation material and other members have contributed to it by giving their feedback and opinion. Public Awareness: Ye Eun Lee has taken initiative in the public awareness campaign to develop information leaflet and lead the distribution of it whenever and wherever possible. She encouraged all members to make general public aware of the risk of MRSA infection by distributing information leaflet on streets and visiting nursing homes. Human Practice: Soo Jung Lee has led the project to visit hospitals and private clinics to explain the hazard of MRSA and to promote thorough treatment of it. Promotion & Sponsor Recruitment: Ji Hoon Kim has taken the lead of promoting iGem by sending information material to many high schools in Korea and contacting possible sponsors. Through well-design plan and presentation, he has greatly contributed to the securing of 5 sponsors. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/t_overvies elan vital safety rule From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Elan Vital Safety Rule Since our lab has been conducting dangerous germs, we have strictly followed domestic regulations established by Korea Centers for Disease Control and Prevention, and Korea Society of Infectious Disease. Thanks to the carful implementation of the safety rules, we have no infection-related health-threating cases reported since our lab has been established. Gacheon University Medical Center has its own biosafety committee and conducts regular investigation for the implementation of safety rules and guidelines. During the audit and investigation, the committee evaluates that all national rules and regulations as well as university imposed rules are well kept and present recommendations. Student experimenting under the safety rules above For the high school iGEM team, we specifically established the following rules & regulations: 1. Do not enter the laboratory or work in the laboratory without an instructor or mentor present. 2. Dress appropriately for lab, which may include wearing gown, long pants, gloves, goggles, and closed shoes. No loose closing. Long hair should be tied back. 3. Do not take food, drink, gum or cosmetics into the lab. 4. Wear appropriate eye protection at all times and inform the instructor if you wear contact lenses. 5. Familiarize yourself with the location and usage of safety equipment in the lab. 6. Notify the instructor or mentor immediately of any unsafe conditions you observe. 7. Always engage in safe behavior in the lab (no playing arond, running, yelling, or throwing things, etc.) 8. Perform only experiments authorized by the instructor or mentor. 9. Follow all written and verbal instructions carefully. If you do not understand a direction or part of a procedure, ask the instructor or mentor before proceeding. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/s_elan_vital_safety_rull miniprep From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Miniprep a. Transfer a single colony from the transformed E.colli to the LB liquid broth and incubate in shaking incubator for 24 hours. b. Centrifuge said E.colli for 15 minutes at 1,500 rpm. c. Dispose of supernatant. d. Add 300μl of solution I* and vortex. e. Transfer the fluid into 2 EP tubes, 150μl each. f. Add 300μl of solution II** in each EP tube. g. Add 200μl of solution III*** in each EP tube and mix by inverting. h. Centrifuge both EP tubes for 10 minutes at 14,000rpm i. Transfer 550μl of supernatant to column. j. Centrifuge at 14,000rpm for 1 minute. k. Dispose of fluid in collection tube, and add 700μl of wash buffer to column. l. Centrifuge at 14,000rpm for 1 minute. m. Dispose of fluid in collection tube, and add 700μl of wash buffer to column. n. Centrifuge at 14,000rpm for 1 minute. o. Dispose of fluid in collection tube. p. Centrifuge at 14,000rpm for 1 minute. q. Add 50μl of distilled water and centrifuge at 14,000rpm for 1 minute. r. Keep filtered solution. * solution Ⅰ 50mM glucose, 25mM Tris•Cl (pH 8.0), 10mM EDTA (pH 8.0) ** solution Ⅱ 0.2N NaOH, 1% SDS *** solution Ⅲ 5M potassium acetate, glacial acetic acid, H₂O ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/l_minipreh promotion From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Promotion of Information Sharing in Hospitals According to a report released by medical news today, around 94,360 invasive MRSA infections are diagnosed annually in the US, with 18,650 associated deaths. Approximately 86% of all invasive MRSA infections are health care associated. In Korea, situation is more serious. Due to the sensitivity of the nature, general hospitals are reluctant to release accurate statistics regarding infection cases and many of the individual clinics do not have proper prevention system. We feel urgency given the current situation to encourage proper management of the infection prevention. By the initiative of Soo Jung Lee, we have visited more than 20 hospitals and clinics and explained the importance of accumulating statistical information and the implementation of proper prevention policy. We have distributed information brochure and requested them to display it so that patients can see it for their interest. Also, Soo Jung conducted an interview with a doctor. It was very useful for all of us to understand the current management status and it was very happy for our team to get support from medical professional in our efforts. Following is the contents of the interview Interview date : May 17, 2014Interviewer : Lee, SoojungInterviewee : Kim, KeonhwaThe Interviewee, Kim Keonhwa, went throught the following education programs: Inje University College of Medicine(2009-Present) Harvard Medical School-Brigham and Women’s Hospital, 221 Longwood Avenue Brigham Dermatology Associates, and Boston Children’s Hospital Division of Rheumatology/Immunology/ Allergy Student Intern (May, 2014) Scheduled MD Degree (Jan, 2015) Question: I have heard there were frequent incidents of infection inside hospitals. Is this true? Yes, infections that occur inside the hospital environment or any type of health care providing service centers are common and also a major problem both inside and outside of the US. The incidents that happen inside these environments are what we call hospital-acquired infections(HAI). There are many types of HAI that are classified by the route of transmission. The most common types, just to name a few, are blood stream infections(BSI), pneumonia, urinary tract infections(UTI) and surgical site infections(SSI). Blood stream infections are mostly due to the excessive drawing of blood for laboratory studies of patients. Contaminated needle tips or insufficient sterilization of the blood-drawn area are the most common causes. Pneumonia is usually caused by patients that are under bed-ridden states with ventilator association. The decreased breathing functions causes aspiration of foreign bodies into the respiratory tract. Urinary tract infections are mostly caused by catheter insertions to patients who have impaired bladder functions. Last but not least, surgical site infections are mostly due to insufficient sterilization and contaminated dressing procedures. The US Center for Disease Control(CDC) estimates around 1.7 million HAI and a total of almost 100,000 deaths due to HAI per year. This is a major problem that is preventable by physicians and health care professionals and should never be overlooked. Question: In case such things happen, what kind of action takes place in the hospital? Sterilization and antibiotic injection is what is mostly done to patients. Most patients recover from these infections if they are not in an immunosuppressed state or have severely impaired immunologic functions. However, the major problem is not only the recovery of infected patients but also the advent of microorganisms that are more highly resistant than the previous types. A couple of examples would be VRSA(Vancomycin Resistant Staphylococcus Aureus) and CRE(Carbapenem Resistant Enterobacteriaceae) which I, myself, have also seen in my university hospital Medical Intensive Care Unit(MICU). If such infections happen, the whole hospital floor or unit is shut down and access is restricted to everyone but health professionals. It is always safest to prevent any HAI by frequent hand washing and preventing redundant contact to patients with any contaminated object. Question: What should the public and government do to prevent reoccurrence? The public should always be aware that infections can be possible in any type of place or situation. The first step is to always keep your hands clean by washing them frequently and thoroughly with antibacterial sanitizers and minimizing infectious contact to others and yourself. The government should always sufficiently subsidize the Center for Disease Control and keep a statistically detailed track of the incidents and death rates due to HAI. Physicians and health care professionals should always be self-conscious about their own hygiene before encountering anyone in the hospital. Abiding by these basic sanitation rules will significantly decrease the rate of any type of infection inside and outside of the hospital. Question: What kind of advice would you give to Lee, Soojung for her future as a medical scientist? First, I would like to say it was a pleasure having this interview with you and I appreciate you taking medical advice from me. There are many different paths in pursuing a career in medical science, it can be a physician who works in hospitals and takes care of patients, a scientist who does research in laboratories for medical technology, a physician assistant, a nurse, an emergency medical technician and etc. But regardless of the field of study you may major or job you may have, anything that is related to medicine or medical science should be for the care and good of the people in need of help, patients. There is a short and famous saying for health care professionals : “Do no harm.” Which means, before you can treat or make a patient feel better, you should at least not do anything that aggravates the patient’s situation. That directly refers to the issue we have just talked about, hospital-acquired infections. Live by this rule, and you’ll see yourself positively contributing to yourself and the people around you in the world of medicine. There’s always much more to say, but I think it’s best to keep an advice simple for it to be followed. Thank you, and good luck with your future. Promotion in hospitals In addition, Ji Hoon Kim visited hospitals in Atlanta, Georgia when he came there and met with local medical professionals to know about the infection control measures. His input was very valuable for us to understand that there exists a big gap in the country specific policies and make us think about the necessity of establishing global standard and management system. Promotion in Crestwood Medical Center Young Chan Kim wrote an essay about MRSA infection statistics in Korea to make a better understanding on the current status of MRSA control system. Introduction MRSA stands for Methicillin Resistant Staphylococcus Aureus. Methicillin is a type of drug used commonly to cure infections, and MRSA is a strain of Staphylococcus that is resistant to Methicillin. MRSA can be quite dangerous, so it is a focus of medical attention today. It can cause a number of infections on soft skin, and can cause death. MRSA also generally exhibits multi-drug resistance in addition to methicillin resistance. In some studies, it was suggested that MRSA acquired from hospitals were more dangerous than MRSA imported from the general public. When I heard this claim, I was interested because that would mean that hospitals are more dangerous for the disease. To test this, I will compare the drug resistance of acquired (in hospital) MRSA and imported (from the general public) MRSA. I expect that acquired MRSA will show more drug resistance than imported MRSA. Materials and Methods Getting data for this project was nearly as impossible, because most of the data were classified for national security reasons. In the end, I asked one of my neighbors who is a medical doctor for help, and acquired several reports of collaborative researches by hospitals on MRSA sponsored by the government: Korea Center for Disease Control and Prevention and Korean Society for Nosocomial Infection Control. It had some raw data I could use. The data was collected from a number of local and general hospitals. To distinguish between imported and acquired MRSA, they counted all MRSA found within 72 hours of hospitalization of the patient to be “imported,” and all MRSA found after 72 hours of hospitalization of the patient to be “acquired.” I ran the Chi-square tests and regression analysis on them. Note that these reports were counted as classified, so you might not be able to access these. Data I acquired a table with data for MRSA resistant to different kinds of drugs. All the data came from the sample collected by the hospitals involved in the study. The total sample was 733 for Acquired MRSA and 354 for Imported MRSA.In this analysis, the source of the MRSA is the independent variable, with nominal scale data, and the percentage of MRSA resistant to drugs is the dependent variable, with ratio scale data. Resistance to drugsAcquired MRSAImported MRSACiprofloxacin457167Tetracycline329118Clindamycin442163Sulfamethoxazole3816Total733354 Statistical Analysis For the project I used Chi-square analysis and Regression analysis. Since I think that Acquired MRSA is more dangerous than Imported MRSA, I hypothesized that Acquired MRSA will also show higher amounts of drug resistance than Imported MRSA. The data and the ratios that resulted from the data seems to suggest this is true, but to make sure this isn’t because of sampling error or bias from personal opinion, I need to run Chi-square tests for association. Regression analysis helps create possible models for linear relationships, and can predict what we would see if we collected some more data. Normally, I would not use regression analysis for such a small set of data, but the Acquired Resistance Rate Vs the Imported Resistance rate ratio stays almost exactly the same for almost all of the data, and since it would make sense that acquired MRSA resistance rate ad imported MRSA resistance rate would be directly related, I found a mathematical model connecting the two values using regression analysis, particularly model 2 regression analysis. First, I noticed that the ratio of resistant MRSA seemed to be higher for Acquired MRSA then Imported MRSA. Therefore, I ran Chi-square test for associations for the 4 different drugs tested. For the different number of drug resistant MRSA, I calculated the number of MRSA sensitive to the drug by subtracting the number of MRSA resistant to the drug from the total. Then I ran Chi-square tests for association for all the different types of drugs. CiprofloxacinAcquired MRSAImported MRSACiprofloxacin Resistant457 (0.6235)167 (0.4718)Ciprofloxacin Sensitive276 (0.3765)187 (0.5282) H0: There is no statistical significance, so there is no difference in the Ciprofloxacin resistance level of imported and acquired MRSA. HA: There is statistical significance, so there is a difference in the Ciprofloxacin resistance level of imported and acquired MRSA. Since the table is 2x2, we have to use Yate’s correction. The degree of freedom is 1. The chi-square value is 22.47>3.84=critical value for 95% confidence. Therefore reject H0, accept HA. There is a difference in the Ciprofloxacin resistance level of importance and acquired MRSA. TetracyclineAcquired MRSAImported MRSATetracycline Resistant329 (0.4488)118 (0.3333)Tetracycline Sensitive404 (0.5512)236 (0.6667) H0: There is no statistical significance, so there is no difference in the Tetracycline resistance level of imported and acquired MRSA. HA: There is statistical significance, so there is a difference in the Tetracycline resistance level of imported and acquired MRSA. Since the table is 2x2, we have to use Yate’s correction. The degree of freedom is 1. The chi-square value is 13.15>3.84=critical value for 95% confidence. Therefore reject H0, accept HA. There is a difference in the Tetracycline resistance level of importance and acquired MRSA. ClindamycinAcquired MRSAImported MRSAClindamycin Resistant442 (0.6030)163 (0.4605)Clindamycin Sensitive291 (0.3970)191 (0.5395) H0: There is no statistical significance, so there is no difference in the Clindamycin resistance level of imported and acquired MRSA. HA: There is statistical significance, so there is a difference in the Clindamycin resistance level of imported and acquired MRSA. Since the table is 2x2, we have to use Yate’s correction. The degree of freedom is 1. The chi-square value is 19.65>3.84=critical value for 95% confidence. Therefore reject H0, accept HA. There is a difference in the Clindamycin resistance level of importance and acquired MRSA. SulfamethoxazoleAcquired MRSAImported MRSASulfamethoxazole Resistant38 (0.0518)16 (0.0452)Sulfamethoxazole Sensitive695 (0.9482)338 (0.9548) H0: There is no statistical significance, so there is no difference in the Sulfamethoxazole resistance level of imported and acquired MRSA. HA: There is statistical significance, so there is a difference in the Sulfamethoxazole resistance level of imported and acquired MRSA. Since the table is 2x2, we have to use Yate’s correction. The degree of freedom is 1. The chi-square value is 0.22 Therefore accept H0. There is no difference in the Sulfamethoxazole resistance level of importance and acquired MRSA. (But I suspect this might be type II error resulting from a sample size not big enough.) Therefore, I concluded that Acquired MRSA generally had more drug resistance then Imported MRSA. After I conducted the Chi-square tests, I noticed that the ratio of resistant Imported MRSA VS the ratio of resistant Acquired MRSA seemed to be constant. Therefore, I ran a regression analysis. Since there were only 4 sets of data, it might not have been such a good idea, but the ratios were almost exactly the same for every drug except for Sulfamethoxazole, which seemed to suggest strong correlation. Since the data was count data, I cannot run any correlation coefficient tests, so I went straight for the regression analysis. Since neither of the variables was controlled and there was no clear dependence relationship, I used model 2 regression. The Standard Deviation of Acquired MRSA: 0.2650 The Standard Deviation of Imported MRSA: 0.1985 b=0.1985/0.2650=0.7491 The mean of Acquired MRSA: 0.4318 The mean of Imported MRSA: 0.3277 Therefore, the regression line is y=0.7491x+0.0042 Discussion I concluded that in terms of drug resistance, Acquired MRSA has a higher rate of drug resistance then Imported MRSA, and is more dangerous, which fit my original hypothesis. I also found the regression line to be y=0.7491x+0.0042, and the data fit very well with the regression equation. If I could do things differently, I would have preferred to get some more raw interval/ratio data about MRSA so that I could analyze the results more closely. Analyzing the data made me want to analyze the effect of MRSA on other drug resistance levels. Does Acquired MRSA always exhibit higher resistance? Or does it happen only with certain drugs? Reference Note: You might not be able to access some of these as they were classified to some extent. But I assure you I didn’t break any laws in accessing these reports. JinHung Yoo. “Analysis of clinical characteristics and assessing the effectiveness of active surveillance/decolonization on the occurrence of nosocomial MRSA”. 2009 Seoul National University Hospital Bundang. “Analysis for changes of epidemiology in Staphylococcus aureus infections and predictive factors for methicillin resistance in Korea”. 2012 Jae-Il Yoo. “Laboratory surveillance system of vancomycin-resistant Staphylococcus aureus”. 2010 Jae-il Yoo, Kisoo Kang. “Characterization of vancomycin intermediate Staphylococcus aureus (VISA) isolated from hospitals”. 2012 Kyungtae Chung. “Monitoring of antimicrobial resistance in clinically important pathogens from non-tertiary hospitals in Korea”. 2011 ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/h_promotioresults and conclusion From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Results and Conclusion We first grew the MRSA in liquid LB broth, and then placed the MRSA in solid LB plate with a variety of antibiotics, and observed its growth. This is the resulting growth of the MRSA. Top petri dishes are the resulting growth of MRSA from patient 7, and the bottom petri dishes are the resulting growth of MRSA from patient 8. The antibiotics used are tetracycline, kanamycin, gentamycin, and ampicillin, left to right. The results show that MRSA has resistance to gentamycin, ampicillin, and kanamycin, but slight or no resistance to tetracycline. Then we wanted to transform the E. coli with the DNA from the MRSA and test its resistance, but first, we had to test the original E. coli in the drugs, as without that, we wouldn’t know if the drug resistance was from the MRSA DNA, or from the E. coli. We placed the E. coli on a LB plate with a variety of antibiotics and observed its growth. This will be the control of this section of the experiment. This is the resulting growth of the E. coli. The results show that the E. coli has no resistance to gentamycin, kanamycin, ampicillin, and tetracycline. Growth of E. coli 24 hours it was placed in different antibiotics (top left: kanamycin, top right: gentamycin, bottom left: ampicillin, bottom right: tetracycline.) with 1/1000 concentrations. Then to test the drug resistance of the transformed E. coli, we placed the E. coli in a LB plate and observed its growth. This is the resulting growth of the transformed E. coli. E. coli transformed with the DNA of the MRSA from patient 7 grown in ampicillin survived in ampicillin E. coli transformed with the DNA of the MRSA from patient 7 grown in kanamycin survived in ampicillin E. coli transformed with the DNA of the MRSA from patient 7 grown in tetracycline survived in ampicillin E. coli transformed with the DNA of the MRSA from patient 7 grown in gentamycin survived in ampicillin E. coli transformed with the DNA of the MRSA from patient 8 grown in gentamycin survived in kanamycin E. coli transformed with the DNA of the MRSA from patient 8 grown in gentamycin survived in gentamycin E. coli transformed with the DNA of the MRSA from patient 8 grown in kanamycin survived in gentamycin E. coli transformed with the DNA of the MRSA from patient 8 grown in kanamycin survived in kanamycin Then we wanted to analyze the DNA. To do that, we preformed PCR with a variety of primers and a variety of DNA. PCR includes florescent dye that activates when it attaches to DNA, so the PCR machine can measure the progress of PCR by measuring the fluorescence. These are the resulting graphs. PCR Program PCR lane placements The amplification chart from the PCR process. The top graph represents lanes 2-4, and the bottom graph represents lanes 6-8 The graph is the amplification chart of the DNA. The x-axis is the number of cycles the DNA went through, and the y-axis is the amount of DNA measured by the fluorescence level. A successful PCR starts to grow exponentially at about 20-25 cycles, while flat lining curves represent DNA that is not copied very much. The melt curves and melt peaks. The top graphs represent lanes 2-4, and the bottom graphs represent lanes 6-8. The left graphs are the melt curves, and the right graphs are melt peaks The graphs are the melt curves and the melt peaks. The x-axis is the temperature, and the y axis is the florescence level which represents the number of associated double strand DNA (as opposed to disassociated single strand DNA). By taking the first derivate of the melt curve, we can get the melt peak chart. Melt curves with steep slopes mean there are more DNA there to be disassociated, which means PCR was successful. Since the melt peaks are the first derivation of melt curves, high melt peaks mean steep slopes, and therefore successful PCR. We decided to conduct gel electrophoresis on the DNA with the highest melt peaks. Then we analyzed the amplified DNA using gel electrophoresis. The primers used in PCR copies the DNA at different sites, so if the bands from DNA amplified using the same primer results in similar bands, that most likely means that the section of DNA is included in both of the stands of DNA. This most likely means that the section of DNA that is shared in the DNA segments with multidrug resistance could code for multidrug resistance. 7 A, G, K, T: DNA of MRSA from patient 7 grown in ampicillin, gentamycin, kanamycin, and tetracycline, respectively. 8 A, G, K: DNA of MRSA from patient 8 grown in ampicillin, gentamycin, and kanamycin, respectively. 7 GA, KA, TA: DNA of E. coli grown in ampicillin, which were transformed with the DNA of MRSA from patient 7 grown in gentamycin, kanamycin, and tetracycline, respectively. 8 GK, KK: DNA of E. coli grown in kanamycin, which were transformed with the DNA of MRSA from patient 8 grown in gentamycin and kanamycin, respectively. 8 GG: DNA of E. coli grown in gentamycin, which were transformed with the DNA of MRSA from patient 8 grown in gentamycin. The results show that wells 2-8 has about 300 base pairs, which means that most likely the type V primers were active in PCR, and the section of DNA between V-F and V-R with about 300 base pairs were multiplied in PCR of 7A, 7G, 7K, 7T, 8A, 8G, and 8K. This most likely means that 7A, 7G, 7K, 7T, 8A, 8G, and 8K all included the section of 300 base pairs that is between V-F and V-R. So, this 300 base pair long section of DNA has a possibility of coding for multidrug resistance. The results also show that wells 9-11 has about 1,000 and 1,600 base pairs, which means that most likely the primers ccrAB-α3 and ccrAB-α4 were active in PCR, and the section of DNA between ccrAB-α3 and ccrAB-α4 with about 1000 base pairs and the section of DNA between ccrAB-α3 and ccrAB-α4 with about 1,600 base pairs were multiplied in PCR of 7K, 8G, and 8K. This most likely means that 7K, 8G, and 8K all included the sections of 1,000 and 1,600 base pairs that is between ccrAB-α3 and ccrAB-α4. So, this 1,000 base pair long section of DNA and the 1,600 base pair long section of DNA has a possibility of coding for multidrug resistance. The results show that wells 12-17 has about 150 base pairs, which means that most likely the primers MecA147 or the primers mecI were active in PCR, and the section of DNA between MecA147-F and MecA147-R or the section of DNA between mecI-F and mecI-R with about 150 base pairs were multiplied in PCR of 7GA, 7KA, 7TA, 8GK, 8GG, and 8KK. This most likely means that 7GA, 7KA, 7TA, 8GK, 8GG, and 8KK all included the section of 150 base pairs that is between MecA147-F and MecA147-R or the section of 150 base pairs between mecI-F and mecI-R. So, this 150 base pair long section of DNA has a possibility of coding for multidrug resistance. The investigation showed that MRSA showed resistance to some of the antibiotics, and some of the transformed E. coli showed antibiotic resistance. That most likely means that the DNA that codes for antibiotic resistance was successfully transported into the E. coli that survived the antibiotics. By running PCR, we were able to amplify some of the DNA in the drug resistant MRSA and transformed E. coli. Since the section of DNA that codes for the multidrug resistance is most likely shared in all the bacterium that survived the antibiotics, by analyzing the DNA through gel electrophoresis, we were able to get an idea about which section of DNA was shared. If possible, the next step forward would have been to analyze the sequence of the section of DNA, but we were not able to precede that far. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/p_results_and_conclusios answers From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Safety Questions Answers Would any of your project ideas raise safety issues in terms of: researcher safety public safety environmental safety Guidence There are three recommended steps in addressing this question. 1. To start, please list organisms you are using and organisms from which your parts are derived, indicating the risk group or biosafety level for each. For help, see Table 1 and 2 of the World Health Organization (WHO) Laboratory Biosafety Manual. You are welcome to use your national standards if you prefer. If national standards do not use the WHO 1-4 scale, please provide a link to an explanation of your standards. We used MRSA (WHO Biosafety Level Risk Group 3) and E. coli (WHO Biosafety Level Risk Group 2) 2. Then consider risks to team members, publics and environment if the project goes according to plan. Please describe risks posed by lab equipment and chemicals as well as biological parts and organisms. How are you addressing these issues in project design and lab work? Have you received biosafety training and other laboratory safety training? If so, please briefly describe the training. If everything goes as planned, there should be minimal biological risk to team members, public, and the environment. 3. Then consider risks to team members, publics and environment if the project does not go according to plan. What are risks if safety measures such as containment procedures go wrong and organisms or parts are released? What are risks to security from malicious misuse? How are you addressing such risks? If things do not go as planned, there could be serious biological risk to team members, but minimal biological risk to the public and the environment. E. coli is categorized as WHO risk group 2, so is unlikely to be a serious health hazard to team members, and does not pose a serious biological risk for the public and the environment. But MRSA is categorized as WHO risk group 3, so it could cause serious health issues for tean members, but does not pose a serious biological risk for the public and the environment. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? If yes Did you document these issues in the Registry? How did you manage to handle the safety issue? How could other teams learn from your experience? Guidence Please reference the biosafety level of parts. If you are working with anything other than a BSL1 organism, take extra care with this question. Your nation regulates handling and transfer of pathogens and parts associated with pathogenicity. For a list of regulated organisms, see the Australia Group website. We did not use the BioBrick parts, and we did not make any devices for the project. We did not have any safety issues. We worked with BSL2 and BSL3 organisms, so we took extra care with the experiments involving these organisms. Look above for detail. Is there a local biosafety group, committee, or review board at your institution? If yes, what does your local biosafety group think about your project? If no, which specific biosafety rules or guidelines do you have to consider in your country? Guidence The iGEM Safety Committee is not a substitute for national and local university institional biosafety committees 1. Does your university have a Biosafety Committee or equivalent? Please provide a link to regulations and local requirements. Gacheon Infection Control Office (Infection Control Committee)http://www.gilhospital.com/eng/e-mail: infe@gilhospital.comphone: 82-32-460-3744 / Fax: 82-32-472-1578 2. Is your project in compliance with national regulations and university requirements? Yes. Look above for university safety guidelines 3. If you are working with any organisms or parts requiring containment arrangements above BSL 1 or equivalent, have you consulted with your Institutional Biosafety Committee regarding your project? Yes. Our instructor, Dr. Hwang, contacted the Gacheon Biosafety committee. They looked at our safety protocols, and found no problems with it: they decided that there should be no significant safety issues as long as we followed the guidelines. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering? Guidence This is an open-ended space for you to consider and suggest ways of improving safety or safety awareness at iGEM and beyond. Some iGEM teams have offered ideas (and sometimes full projects) to limit gene flow, to create software for screening pathogens, and to reduce reliance on antibiotic resistant markers. Other iGEM projects have discussed concerns that might arise if the project succeeded and became widely used, as commercial product or other means of distribution. Some iGEM projects have discussed risks that might materialize if the knowledge generated or methods developed were to become more widely available. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/s_answes first From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Safety First! Saving the world starts with saving yourself. You cannot save the world unless you’re in good shape. Safety rules for MRSA project can be complicated. The safety rules must not be confusing; they should be straightforward, consistent and intuitive to ensure that members follow them, because how can you expect anyone to follow the rules they do not understand? The rule of thumb is: 1. Start with reading Safety Questions for overview. 2. Follow Elan Vital Safety Rules all the time. 3. Before working with MRSA, read WHO BSL3 Guidelines again and strictly follow them. 4. Before entering the laboratory, read GMS General Safety Rules and GMS Extra Rules again and follow them (Remember, this is a real-life medical lab of Gacheon Medical School, with all sorts of cutting-edge, mission-critical equipment. Here, rules are MEANT to be followed!) 5. If you are unsure of the rules or confused, ASK COACH and INSTRUCTOR before doing anything. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/s_firsl transformation From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Transformation a. Take out 50㎕of component cell* kept in 80 degrees below zero and melt it in ice. (We used the compotent cell of One Shot Top 10 Chemically compotent E. coli from Invitrogen.) b. Add 1㎕ of plasmid DNA** to each of the tube. c. Mix well and place them in ice for 30 minutes. d. Place plasmid DNA in the compotent cell and keep it in 42℃ for 30 seconds and place it in ice for 2 minutes. e. Add 250㎕ of liquid LB broth in each of the tube and grow it in 37℃ shaking incubator for an hour. f. Leave 100㎕ and dispose. g. Resuspend the cell, and spread 25㎕ in each of the ampicillin, kanamycin, gentamycin, tetracycline LB solid plate. h. Spread 25㎕ of competent E. coli in LB solid plate with the same antibiotics. i. Wait until the E. coli bacteria smears into the plate. j. Grow it in the 37℃ incubator for a day and observe. k. Go through miniprep again. *We used the competent cell from ‘One Shot® TOP10 Chemically competent E. coli’ from Introgen. **We used the plasmid DNA we got from miniprep previously. How transformation works Incubator ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/l_transformatioh public awareness From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Public Awareness Campaign A medical thriller “Outbreak” written by Dr. Robin Cook is popular among the Korean general public. With the influence of such books and movies, people often develop misunderstandings about the characteristics of pathogens: confusing viruses with bacterium, for example. Only a handful of people know about MRSA, but most people probably heard of the term ‘super bacteria’ but when they hear super bacteria, things like Ebola virus: exotic, fatal, incurable disease causing pathogens. But people rarely think about anything that they might ever be infected by in everyday life. We captured the misunderstanding and lack of information on MRSA and its dangers, and decided to launch a public awareness campaign focusing on enlightening people by giving easy and crucial information about MRSA and methods to prevent infections. Ye Eun Lee, Ji Yong Hong and Young Chan Kim were involved in the public awareness campaign first-hand. We developed pamphlets with short summaries about MRSA, Superbug and simple tips of prevention measures. We visited places where many people gather such as bus terminals and baseball park and schools to inform the public of the dangers of MRSA. Also, we visited a long term care center for the seniors. They live together in the care center, and seniors have weakened immune systems, so they are quite susceptible to MRSA infections. We gave out information about MRSA and prevention actions necessary to prevent infection. We emphasized that simple actions such as regular hand washing can reduce risk of CA-MRSA (community-associated MRSA). We aked the public to perform the following actions on a daily routine. Regular hand-washing. Fingernails cut short. Do not share products such as soaps, lotions, creams and cosmetics with others. Avoid sharing unwashed towels. Avoid sharing personal items such as razors, nail files, toothbrushes, combs or hairbrushes. Introducing MRSA to people FAQ What is the difference between virus and bacteria? Bacteria are unicellular microorganisms typically a few micrometers long. They are included in the category of living organisms. They have all the characteristics of life such as development, reproduction, homeostasis, etc. However, a virus cannot be strictly categorized as a living organism. Although it exhibits some of the characteristics of life such as having genetic materials, it does not show many of the crucial requirements of life such a s growth and development or (independent) reproduction. While bacteria can survive and reproduce on its own, viruses will quickly be wiped out in the absence of host cells. Viruses require host cells in order to reproduce. What is super bacteria? Super bacteria generally refer to pathogenic bacterium that are resistant to many of the standard drugs used in hospitals. Having this reistance makes it harder to cure the bacterial infection with medicine. Because of its ability to survive in the presence of these drugs, it is called a super bacteria. What is MRSA? MRSA, which stands for methicillin resistant staphylococcus aureus, is a strain of the bacteria staphylococcus aureus that shows reistance to beta lactam antibiotics. It is quite difficult to cure because of this antibiotic resistance. It can cause a variety of disease from minor skin infections to serious, life threatening disease. Because of the antibiotic resistance and the difficulty in treating it, MRSA is often called a super bacteria. Who is at risk? MRSA infections are especially common in hospitals. MRSA is easily transmitted by contact, and without the proper safety procedures, MRSA can easily spread to other patients, caretakers, and doctors. Patients often have weakened immune systems, which makes them an easy target for MRSA, and open wounds, where MRSA can infect the open tissue. How do people get infected by MRSA ? The spread of MRSA relies on skin-to-skin contact with an individual who either has MRSA and/or colonized by the bacteria MRSA has an aptitude to survive for extensive periods on surfaces and objects including door handles, floors, sinks, taps, cleaning equipment and fabric. It is very important to maintain thorough contact control and meticulous disinfection procedures to limit spread of bacteria. Inappropriate use of antibiotics can cause MRSA to develop antibiotic resistance instead of curing the disease. Inappropriate use of antibiotics is thought to have initiated the development of resistance. Inappropriate use and prescribing includes: Failing to complete a course of antibiotics as prescribed. Doses of antibiotics being skipped. Failure to take antibiotics at regular intervals. Saving antibiotics for a later date. Unnecessary prescription of antibiotics. Improper use of broad-spectrum antibiotics. Inappropriate selection and dose duration of antibiotics. Signs and symptoms of MRSA MRSA signs and symptoms can vary greatly depending on the situation. Although countless people carry MRSA bacteria in their mucosa (inside the nose), they may never display any symptoms whatsoever. But, MRSA may cause minor skin infections that appear as a bump or sore area of the skin that could be mistaken for an insect bite. The infected area might be red, inflamed, painful, hot to the touch, full of pus or other liquid and accompanied by a fever. MRSA is known to cause more serious problems when it penetrates the bloodstream or deep body tissue. These symptoms include fever, chills, malaise, dizziness, confusion, muscle pains, swelling, chest pains, coughing, breathlessness, headache, rash,etc. How to avoid infections? Wash your hands: Careful hand-washing is your best defense against pathogens. Scrub hands briskly for at least 15 seconds, then dry them with a disposable towel and use another towel to turn off the faucet. Carry a small bottle of hand sanitizer containing at least 62 percent alcohol for times when you don't have access to soap and water. Keep wounds covered: Keep cuts and abrasions clean and covered with sterile, dry bandages until they heal. Naked wounds could provide MRSA easy access to your body, letting them pass the the body’s first line of defence (the skin) with no problem. Also, if you already carry dormant MRSA, The pus from infected sores may contain MRSA, and keeping wounds covered will help keep the bacteria from spreading. Keep personal items personal: Avoid sharing personal items such as towels, sheets, razors, clothing and athletic equipment. MRSA spreads on contaminated objects as well as through direct contact. Shower after athletic games or practices: Shower immediately after each game or practice. Use soap and water. Don't share towels. Sanitize linens: If you have a cut or sore, wash towels and bed linens in a washing machine set to the hottest water setting (with added bleach, if possible) and dry them in a hot dryer. Wash gym and athletic clothes after each wearing. Poster We Used For Public Awareness In Korean 슈퍼박테리아가 무엇인지 아세요? 마이클잭슨이 살아있을 때 감염되었던 슈퍼박테리아! 슈퍼박테리아는 항성제에 내성을 가진 박테리아를 부르는 말인데 슈퍼박테리아에 일단 감염되면 항성제가 소용이없어 치료가 어려운 무서운 균입니다.또 한가지 황당한 점은 슈퍼박테리아는 병을 고치러 가는 병원내에서 감염되는 경우가 많다는 것입니다. MRSA는 슈퍼박테리아 중에도 아주 위험해 지금도 많이 거론되고있는 박테리아의 한 종류입니다. MRSA란 포도상구균중에서 베타-락탐 계열의 항생제에 내성을 갖게된 박테리아를 말합니다. 이 MRSA는 페니실린 계통의 항생제를 포함한 아주 많은 종류의 항생제에 내성을 지니고 있어 치료하기가 까다롭습니다. MRSA는 어떤 박테리아인가? MRSA 균 위의 그림은 에서 보는 것처럼 노란 공처럼 생긴 세균들이 포도송이처럼 뭉쳐 있는 게 황색포도상구균입니다. 이 균은 피부염증이나 폐렴 등을 일으키는데 메티실린이라는 항생제를 넣으면 죽습니다. 그런데 이 항생제에 죽지 않는 황색포도상구균이 MRSA이며 이것이 돌연변이를 일으키면 수퍼박테리아가 됩니다. 한마디로 슈퍼박테리아는 어떤 항생제에도 듣지 않는 세균을 뜻합니다. 수퍼박테리아 감염을 막으려면 우선적으로 MRSA 감염을 막아야 합니다. MRSA는 주로 척추,관절,눈,뇌,심장 등 신체의 무균 부위를 수술하거나 요로·정맥·호흡기 등을 치료하기 위해 인체에 관을 삽입할 때 발생합니다. MRSA 감염경로 MRSA에 감염된 환자 모습 우리나라에서는 MRSA에 감염되어 사망하는 사람이 많을 것으로 추정하고 있지만 아직 병원감염의 부작용을 겪고 있는 환자나 사망자의 수조차 정확하게 파악되고 있지 않은 실정입니다. 따라서 정부는 각급 병원 규모에 맞는 감염관리 프로그램을 실시하고, 현장의 감염관리 실무능력 향상을 위한 교육프로그램을 개발하는 등 의료현장의 역량 강화를 위한 지원을 확충해야 합니다. 병원감염 예방을 위해서는 손소독제 및 보호구 등에 대한 체계적이고 철저한 관리 체계가 수립되어야 합니다. MRSA 감염을 막기 위해선 무엇을 해야 할까요? 제일 먼저 손 씻기가 생활화되어야 합니다. 또한 환경소독을 적절히 실시해서 청결을 유지해야 합니다. 보다 전문적으로는 항생제를 남용하지 않도록 해야하며 감염위험이 있는 환자들을 특별 관리해야 합니다. 특히 MRSA 가 정착되거나 감염된 경력이 있는 환자, 다른병원이나 요양원등에서 이송된 환자, 최근 3개월 이내에 입원한적이 있는 환자 등을 병원에서 특별 관리를 통해 MRSA 감염 방지 노력을 해야 합니다. 뿐만 아니라 중환자실,장기이식 센터, 흉부외과,정형외과,혈관외과, 기타 심각한 MRSA 감염증(균혈증,폐렴,수술부위감염등)이 발생하는곳에 대해서는 의료기관의 철저한 관리가 이루어져야 하고 정부의 지원과 감독이 꼭 필요합니다. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/h_public_awarel pcr From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun PCR a. Add 10μl of syber green to each PCR tube. b. Add 0.3μl of each primer to each PCR tube according to the picture. c. Add distilled water until the mixture reaches 19.5μl. d. Add 0.5μl of sample DNA to each PCR tube according to the picture. e. Go through PCR. How PCR works PCR machine PCR program PCR tube mixture 7 Amp, Genta, Kana, Tetra: DNA of MRSA from patient 7 grown in ampicillin, gentamycin, kanamycin, and tetracycline, respectively. 8 Amp, Genta, Kana, Tetra: DNA of MRSA from patient 8 grown in ampicillin, gentamycin, kanamycin, and tetracycline, respectively. 7 AA, GA, KA, TA: DNA of E. coli grown in ampicillin, which were transformed with the DNA of MRSA from patient 7 grown in ampicillin, gentamycin, kanamycin, and tetracycline, respectively. 8 GK, KK: DNA of E. coli grown in kanamycin, which were transformed with the DNA of MRSA from patient 8 grown in gentamycin and kanamycin, respectively. 8 GG, KG: DNA of E. coli grown in gentamycin, which were transformed with the DNA of MRSA from patient 8 grown in gentamycin and kanamycin, respectively. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/l_pcs general safety rule From 2014hs.igem.org Project Background Purpose Process Results & Conclusion Notebook Protocol MRSA Drug Resistance Miniprep Transformation PCR Gel Electrophorosis Team Human Practices Overview Public Awareness Promotion In Hospitals iGEM Promotion Safety Safety First! Safety Questions Elan Vital Safety Rules WHO BSL3 Guidelines GMS General Safety Rules GMS Extra Rules Fun Gacheon Medical School General Lab Safety Rule We clearly understand that MRSA-related research can be very dangerous. In order to prevent any harmful result, we have implemented the most strict safety rules and guidelines and carefully reassured that all the procedures are well observed. Since our lab is a part of Gachon Medical School & Hospital, we followed the safety guidelines provided by the university. General Safety Rules and Regulations are as follows: Labs should be maintained in clean, organized and orderly manner. All participants including students and instructors must understand and observe safety rules and guidelines. Before the lab activities, lab safety education must be taken in which contents of lab experiments, operation methods of lab equipment, rules and guidelines, and procedures in case of accidents must be properly understood. Everyone must follow instructions of the responsible person in the lab and should not perform any activities which may cause any danger. Everyone must understand emergency behavior guidelines. Emergency includes fire, emergency patient and other dangerous situation. Everyone must know the location of emergent phone calls, fire alarm, and power disconnecting switch, etc. Everyone must understand how to use fire extinguisher and check the status of it. Smoking, eating, drinking, and playing are prohibited in the lab. All participants must be properly dressed in the lab with the safety consideration. They should wear safety goggles, dust & poison protection mask, and gloves, etc. During the operation of the equipment and during the experiment, at least one person should attend and observe the situation. Person who leaves the lab last must make sure that power is disconnected, flammable material is isolated, dangerous material is safely kept, water is disconnected, lab is cleaned and locked, and order of the lab must be double-checked. Power wiring appliance must be properly installed to accommodate the experiments. Cables, and lines must be checked regularly to confirm that they are capable of handling the electrical load. All equipment must be connected with the proper power cable and must not be connected in a multiple power socket. Flammable material must not be used near firearms and must be kept far away from the electric socket, air conditioner and/or heaters. Heaters and other heating devices that are not related to experiments must not be used in the lab. Places where dangerous materials are kept must be identified with labels including the name of the material and the level of danger. (Explosiveness, spontaneous combustion, flammability, toxicity, water reactivity, oxidation, contagiousness, radioactivity, corrosiveness and other dangerous material.) Chemicals must be kept in isolation in consideration of flammability, corrosiveness, and other chemical features. When conducting chemical experimentation, tasting and smelling must not be allowed. Pipette using mouth must not be allowed. Inflammable chemicals must be purchased in small amount when necessary. Inflammable chemicals including acetone, oil, or gas must be kept in the place where ventilation is easy and access is infrequent. Importing and exporting of high-pressure gas container must be done using transportation equipment. When storing high-pressure gas containers, they must be safely and firmly fixed, isolated from flammable substances and inflammatory materials. Experiments using flammable, explosive, toxic, or volatile gases or vapors must be carried out in a hood. Reagents container or cabinets for storing reagents must be kept in cool, well-ventilated places without direct sunlight, and far away from fire and heat sources. Experimental drugs shall be stored in a way not to be shaken by outside shock, and so that its storage bottles do not fall. Toxic materials shall be handled using protective films or other safety tents to prevent damages from splatter, heating or explosion so as to ensure the safety of the experimenter. Containers for chemical wastes shall not be left in the hallway or on the stairs, and shall not be stored in the corners of the laboratory or other invisible places. When performing microbiological tests, gloves shall be worn, and after the experiment, the used glassware shall be washed through sterilization, and disposable supplies and medium shall be disposed of separately from general garbage. Periodic inspection must be performed for fume hoods, benches, exhaust facilities, sinks, laboratory sinks, and waste water piping, etc. Chemical reagents shall not be stored in large volumes inside the laboratory, but shall be purchased from the reagent centers in small quantities. Laboratories shall be well ventilated to expel harmful gases, and the concentrations of hazardous substances must be gauged to ensure the safety of laboratory workers. ©2014 Elan Vital Retrieved from "http://2014hs.igem.org/Team:Elan_Vital_South_Korea/s_general_safety_rul1280SMTexasSt. Mark's School of Texas Dallas, TX, USA www.smtexas.orgHigh SchoolVOColi: Detecting Lung Cancer BiomarkersInspired by the olfactory ability of canines to detect diseases, we will pursue a long-term project that revolves around the creation of a minimally invasive and inexpensive detection system for lung cancer, through the identification of exhaled biomarkers. Lung cancer is the leading cause of cancer-related death around the world. Twenty-two volatile organic compounds (VOCs) have been distinctly found in the breath of affected patients, creating a viable ?fingerprint? for reliable detection (Horvath et al.). This year we focused on creating biosensors for three VOCs: ethanol, formaldehyde, and xylene. We plan to create genetic circuit systems for aldB induced by ethanol, frmR recognition of formaldehyde, and xylR activated by xylene. Our current device will utilize three reporter proteins (CFP, GFP, RFP) to indicate the concentrations of the three VOCs present in an exhaled sample. This research will eventually go into creating a conclusive test for use in the developing world.1601BBa_K1280999http://2014HS.igem.org/Team:SMTexasBBa_K1280000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=164220140http://2014hs.igem.org/files/poster/SMTexas.pdfHigh SchoolLog in   Team:SMTexas/Notebook From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Notebook Table of Contents Leadership Policy Budget Letter to Admin August-December March April May June Leadership Policy The Budget Letter to Administration AUGUST-DECEMBER The founding of the St. Mark's School of Texas iGEM Team required overcoming many administrative and logistical hurdles from funding to lab space. Through garnering the support of the science department, we were fortunate enough to have a laboratory set aside for us Fundamental Research: Go over http://parts.igem.org/Help:Learn, the folder "Learn" in Dropbox Applied Research: Organize and store DNA Distribution kit materials, (time permitting) go over pipetting with Mr. Adame Outreach: brainstorm ideas about getting more Marksmen interested in iGEM, plan to make a brochure, determine a date to display the posters Killian created around the campus Web Development: go over wiki design/layout, ask Mr. Rummel if you all are having trouble October 2, 2013: Our first meeting will be on October 17th during the 10:30 period. We'll be introducing iGEM, an exciting synthetic biology and genetic engineering competition. October 8, 2013: Stanford and Brown astrobiology project was presented to give some perspective on what we could do. October 16, 2013: Hey guys, I'd just like to remind you all that our first meeting is tomorrow during the 10:30 period. Unless we have an assembly, we will be talking about iGEM and possible projects we'll be doing. October 18, 2013: Here are the assignments. You can find team wikis at http://igem.org/Team_Wikis?year=2013&division=high_school. Remember that for now, our meetings will be every Thursday during the 10:30 period in Mr. Adame's room. For our next meeting, please come with information about your team's project. Be able to explain what, why, and how. This will help immensely when we start brainstorming ideas for our own iGEM project. October 24, 2013: We had a very productive meeting today. Thanks for spending time thoroughly researching your assigned team. You all knew details of your projects very well! For our next meeting, continue looking into your assigned team and focus primarily on cost and parts. See what BioBricks or other resources they utilized to create their project. This next meeting will be the last one focused solely on what other teams have done in the past. We need to start generating ideas for our own project. You can see how one collegiate team that worked on converting CO into CO2 biologically came up with their idea here: http://2012.igem.org/Team:METU/Brainstorm November 2, 2013: Hey guys, At our next meeting, we'll start brainstorming iGEM projects to pursue. I've seen some great ideas from the leadership applications we've received. Please continue exploring topics we could look into. Science magazines, journals, and blogs are great sources for inspiration. Even the news is a viable source. The World iGEM Collegiate Championship Jamboree is also live right now!! You can check out the live videos or the archives of specific teams. Listening to the presentations should give us some valuable insight. November 6, 2013: Hey all, We have a meeting tomorrow at 10:30. Please bring some iGEM project ideas and if you'd like to apply for a leadership position, remember that the deadline is next Friday, November 15. If you have any questions, please let me know. November 7, 2013: I think Gopal mentioned producing bacteria to create O2 to promote healing in bandages. Well Thomas Jefferson High School studied a similar project last year, check it out here: http://2013hs.igem.org/Team:Jefferson_VA_SciCOS/Project. Notice how they took a simple idea and expanded upon it. Here are some of the ideas we generated today. Mr. Adame also sent us a link to a great article on treating contaminated water. Check it out below! Remember, we don't have to come up with a completely original idea for our project. We can take a project someone else has worked on and modify it. November 10, 2013: Hope you all had a great weekend! Go Lions! I'd just like to update you guys on the direction of our iGEM project. Aarohan, Jeffrey, Nikhil, and I thoroughly considered all of the great ideas you all proposed. This afternoon, we made a final decision; we are pursuing a project centered on probiotic production. After consulting with Dr. Sandeep Burma, we found that creating bacteria that would facilitate enzyme production and promote healing in certain populations would not only have useful applications, but also be practical for us accomplish with the resources we have available. November 12, 2013; Hey guys, We'll be going over gene expression/regulation during Thursday's meeting. To help lay the groundwork, I have sent you all two PDFs from chapters of the AP Biology textbook on the basics of DNA, transcription, and translation (check your e-mail). I hope you all will scan them over. The two chapters go in depth on what you learned in first year biology. If you're thinking about competing in the USA Biology Olympiad in the spring, Campbell's Biology (the text used in AP Biology) is the official textbook and most questions come from material covered in the book. November 17, 2013; Hey guys, Check out this TED talk if you have a chance. Here's a little intro from TED.com: "Once it's created, plastic (almost) never dies. While in 12th grade Miranda Wang and Jeanny Yao went in search of a new bacteria to biodegrade plastic -- specifically by breaking down phthalates, a harmful plasticizer. They found an answer surprisingly close to home." November 20, 2013: If you're interested in iGEM, find some time to go over Unit 1: Biotechnology Techniques on http://www.nature.com/scitable/ebooks/intro-to-biotechnology-techniques-and-applications-16570330/contents. You must know these basic techniques before you can do anything biotechnology related. The tutorial covers restriction enzymes, PCR, and the cloning of expressed genes. If you have any questions, feel free to message me this Thanksgiving break. December 4, 2013: Hey guys, Just a reminder that we are having a regular club meeting tomorrow. We have read through all your applications and have determined the remaining leadership positions for this year's Biology Club and iGEM team. We'll announce those and talk about the direction of our iGEM project. December 11, 2013: Hey guys, We have an important meeting tomorrow. Financial support for our iGEM project is contingent upon your attendance at the next two meetings. But sadly, seniors have a conflict with the meeting time again. Fortunately, Raymond Guo has agreed to lead this meeting. Please try to get to the next two meetings promptly at 10:30. December 18, 2013: Hey guys, We have a really important meeting tomorrow. Please be there promptly at 10:30. Be sure to give a warm welcome to our three incredible guests tomorrow. December 19, 2013: The help and support we have received for our biotechnology initiative is truly incredible. Now with the school's financial backing, we will be able to pursue new frontiers of research and discovery through the iGEM competition. To finalize the details of our project, I've created a new Dropbox folder specifically for the iGEM Team. We will start reviewing literature on probiotics over winter break. If you're on the iGEM team, please be on the lookout for documents placed in the iGEM Dropbox folder along with e-mails regarding our project and your individual roles. So far, I have contracts of commitment from the following guys. You will receive an invitation to join the folder from the e-mail address below. If you'd like me to send the invitation to an alternate e-mail, please let me know. December 21, 2013: I'm really excited to announce that we are now an official iGEM team. You can see our team page and our fellow competitors from around the world at http://igem.org/Team_List?year=2014&division=high_school. Our team name is SMTexas. DEVELOPMENTAL PHASE WEEK OF MARCH 17 General Meeting: Captain introduces details of iGEM to new members, reveals the project direction to all members, and introduces the creation/makeup of the Committees (30 min), divide the team into groups, have first group meetings, discuss having an additional Tuesday or Wednesday after school meeting Fundamental Research: Go over http://parts.igem.org/Help:Learn, the folder "Learn" in Dropbox Applied Research: Organize and store DNA Distribution kit materials, (time permitting) go over pipetting with Mr. Adame Outreach: brainstorm ideas about getting more Marksmen interested in iGEM, plan to make a brochure, determine a date to display the posters Killian created around the campus Web Development: go over wiki design/layout, ask Mr. Rummel if you all are having trouble March 19, 2014: Official unveiling of our project direction tomorrow after school. Committee sign-ups will begin as well. I'd like to encourage you all to recruit your friends and peers. We need all the guys we can get, and this is the perfect time for them to join. March 22, 2014: Detecting cancer will be challenging, and we need all the help we can get. Please peruse the information in the packet I handed out last Thursday. Committee sign ups have begun. If you weren't able to make Thursday's meeting, please fill out this online form. https://docs.google.com/forms/d/19b9k1h3Tb8ORI5F0efW-5QO1xOht0JdmT9-zugtR7lU/viewform. Every member of the iGEM Team must be on a Committee. You can find the schedule for the next few weeks at bit.ly/smbiology. As a Committee member, you will be responsible for completing the weekly goals. I will send out e-mails regarding the leadership team and the responsibility of your chosen Committee in the next few days as you all sign up. Hope you guys have a nice weekend. WEEK OF MARCH 24 General Meeting: Captain introduces Gene Designer (20 min) Fundamental Research: Start designing in Gene Designer by looking at possible promoters & repressors, peruse: http://www.biotechniques.com/news/University-of-Cambridge-team-wins-iGEM-synthetic-biology-competition/biotechniques-180278.html & http://biospot10.blogspot.com/2011/07/igem-grand-prize-2009-biosensors-and.html Applied Research: Work with the Fundamental Research group to develop a streamlined construct, go over best practices in the lab with Mr. Adame Outreach: start posting posters, print out 300-400 copies of the brochure depending on cost, plan to distribute the brochures after Friday's Upper School Assembly (April 11) Web Development: follow iGEM Wiki guidelines and create relevant content, make sure to work on the team page March 24, 2014: Meetings Wednesdays and Thursdays after school this week. If you haven't done so already, please sign up for a Committee. Not doing so would infringe the Contract of Commitment you signed in the fall last year. iGEM Committee Online Sign Up Form: https://docs.google.com/forms/d/19b9k1h3Tb8ORI5F0efW-5QO1xOht0JdmT9-zugtR7lU/viewform If you weren't able to make the meeting last week, be sure to peruse the packet I attached in the e-mail I sent you all. It covers the basics of synthetic biology. Try to understand the information and ask any questions you may have on Wednesday. March 27, 2014: Looking at our competition, we began to get behind some of the highly competitive teams, which mostly have University support. Worked extra hard. To this end, if you all have a chance, please download these two programs and watch the following tutorial video. WEEK OF MARCH 31 General Meeting: Presentation by the Fundamental Research Group on their findings & in-development construct Fundamental Research: Worked with the Applied Research Group to start constructing the plasmid, continue research Applied Research: Found protocols for implementing the construct, identify problems and consult the Fundamental Research Group Outreach: Brainstormed ideas on promoting the biotechnology further, esp. ideas for getting additional funding, possibly think about hosting parents for a luncheon, e-mail Dr. Burma and any other professors at UTSW for lab tour/advice/guidance Web Development: Documented progress and work with the Outreach Group POSTPONED B/C of ISAS Mandatory Meeting: APRIL 2 Evaluation: Meeting with Mr. Adame, Mr. Rummel, and Committee Leaders ISAS Commitments are taken into consideration CONSTRUCTION PHASE WEEK OF APRIL 7 General Meeting: Captain spoke on the leadership policy, Evaluation of Committees by Captain/Faculty Sponsors Fundamental Research: Continue research; Finalized research on genes for the detection of VOC biomarkers Applied Research: 3A Assembly; Worked with the Fundamental Research Team on finding the DNA sequences, reinforced laboratory skills, calculated potential budget for future years Outreach: Finalized Brochure; Placed Posters around campus, printed out 200 copies of the brochure and distributed to interested science instructors, began development of a more formal packet of info for the administration Web Development: Continued work on the wiki April 7, 2014: Made the goals for the week. Held Regular meetings Wednesday and Thursday. Captain seeks new recruits for next year's iGEM Team; reveals ultimate vision of the Project to the Upper School; sends the Biotechnology Initiative brochure to underclassmen WEEK OF APRIL 14 General Meeting: Committee Meetings Fundamental Research: Finalized research on genes for the detection of VOC biomarkers Applied Research: Worked with the Fundamental Research Team on finding the DNA sequences, reinforced laboratory skills, calculated potential budget for future years Outreach: Placed posters around campus, print out 200 copies of the brochure and distributed to interested science instructors, began development of a more formal packet of info for the administration Web Development: Assisted Outreach, finalized at least half of the wiki CANCELED UNTIL CONSTRUCT DEVELOPMENT IS COMPLETE: Plan for a UTSW visit April 18 WEEK OF APRIL 21 General Meeting: Brief presentation by Web Development on Thursday Fundamental Research: Began creating construct in Gene Designer if necessary; looked for options for the purchase of these sequences from biotech companies, check iGEM Registry for existing BioBricks Applied Research: Finalized budget, continue refining laboratory skills, assist the Fundamental Research Team Outreach: Finalized packet for the admininstration, print 10 color copies Web Development: Finalized wiki with detailed content & designs, brief presentation to the Team on how to add/modify content WEEK OF APRIL 28 General Meeting: Presentation on established construct by the Fundamental Research Team Fundamental Research: Order sequences, collaborate with the Applied Research Team to address possible issues during the actual creation of the construct Applied Research: Collaborate with the Fundamental Research Team to identify room for error Outreach: Design iGEM wearable gear (t-shirt, sweatshirt, and/or hat) and list cost in a document Web Development: Address iGEM requirements, including Human Practices WEEK OF MAY 5: AP EXAMINATIONS General Meeting: Meeting Cancelled Fundamental Research: N/A Applied Research: N/A Outreach: N/A Web Development: N/A WEEK OF MAY 12: AP EXAMINATIONS General Meeting: Meeting Cancelled Fundamental Research: N/A Applied Research: N/A Outreach: N/A Web Development: N/A May 16, 2014: We're planning to order iGEM Shirts regardless of whether we attend the Jamboree. If you would like one, please fill out this quick online form. WEEK OF MAY 19 General Meeting: Fundamental Research: Prepared to finalize gene constructs; Looked for any last minute corrections and do any other research that will aid in later implementation. Applied Research: Continued working with 3A assembly; Worked with basic research team and prepare to implement concepts and ideas of basic research team. Outreach: Continued working on human practices, including community outreach and global outreach; Continued filming as well Web Development: Worked extensively on the wiki; write more content; Worked with other committees to do so WEEK OF MAY 26 General Meeting: Evaluate progress Fundamental Research: Continued piecing together constructs and preparing for implementation Applied Research: Prepared lab space and order necessary materials and do orientation for summer wet lab work and research Outreach: Continued human practices as mentioned above; Worked with other committees as well Web Development: Worked extensively on the wiki and worked with other committees MAY 30: REGULAR REGISTRATION FOR HIGH SCHOOL JAMBOREE CLOSES, JAMOREE ATTENDANCE FEES DUE WEEK OF JUNE 2 General Meeting: Evaluate progress Fundamental Research: Researched novel solutions to the genetic construct, found outstanding research paper, continued research on potential VOC biomarkers; Arrived after lunch, researched solutions to mechanism discrepancies, Aste gene; gene sequence, researched solutions to constructing genetic circuits, Applied--developed prototype genetic circuit system, assigned special duties, specified equipment available, purchased additional equipment and poster; Applied Research: Did another 3A Assembly and analyze possibility of Gibson Assembly. Worked with Fundamental Research to help identify genes. Made bacteria cultures, prepared more ampicillin and kanamycin solutions, found gene sequence for RFP; applied research: prepared more ampicillin and kanamycin bacteria cultures, miniprep; Did gel electrophoresis previous day's miniprep; all were successful. Worked with Basic Research to map and understand a potetnial mechanism for the expression of the aldb gene; Arrived after lunch, helped research potential genetic circuit solutions; developed prototype genetic circuit system, assigned special duties, specified equipment available, purchased additional equipment and poster Outreach: Created a detailed production timeline for the first week, brainstormed and came up with a mind map for the film, help decide the wiki design layout; Web Development: Worked extensively on the wiki, added interactive elements and modified existing layout significantly JUNE 8: TRACK SELECTION DUE, PROJECT ABSTRACTS DUE, TEAM ROSTERS DUE WEEK OF JUNE 9 General Meeting: Evaluate progress Fundamental Research: Helped applied research as well as other committees that require help Applied Research: Finished and analyze 3A Assembly and finish work with Fundamental Research Committee; Also worked with Outreach Committee on Poster Outreach: Continued work with human practices Web Development: Continued work on wiki and work with other committees WEEK OF JUNE 16 General Meeting: Evaluated progress Fundamental Research: Helped other committees Applied Research: Worked with Outreach Team. Outreach: Worked on human practices (extensively) Web Development: Worked on wiki (extensively) JUNE 20: WIKI FREEZE AT 11:59PM BOSTOM TIME (SEE: WIKI REQUIREMENTS AND SAFETY QUESTIONS) WEEK OF JUNE 23 Practice presentation, polish poster, submit part to iGEM JUNE 28: JAMBOREE! Retrieved from "http://2014hs.igem.org/Team:SMTexas/NotebookBrendanCourt From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Our Work with Brendan CourtBrendan Court is an enrichment program in which underprivileged students who are entering the seventh and eighth grades are given the opportunity to study mathematics, science, humanities, and the arts at St. Mark’s School of Texas during summer break. As an iGEM Team, we sought to teach and inspire the students in synthetic biology. We presented informative and engaging presentations that explained basic biotechnology concepts along with compelling and visual demonstrations that captivated the students.The team has also been working with many programs to help promote the spread of biotechnology, especially for the youth. One of the programs that we worked with is Brendan Court. Brendan Court is a four-week, tuition-free enrichment program for middle school boys that is hosted annually at St. Mark's School of Texas. These students are exposed to many opportunities that may not be available at their own schools, such as photography, computer and word processing, physical education, poetry workshops, model rocket building, and more. The St. Mark's iGEM team wanted to add bioengineering to this list, and the team worked exclusively with the students for one day. We divided the forty-four students into four groups, and we worked with two of the groups at a time. In one room, one of the two groups received a presentation about synthetic biology, smoking, as well as a tour of the lab while the other group in another room watched science demonstrations. Using a straw, the students blew into a cup with a bromothymol blue and sodium hydroxide solution and observed effects of the carbon dioxide they exhaled. Bromothymol blue is a pH indicator that turns blue in a base and yellow in an acid. When the students exhaled carbon dioxide into the solution, the carbon dioxide reacted with the water to form carbonic acid which disassociated to hydrogen ions and bicarbonate. This makes the solution acidic, and the color of the solution changes. We also showed them how to make elephant toothpaste using dishwasher soap, 30% hydrogen peroxide solution, and saturated potassium iodide solution. We first mixed the hydrogen peroxide and the soap, and after adding the potassium iodide, the chemicals react to expand and form a gooey paste. We also prepared microscopes and some interesting slides, such as some tissue or cells, for the students to observe. Finally, we made "super-tang," an orange-soda drink by adding dry ice to an orange-flavored liquid. The dry ice carbonates the drink, giving it the fizzy taste. After ten minutes, the two groups would switch rooms and receive the other presentation. Once the groups received both presentations, they switched out with the other two groups with whom we had not yet met.Our Powerpoint on SmokingCreated by Kevin WuPicturesBrendan Court's Thank You PosterRetrieved from "http://2014hs.igem.org/Team:SMTexas/BrendanCourappliedresearch From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Our Applied Research CommitteeThe work of the Applied Research Committee has worked to turn the ideas of the Fundamental Research Committee into reality. We worked to perfect the 3A Assembly and later used this experience to use in the incorporation of our basic ideas into a potential lung cancer screening device. We also worked with the Fundamental Research Committee to research genes that would be indicators of the presence of these VOCs. This committee has been very multifaceted, with the members taking up various aspects of our project and working to accomplish them. Team Members: Rohin Maganti Eric LiShaheer Khan Gopal Raman Mitchell So Abhi Thummala Kevin Wu (Director) Akshay Malhotra (Director)Retrieved from "http://2014hs.igem.org/Team:SMTexas/appliedresearcConcept From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices The ConceptEarly detection of lung cancer is vital to increasing the success rate of current treatment options. Current diagnostic methods, including chest radiographs and CT scanning, are expensive, unreliable, and potentially dangerous. Our proposed alternative was inspired by observing canines, and their ability to detect noncommunicable diseases quickly and accurately without harm to the patient. According to leading research Rainer Ehmann’s study on canine olfactory screening of lung cancer, “[Lung cancer] was identified with a sensitivity of 90% and a specificity of 72%” (3). Training dogs for this purpose, however, is expensive and time-consuming. ReferencesMcCullough, M., Turner, K., & Broffman, M. (2012). Lung cancer detection by canine scent: will there be a lab in the lab?. European Respiratory Journal, 39, 511-512. doi: 10.1183/09031936.00215511Ehmann, R., Boedeker, E., Friedrich, U., Sagerta, J., Dippon, J., Friedel, G., & Walles, T. (2011). Canine scent detection in the diagnosis of lung cancer: Revisiting a puzzling phenomenon. European Respiratory Journal, 39, 669-676. doi: 10.1183/09031936.00051711Retrieved from "http://2014hs.igem.org/Team:SMTexas/ConcepSponsors From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Please check out our sponsors:New England BioLabs IncorporationBio-Rad Laboratory IncorporationBio Basic IncorporationRetrieved from "http://2014hs.igem.org/Team:SMTexas/SponsorsDesign From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Our Genetic Constructs aldB Gene (Detects Ethanol) The aldB gene codes for a functional aldehyde dehydrogenase, which is directly induced by ethanol. Metabolism of the alcohol consists of its transformation into an aldehyde and then into a carboxylic acid in activating several related pathways. The acid reacts with the BarA histidine sensory kinase, a signalling enzyme involved in a two component signal transduction system present in E. coli, to catalyze the breakdown of various carboxylic acids. The kinase is additionally responsible for the induction of RpoS, a regulatory gene of aldB that directly opposes fis during the activation of the aldB operon. Eventually, BarA triggers a series of vital reactions that affect the Crp-cAMP regulatory mechanism, a dual complex that controls the expression of the aldB coding sequence. In the complex, cAMP conforms the shape of Crp, also known as CAP (catabolite activator protein). This newly conformed Crp then attaches to the promoter and contributes to the initiation of transcription of the aldB operon. Downstream of aldB, CFP (cyan fluorescent protein) is expressed and the bacteria exhibits cyan fluorescence. XylR Gene (Detects Xylene) The genetically-related expression of the XylR gene consists of promoters, regulator complexes, and proteins that all aid in the expression of fluorescent proteins. The initial DNA sequence Pr promotes the expression of XylR gene exon itself. Shortly after, it is followed by a ribosomal binding site that orchestrates the timing and efficiency of translation. The naturally expressing XylR sequence succeeds the RBS and undergoes strict regulation of the Pr promoter. Because this protein triggers a secondary response in the bacterium which is vital to Xylene detection, a double termination sequence is essential to the discontinuation of sequences downstream of the XylR coding region is not expressed which can disrupt the reactions involved in the detection system. These stop codons, which are short and effective, operate with a stem-loop that possesses both forward and reverse termination mechanisms. The expressed XylR protein then reacts with xylene and is conformed to accommodate a secondary gene sequence. This newly conformed version of the protein can then bind to the Pu promoter. After a second ribosomal binding site (strong) is subsequently intiated and YFP is expressed, which supersedes the RBS, the bacteria will exhibit yellow fluorescence to indicate a positive test. frmR Gene (Detects Formaldehyde) Formaldehyde induces the frmR gene, functioning as a regulatory gene of Green Fluorescent protein. When transcribed, it expresses a regulatory protein that binds to an downstream operator that prevents the movement of RNA polymerase. Under typical conditions, the promoter downstream of the regulatory gene increases the affinity of RNA polymerase to the DNA strand, but the transcription enzyme cannot bypass the operator and transcribe GFP, the gene that is ultimately under regulation. In such a scenario, the regulatory protein that frmR expresses functions as a repressor and effectively inhibits transcription of the coding sequence. Formaldehyde, on the other hand, induces the transcription of GFP and ultimately causes bacterial fluorescence. Acting as a corepressor, the VOC binds to the regulatory protein and conforms it into an inactive shape, allowing for the passage of RNA polymerase through the operator and transcribe the GFP protein.ReferencesGeneral:Society of Thoracic Surgeons. (2014, January 28). Exhaled breath may help identify early lung cancer. ScienceDaily. Retrieved June 15, 2014 from www.sciencedaily.com/releases/2014/01/140128094145.htm American Lung Association. (2012, April). Providing Guidance on Lung Cancer Screening To Patients and Physicians. Retrieved from http://www.lung.org/lung-disease/lung-cancer/lung-cancer-screening-guidelines/lung-cancer-screening.pdfPfizer Oncology. Lung Cancer and Biomarkers. Retrieved from http://www.lungcancerprofiles.com/lung_cancer_and_biomarkers.Hakim, M., Broza, Y. Y., Barash, O., Peled, N., Phillips, M., Amann, A., & Haick, H. (2012). Volatile Organic Compounds of Lung Cancer and Possible Biochemical Pathways. Chemical Reviews, 112(11), 5949-5966. doi: 10.1021/cr300174aFu, X., Li, M., Knipp, R. J., Nantz, M. H., & Bousamra, M. (2013). Noninvasive detection of lung cancer using exhaled breath. Cancer Medicine, 3, 174-181. doi:10.1002/cam4.162Mazzone, MD, MPH, FRCPC, FCCP, (July 2008). Analysis of Volatile Organic Compounds in the Exhaled Breath for the Diagnosis of Lung Cancer. Journal of Thoracic Oncology . 3 (7), pp.774-781Phillips, M., Gleeson, K., B Hughes, J. M., Greenberg, J., Cataneo, R. N., & Baker, L. Volatile organic compounds in breath as markers of lung cancer: a cross-sectional study . Early Report, 353, 1930-1936.Fuchs, P., Loeseken, C., Schubert, J. K., & Miekisch, W. Breath gas aldehydes as biomarkers of lung cancer. International Journal of Cancer, 126, 2663-2670.Bajtarevic, A., Ager, C., Pienz, M., Klieber, M., Schwarz, K., Ligor, M., et al. Noninvasive detection of lung cancer by analysis of exhaled breath. BMC Cancer, 9.Wang, Y., Hu, Y., Wang, Di., Yu, K., Wang, L., Zou, Y., Zhao, C., Zhang, X., Wang, P., & Ying, K. The analysis of volatile organic compounds biomarkers for lung cancer in exhaled breath, tissues and cell lines. Cancer Biomarkers, 11, 128-137. doi:10.3233/CBM-2012-00270.Clinical Trials. (2012). Exhaled Breath Biomarkers in Lung Cancer. Retrieved from http://clinicaltrials.gov/show/NCT01386203xylR gene: EcoCyc (n.d. )Escherichia coli K-12 substr. MG1655 Polypeptide: XylR transcriptional activator. Retrieved from http://ecocyc.org/ECOLI/NEW-IMAGE?type=ENZYME&object=EG20253-MONOMERNucleic Acids Research 39:D583-90 2011 WikiGenes (n.d.) xyIR-xyIR Pseudomonas Putida. Retrieved from http://www.wikigenes.org/e/gene/e/1218757.htmlSmoColi. (n.d.) How we calculated the concentration of m-xylene in the medium…. Retrieved from http://2011.igem.org/Team:ETH_Zurich/xylenealdB gene: Xu, J., Diderichsen, B., Ho, K. K. Wiki Genes. aldB - aldehyde dehydrogenase BEscherichia coli str. K-12 substr. MG1655. Retrieved From http://www.wikigenes.org/e/gene/e/948104.html Xu, J., & Johnson, R. C. National Center for Biotechnology Information. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. Retrieved From http://www.ncbi.nlm.nih.gov/pmc/articles/PMC177007/EcoCyc. (n.d.). Escherichia coli K-12 substr. MG1655 Enzyme: acetaldehyde dehydrogenase. Retrieved From http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=EG12292PortEco. (2013). aldB:gene. Retrieved from http://ecoliwiki.net/colipedia/index.php/aldB:GenefrmR gene: Tokyo Metropolitan University. (n.d.) Parts. Retrieved from http://2012.igem.org/Team:TMU-Tokyo/Parts PortEco. (n.d.) Retrieved from http://heptamer.tamu.edu/fgb2/gbrowse/MG1655/?plugin=FastaDumper&q=NC_000913:378830..379105&plugin_action=Go PortEco. (2013). frmR gene. Retrieved from http://ecoliwiki.net/colipedia/index.php/frmR:GeneWang, S., Deng, K., Zaremba, S., Deng, X., Lin, C., Wang, Q., et al. (2009, August 7). Transcriptomic Response of Escherichia coli O157:H7 to Oxidative Stress. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2753066/ EcoCyc. (n.d.) Escherichia coli K-12 substr. MG1655 Polypeptide: regulator protein. Retrieved from http://ecocyc.org/ECOLI/NEW-IMAGE?type=GENE&object=G6209Retrieved from "http://2014hs.igem.org/Team:SMTexas/Desigproject From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices VOColi Detecting Lung Cancer Biomarkers Inspired by the olfactory ability of canines to detect diseases, we will pursue a long-term project that revolves around the creation of a minimally invasive and inexpensive detection system for lung cancer, through the identification of exhaled biomarkers. Lung cancer is the leading cause of cancer-related death around the world. Twenty-two volatile organic compounds (VOCs) have been distinctly found in the breath of affected patients, creating a viable “fingerprint” for reliable detection (Horvath et al.). This year we focused on creating biosensors for three VOCs: ethanol, formaldehyde, and xylene. We plan to create genetic circuit systems for aldB induced by ethanol, frmR recognition of formaldehyde, and xylR activated by xylene. Our current device will utilize three reporter proteins (CFP, GFP, RFP) to indicate the concentrations of the three VOCs present in an exhaled sample. This research will eventually go into creating a conclusive test for use in the developing world. The Concept The future of screening for diseases. More The Design The genetic circuits we have created. More Future Implementation Our plans for the future. More Retrieved from "http://2014hs.igem.org/Team:SMTexas/projecAttributions From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Our Faculty SponsorsMark Adame teaches Freshman and A.P. Biology at St. Mark's School of Texas. He graduated with a Bachelor of Science in Marine Biology in 1993 from Texas A&M University. Mr. Adame was a research assistant at the university of Arkansas. He is the main faculty sponsor for the iGEM Team, and he has played an instrumental role for our success. Mr. Adame has helped the team receive funding while teaching the team advanced laboratory procedures currently used in many laboratories around the worldDoug Rummel is a current Physical Science instructor at St. Mark's School of Texas. He graduated with a Bachelor of Science degree from Oregon State University and a Master of Arts in Teaching Degree from the University of Texas at Dallas. He has been the faculty sponsor for the Robotics Team for a decade. Mr. Rummel worked with the Fundamental Research Committee to help the genes for the individual VOCs and offering his advice.Ken Owens graduated from St. Mark's in 1989 and returned to teach in 1997. The eight years off-campus were spent at Rice University (B.A), the University of Michigan in Ann Arbor (M.S.), and The Oakridge School in Arlington, TX. He currently teaches Chemistry and A.P. Chemistry. He has also taught Applied Chemistry, Physical Science, Science Fiction, and Life Science at St. Mark's, and various science and mathematics courses at Oakridge. He serves as the head counselor for the Chemistry Camp at St. Mark's during the summer. Mr. Owens is a Freshman/Sophomore class sponsor and advisor. He is also the coach for the JETS/TEAMS science competition. He serves on the Faculty Advisory Committee and the Safety Committee for the school. Mr. Owens helped answer many of the team's chemistry-related problems.Retrieved from "http://2014hs.igem.org/Team:SMTexas/AttributionFrom 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Fundamental Research Explore how our Fundamental Research Team fueled our project development and research. More Applied Research Explore how we have made biobricks that can detect lung cancer in a simple, inexpensive, and noninvasive way. More Outreach Educating our community and gathering public opinion on modern biotechnology. More Videos Watch the video explaining our project in detail as well as numerous informative tutorials. More Safety Our safety precautions. More Attributions Meet the faculty members who played an instrumental role to our success. More Human Practices Our project in the real world. More Sponsors Meet the corporations that played an instrumental role in our success. More Biotechnology Initiative The first iGEM high school team from the state of Texas, the St. Mark’s iGEM Team was established as part of a Biotechnology Initiative begun in September of 2013 by our school’s Biology Club. This Initiative, spurred on by iGEM, revitalized our Club and helped generate significant interest in synthetic biology from the student body, faculty, and school administration. In our first year we achieved regular access to a radiation oncology lab at the University of Texas Southwestern Medical Center, $1500 in funding from the school to cover the registration fee for the iGEM Competition, exlcusive laboratory space at the school, and a more than four-fold increase in membership of the Biology Club to twenty-two members.Our iGEM Team blends the setup of a traditional club with the highly efficient structure of a professional research laboratory. The Team is divided into four Committees: Fundamental Research, Applied Research, Outreach, and Web Development. This allows us to work more efficiently and cohesively. We have had to lay the foundation of our iGEM Team this year by informing the school community on synthetic biology, seeking financial support, and establishing laboratory resources for our project. But it was our infectious enthusiasm and indefatigable determination for synthetic biology that allowed us to overcome administrative hurdles and a steep learning curve. Through our journey this year, we have proven that a school with limited resources in biotechnology can make significant strides, effectively pioneer investigations in the field, and shown our community the tremendous potential we have in the future. About Us Our Mission Statement: To improve the community by pioneering investigations into bio-technology by solving real-world problems in order to promote scientific inquiry and educate the future leaders of biological engineering. About St. Mark's: St. Mark's School of Texas is a non-sectarian, college-preparatory, independent day school for boys in grades one through twelve. The School's charter states that it is "designed to afford its students well-rounded physical, intellectual, moral, and religious training and instruction." The School is intended to be a diverse community of teachers and students who share a love of learning and who strive for high achievement in whatever they undertake. Visit Our SchoolThe Team Follow us on: Facebook Twitter Instagram Youtube Retrieved from "http://2014hs.igem.org/Team:SMTexahttp://2014hs.igem.org/wiki/images/d/d6/Smigemlogo.pngbasicresearch From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices The Basic Research CommitteeThe Fundamental Research Committee was responsible for designing the proposed construct at the theoretical level. Working with the Applied Research Committee, the Fundamental Research Committee helped implement the actual biological system. The Team conducted extensive literature reviews, contacted professionals working in the field, and presented findings to the team on a regular basis.The Committee researched volatile organic compounds (VOCs) that would be present in the exhaled breaths of people with lung cancer. We also researched genes that would indicate the presence of these VOCs. This year we focused on detecting ethanol, formaldehyde, and xylene. The associated genes are aldB, frmR, and XylR, respectively.Team Members:Ali Ahmed Raymond Guo (Director) Rohin Maganti (Director) Rohan Pinto (Director) Our Drawing Boards:Retrieved from "http://2014hs.igem.org/Team:SMTexas/basicresearcHuman Practices From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Promoting Synthetic BiologyVideosBrendan Court#InspiringTomorrowThe St. Mark’s iGEM Team is committed to serving the community through innovative research and engaging outreach. Our mission is in line with that of iGEM’s by promoting and fostering scientific research in our school and beyond. We strive to engage with social, cultural, ethical, philosophical, environmental, political, legal, and economic dimensions of synthetic biology. In our first year, we developed comics that would integrate synthetic biology concepts into the plot and artwork to bring youth awareness to our Biotechnology Initiative, created engaging YouTube biotechnology tutorial videos as we strove to give a global online audience access to quality instructional resources in synthetic biology, launched our school’s greatest social media campaign through Facebook, Twitter, Instagram, and YouTube, and created a professional film to showcase our Biotechnology Initiative. Our Comic:Designed and Created by Abhi Thummala Retrieved from "http://2014hs.igem.org/Team:SMTexas/Human_PracticeImplementation From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices The ImplementationWhile the development of mechanical and electronic noses have been in progress for many decades, we propose that a circuit of genetically-modified E. coli used in conjunction with a mechanical contraption has the potential to be used a preliminary diagnostic for lung cancer. We propose a device similar to a breathalyzer that utilizes a cylindrical container with our genetically engineered E. coli systems at the bottom of the aluminum shell. To prevent the possibility of contaminants, an adapter would attach to the top of the cylindrical shell for the user to breath into. The user would fasten the adapter onto the shell and exhale as much as possible. Upon exhalation, the user would immediately detach the adapter to secure the system designed to automatically shut off after the removal of the adapter and the shell. The genetic systems would be underlain with a sensitive paper that would be dyed upon the expression of the fluorescent proteins. We would utilize Cambridge 2009 iGEM Team’s E. Chromi color generator to verify and differentiate the identities of the VOCs present in the user’s breath sample. Currently under development by our team, this device has the potential to be an inexpensive, sensitive, and noninvasive solution to early screening of lung cancer.Retrieved from "http://2014hs.igem.org/Team:SMTexas/Implementatiosafety From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices Safety is our Priority1. Would any of your project ideas raise safety issues in terms of researcher safety, public safety or environmental safety?For our project, the only live organism we are using is the K12 MG1655 strain of E. coli, and all of our parts are also derived from this organism. This organism falls under Biosafety Level 1. Neither our strain of E. Coli nor the genes (aldB, frmr, xylr) are dangerous to humans, publics, or the environment. Only a very few strains of E. coli are dangerous, such as the O157: H7, but our particular strain, K12 MG1655, is not dangerous. Our strain is only capable of causing a mild stomach ache if ingested. E. Coli is also very common in the environment since it is found in the fecal matter of animals. However, soil, sand, and sediments in the environment can naturalize the E. coli. In our lab, we have an autoclave, incubators, Bunsen burners, and a thermocycler which can reach very high temperatures and is handled with extreme care. Only members of the faculty are permitted to use an autoclave, and although our lab is fitted to be a Level II lab, the students only use Level I materials and handle Level I organisms. Ethanol and bleach may damage the skin, so latex gloves should be used when handling these substances. In addition, ethanol is very combustible. We address these problems by always wearing proper safety equipment (lab coat, goggles, gloves, etc.) while being very careful around all laboratory equipment. Horseplay is not tolerated. Mark Adame, our faculty sponsor, requires all team members to read the CDC requirements and regulations for a Level I lab while showing them how to use Level I lab equipment. No harm to humans, publics, or the environment would occur if containment of the E. coli was lost. The risks from malicious misuse are very small; the only harm (a mild stomach ache) from E. coli would occur only if it is ingested.2. Do any of the new BioBrick parts (or devices) that you made this year raise safety issues? Unfortunately, due to the lack of funding, our team was unable to submit any standard biological parts, we but are close to finalizing biobricks that we will submit to the registry in the near future. We currently have three theoretical constructs of devices that pertain to three genes in E. coli that detect respective volatile organic compounds.3. Is there a local biosafety group, committee, or review board at your institution? There is no official Biosafety Committee or equivalent at our school. However, the team has followed all of the national regulations written by the CDC for a Level 1 lab. 4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?In the future, we plan to create a biosafety review board at St. Mark’s to deal with any potential safety issues. This board will consist of the most knowledgeable and responsible faculty members who will know how to deal with any biosafety hazards and make decisions regarding the safety of procedures for the environment, the community, and the researchers.Our Safety ProtocolsIn humans, E. Coli is isolated in the intestines where it helps with the digestive process; however, when ingested or inhaled, E. Coli can cause severe sickness and, in some cases, hemolytic uremic syndrome, a severe form of kidney failure. To prevent this outcome, many lab safety techniques and guidelines are used.Before dealing with the E. coli, students will wash their hands thoroughly with water and soap. Afterwards, the students will put on their gloves, goggles, and lab coats. Before we can experiment with E. Coli, we must grow it. The first step of growing bacteria is sterilizing all equipment that will be used which includes pipettes and petri dishes. We also place the petri dishes in autoclaves to ensure that all other microorganisms are eliminated.Afterwards, we sterilize a test tube with alcohol and then fill it with heated agar, which we pour into the petri dish. For inoculation we sterilize the inoculating loop with bleach and then heat it. We then use the inoculating loop to extract E. Coli from another dish and place it into the agar petri dish using the streaking method. We streak the agar petri dish multiple times with the inoculating loop, heating, sterilizing, and picking up more E. Coli each time. Then we will take the bacteria and incubate it in an incubator. After dealing with the E. coli the students will take their gloves and throw them away into a special bin for gloves and wash their hands thoroughly again.In the lab we have a fire extinguisher, fire alarm activators, and fire blankets dispersed widely for easy access in case of a fire. In addition, if students accidentally get dangerous chemicals in their eyes or on their bodies, they can run to a shower where multiple gallons of water will be dumped on the student and sprayed into the student's eyes.Retrieved from "http://2014hs.igem.org/Team:SMTexas/safetteam From 2014hs.igem.org HomepageProjectTeamNotebookHuman Practices From left to right:Top Row: Kevin Wu, Rohan Pinto, Vik Reddy, Halbert Bai, Akshay Malhotra, Killian Green, Mark AdameBottom Row: Tim O'Meara, Ali Ahmed, Abhi Thummala, Kevin Choi, Mitchell So, Rohin Maganti, Johnathan Ng, Jeffrey Wu, Vishal Gokani Founder and Captain: Halbert BaiVice-Captain: Tim O'MearaFaculty Sponsors: Mark Adame & Doug Rummel Fundamental Research Directors: Rohin Maganti, Raymond Guo, and Rohan PintoMembers: Ali Ahmed, Jonathan Ng, Vishal Gokani More Applied Research Directors: Akshay Malhotra and Kevin WuMembers: Michell So, Rohin Maganti, Eric Li, Shaheer Khan, Gopal Raman More Outreach Directors: Killian Green, Abhinav Thummala, and Jonathan NgMembers: Jeffrey Wu More Web Development Director: Vikrant ReddyInterim Director: Kevin WuMembers: Kevin Choi Halbert Bai is a Benjamin Franklin Scholar at the University of Pennsylvania. He recently graduated as the valedictorian of his class at St. Mark’s School of Texas. Highly interested in science and art, he has pursued laboratory research experiences, collaborated with professional artists across creative disciplines, and sought related extracurricular work. In high school, he was inducted into the Cum Laude Society, the Co-Editor-in-Chief of The Marque Magazine of Arts and Letters, and a member of the Community Service Board. He spearheaded a biotechnology initiative at St. Mark’s through iGEM, an international synthetic biology competition. Artistically, Halbert focuses on photography, video production, and graphic design. He has been recognized as one of the top young photographers in the United States by the National YoungArts Foundation and the Alliance for Young Artists & Writers. Halbert specializes in creating images that capture the beautiful bond between man and nature while documenting the stunning magnificence of light and atmosphere. His photographs have also been exhibited in eight countries on five continents. His videos and designs are characterized by beautiful aesthetics and evocative visual narration. Halbert is the Founder and Captain of the St. Mark's iGEM Team. He combines an unwavering commitment to the Team with a genuine passion for serving humanity through innovative and original research. Under his leadership, the Biology Club was completely revitalized and the St. Mark's iGEM Team catalyzed a movement toward more independent research on campus and brought awareness to synthetic biology.Tim O'Meara is a rising Junior at St. Mark's who enjoys learning more about developments in biology. As the team's vice captain, Tim focused on many aspects of the project including research, outreach, and the film. A 2014 USA Biology Olympiad (USABO) semi-finalist, Tim is engaging in research in a pediatric oncology lab at UT Southwestern Medical Center this summer. Tim has a variety of interests outside of biology. He is a captain of the math team, member of the quiz bowl team and philosophy club, photographer for the school’s newspaper and yearbook and a two-time letterman on the varsity golf team. Tim also mentored a group of 6th grade students through the Telos leadership and ethics program, coached MathCounts for middle school students and is an Eagle scout.Rohan Pinto is a senior who likes to play the violin, learn Chinese, and tutor kids. Rohan is the President of the Chinese Club, the Junior Assistant Scout Master of Troop 730, and Treasurer for the Biology Club. He has made All-State three times and was assistant concertmaster for the 2013-2014 All-State Orchestra. Rohan has also received the Upper School Orchestra Award. Rohan is the Director of Basic Research for the iGEM team and helps with creating and discovering various mechanisms that detect VOCs in Lung Cancer patients. In addition, he also assists with the team poster and teaching basic DNA science concepts.Rohin is a rising sophomore at St.Mark’s School of Texas. He plays the violin in the upper school orchestra and made first violin in the high school region orchestra. In sports, he is member of the swimming team and the junior varsity water polo teams. Outside of the school community, he is an eagle scout and assistant senior patrol leader in Troop 730 at St. Mark's. In iGEM, he worked to find gene mechanisms to detect VOCs and was a director of fundamental research. He has also worked to edit a few tutorial videos for the iGEM team. Finally, Rohin is also a member of the leadership and ethics council and investment club, the JETS annual competition, and the quiz bowl team.Raymond Guo is a rising senior at St. Mark’s School of Texas. Very interested in math and science, he is both a United States of America Junior Mathematical Olympiad (USAJMO) Qualifier and a United States of America Physics Team Semifinalist. Captain of both the math and physics teams, member of the Cum Laude Society, Harvard Prize Book Award Winner, and a Rensselaer Medalist, his passion for learning is clearly evident in his endeavors. In addition to the sciences, he loves music; he has played piano for eleven years, is a member of the All-State Choir, and has received the James Livengood Upper School Chorus Award. He is a co-director of the Basic Research Committee on the iGEM team. Working with Rohan and Rohin, he has done research on potential VOCs in lung cancer patients and on genes that could indicate the presence of these VOCs.Akshay Malhotra is a rising junior at St. Mark’s School of Texas. Aside from working on iGEM and biology, in which he received a Certificate of Excellence for Highest School Score in the University of Toronto National Biology Competition, he plays chess. He is a US chess national master, who holds four national titles, including the reigning US Expert Champion title, and ten Texas state titles. He also tied for third place in last year’s World Amateur Chess Championship in Romania. He uses his chess skill to serve the community as he teaches chess to elementary students. Akshay is the Projects Manager of the Biology Club and the Director of Applied Research of the iGEM Team. He has worked to engineer the bacteria to be able to detect lung cancer VOCs.Kevin Wu is a rising junior at St. Mark's School of Texas. He swims year-round and trains over ten hours a week. Kevin is also the co-president of the Classics Club and the Director of Calender on the Community Service Board. Kevin is very active in helping the community, earning the bronze presidential award in freshman year and the gold presidential award in sophomore year. He has been chosen by his peers to lead the class as a co-chair in the annual McDonald's Week Fundraiser which raises money for the Austin Street Homeless Shelter. He is the director of applied research on the iGEM team as well as the interim director of web development. Kevin has worked with Akshay to understand the different methods of engineering bacterial while working on the wiki after the website crashed.Killian Green is a rising sophomore at St. Mark’s School of Texas. He is the Director of Outreach for the SMiGEM team. Killian plays the cello in the Upper School Orchestra and the piano. He enjoys using his literary expertise and graphic design skills as an involved member of The Marque, the St. Mark’s Upper School Literary Magazine, as well as the Marksmen yearbook. Killian is a member of the Classics Latin Club and has helped his team in bringing home gold medals in the Texas State Junior Classical League. He is also a member of the Varsity Crew and volleyball teams. He enjoys volunteering at the Jubilee Center, tutoring inner-city and often underprivileged kids.Abhi Thummala is a rising junior at St. Mark's School of Texas. In addition to his contributions as Director of Outreach on the iGem team, Abhi will compete on the volleyball and track and field teams. Abhi is the Graphics Director on the school newspaper, the ReMarker, which he writes and designs graphics for. He is also involved in the medical club, Caduceus Club, the Leadership and Ethics Committee, and community service. In addition to his work on campus, Abhi is working on his AP art portfolio. Abhi has worked with Killian Green to create graphics, draw a comic, and spread awareness about biotechnology.Vikrant Reddy is a rising sophomore at St. Mark’s School of Texas. He is the director of web development for the SMTexas iGEM team. He has designed and created the wiki. Vikrant is also a member of the St. Mark's debate team. Some of his other interests include medicine and crew. He served as the class secretary and is also involved in the Caduceus Club and the Visiting Scholar Advisory Board. Vikrant also assisted the robotics team in the beginning of the year. Jeffrey Wu recently graduated from St. Mark’s School of Texas and will attend Duke University this fall. He has a passion for science, community service, and baseball. A 3-time Gold Presidential Volunteer Service Award winner and member of the community service board, Jeffrey completed more than 900 hours of community service throughout high school. He was Vice-President and Vice-Captain of the Biology Club and the iGEM team. In addition, Jeffrey holds 6 varsity letters in swimming and baseball. Jeffrey is part of the Fundamental Research Team and helps with creating and discovering various mechanisms that detect volatile organic compounds in lung cancer patients.Ali Ahmed ’14 recently graduated St. Mark’s. Currently, Ali plans on dual majoring in Finance and Electrical Engineering at the University of Texas at Austin. He hopes to pursue a career in venture capital in Silicon Valley. Ali enjoys engineering, playing sports, and reading about current events. While at St. Mark’s, Ali was captain of the robotics team, the rocketry club. Ali was brought on to the iGem team to help engineering design and genetic circuits. Jonathan Ng is a recent graduate of St. Mark’s School of Texas and will attend Yale University in the fall of 2014. As a student at St. Mark’s, he was the Co-Editor-in-Chief of the Marque Magazine of Arts and Letters, the St. Mark’s Community Service Board Drives Chair, and the President of the Caduceus Medical Club. In the future, Jonathan hopes to combine his love of the humanities and sciences in his undergraduate studies.As a member of the iGEM team, Jonathan enjoys the precise nature of research and how these minute details can lead to a beautiful and influential result. He looks forward to seeing the iGEM project progress and influence the St. Mark's and greater Dallas community.Vishal Gokani '14 is a 12-year Marksman who strives to find ways to contribute to the scientific community at St. Mark's, even assisting a substitute science teacher who had to take on a last-minute class. He enjoys supporting his longtime friend and fellow student Halbert Bai, president of the team, and discussing the group's dynamics and progress. Vishal is honored to be a member of this new group and is eager to see its progress as younger students take the reins in future years.Luke Williams just graduated from St. Mark’s and is heading to the University of Virginia as a Jefferson Scholar. Basketball, football, and leadership are Luke's biggest hobbies. Luke joined the biology club because he liked the leadership initiative, and he helped to write the mission statement.Mitchell So is a rising Junior at St. Mark’s school of Texas. He has been in the St. Mark’s Orchestra for 6 years. Mitchell is also a year-round fencer, representing the US in the 2013 Cadet World Cup in Konin, Poland and winning the 2013 Louisville Y-14 North American Cup. Mitchell is also very involved in Community service. In addition to being a member of the Drives Committee, Mitchell also earned the Presidential Bronze award for community service freshman year and the Silver award sophomore year. Mitchell is a member of the Applied Research Team in iGEM.Shaheer Khan is a sophomore at St. Mark’s School of Texas. He is heartfelt in science, especially in biology, math, and journalism. He will be working on the school’s yearbook called the Marksmen for the next three years. In addition to academics, Shaheer shows passion in basketball, which he plays almost all year. He is also involved in the caduceus club, investment club, and first aid club. He loves to volunteer, especially activities that involve children or young kids. Shaheer works in the Applied Research team in iGEM.Eric Li has been at St. Mark’s since the fifth grade and is a rising junior. Eric is passionate about swimming. He has been a part of the Varsity Swimming Team since freshman year. Eric has also played violin in the Upper School Orchestra since freshman year, and was selected to the Texas All-State Orchestra for the past two years. In addition to swimming and orchestra, he is passionate about volunteering and community service. Since his freshman year, he has been a part of the palliative/pastoral care team at Baylor Medical Center at Garland and provides music therapy for patients by playing the violin. Eric is also passionate about the sciences, especially about biotechnology and biomedical research. During his free time, he has been working in the laboratory of University of Texas at Dallas, focusing at nanotechnology in cancer diagnosis and treatment.Gopal is a freshman at St. Mark’s and served as the Projects Manager of the Biology Club. In iGEM, he worked under the Applied Research Division. He also plays tennis for the Varsity team and is one of the Co-Presidents of the Philosophy Club. Other than science, he loves the literary arts and is the student vice-chair of the Literary Festival. He also writes for the Marque and The ReMarker. In addition, he serves on the Community Service Board. He also works with younger students in Telos 4.Kevin Choi is a rising sophomore at St. Mark's School of Texas. He likes math and playing frisbee and pool. He also recently qualified for the United States Junior Math Olympiad (USAJMO). Kevin Choi has only recently joined the iGEM project, but as a member of the web development team, he aids the progress of the technological parts of the projectKunal Dixit is a rising senior and 12-year Marksman who enjoys learning about biology and interacting with younger kids to help spark scientific interest in future generations. A member of the Community Service Board, Kunal enjoys the community outreach and education portion of the iGEM project. A recent member of the iGEM Team, he has also helped produce the tutorial videos and a film about the iGEM team, its goal, and its future.Retrieved from "http://2014hs.igem.org/Team:SMTexas/tea1251CSWProteensCambridge School Of Weston 45 Georgian Road Weston, MA 02493 USA Web. csw.orgHigh SchoolPlantiFreezeFrost damage to crops causes loses of billions of dollars annually. Currently, farmers? attempts to prevent frost damage are decidedly low-tech and only marginally effective. A synthetic biology solution to this problem is a spray-on slurry of a nonpathogenic strain of E. coli that covers the crop with a biofilm containing a secreted antifreeze protein (AFP). AFP's possess the ability to bind to the ice crystals on the plant surface, inhibiting the formation of ice. RiAFP refers to an AFP produced by the Rhagium inquisitor long horned beetle. This protein is particularly attractive for recombinant expression and biotechnological applications.1101BBa_K1251999http://2014HS.igem.org/Team:CSWProteensBBa_K1251000http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=160520140http://2014hs.igem.org/files/presentation/CSWProteens.pdfhttp://2014hs.igem.org/files/poster/CSWProteens.pdfHigh SchoolLog in   Team:CSWProteens/Attributions From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmail A T T R I B U T I O N S Micah Micah was on both the design and fundraising committees. He frequently worked in the lab and helped write fundraising letters. Summer Summer was on the fundraising committee and was in charge of writing all fundraising letters. Ben Ben was on the communications committee and helped design our Facebook page. He also spent a lot of time in the lab building our project. Matt Matt was on the design committee and did preliminary research specific to insect antifreeze. He also kept track of our inventory. Lilly Lilly was the head of communications committee and designed our posters, Tumblr, and took almost all of our pictures. Howard Howard is our faculty advisor and helped us in many different ways! In addition to keeping us organized and on track, Howard aided in our expression plasmid design and made sure that we got everything done in time. He was always present in the lab. Joey As Team Leader, Joey did a bit of everything. He aided in expression plasmid design, was present frequently in the lab, and helped to design the wiki and poster. Thomas Thomas was on design committee but was also in charge of modelling our dual plasmid system. He worked frequently in the lab. Noa As Project Manager and a member of both design and communications committees, Noa always had her plate full. She kept everyone on track, did extensive work on the wiki and presentation, and evaluated our expression plasmid sequence. Nate Nate was always busy during iGEM. He showed up to work in the lab very often, helped to organize our Illuminarium presentation, was on design committee, and did research on Western Blots so that we can analyze our final product. Mason Mason was in charge of creating the wiki. He was also on the design committee. Mason coded the structure for the wiki and many of the finer details. Liam Liam was on design committee and Plantifreeze was his idea! Jenny Jenny was on the design committee and was responsible for a large amount of research on E. Coli. She also spent a lot of time working in the lab Ellie Ellie was in charge of our presentation at Illuminarium and helped design our posters. Aiden Aiden was a common sight in the lab, as he worked there frequently. He also did a great deal of research on Plantifreeze and was very involved on fundraising committee. He made our project gif that can be seen in the top left corner. Retrieved from "http://2014hs.igem.org/Team:CSWProteens/Attributionsproject From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailP R O J E C T   D E S C R I P T I O N Annually, about 5% to 15% of agricultural produce is lost due to frost. The formation of ice damages plants by rupturing cells and also through dehydration as water molecules are drawn out of the tissue. Current solutions to this problem - such as using heat or covering crops with protective material - are cumbersome, costly, and not fully preventative. Synthetic antifreeze chemicals have not been proven to work. Even if they did, they would be need to be applied repeatedly, at great cost, and may also leave residues in the environment.The project undertaken by the Cambridge School of Weston (CSW) 2014 iGEM team aims to design a synthetic biology solution to this problem by building upon the work of the 2009 Utah State team that developed a protein secretion mechanism and that of the 2011 Yale team which synthesized an “antifreeze” protein (RiAFP) isolated from a cold-tolerant beetle called Rhagium inquisitor. This is the most potent antifreeze protein so far studied. Our device (Plantifreeze) is designed to mitigate crop damage caused by ice crystal formation on the plant surface. Plantifreeze is a dual plasmid system. Transport of RiAFP by the bacterium: We quickly realized that expression of RiAFP in the cytoplasm was only half the functionality needed. The protein had to be secreted outside the cell envelop of the bacterium, so the surface of the crop would be protected from damage by ice crystals. E.coli have two membranes: inner membrane and outer membrane. To transport RiAFP protein to outside of E.coli, the protein has to pass through the two membranes. However, we were puzzled by how we would get the individual bacterium to transport the protein out of the bacterial cell. We could lyze each bacterium but this seems not to be a very elegant solution. So we needed to find a transport system for RiAFP.Our research into the transport of proteins in E coli was very interesting. E. coli normally does not secrete proteins extra-cellularly except for a few classes of proteins such as toxins and hemolysin (certain proteins and lipids that cause lysis of red blood cells by damaging their cell membrane). There are six pathways for secretion of recombinant proteins in E coli, numbered I through VI. While all of these pathways differ mechanistically, they each promote secretion while maintaining the integrity of the cell structure. Types I and II are the most common pathways for recombinant protein secretion.Utah State to the rescue. Further review of past iGEM projects led us to the work of the Utah State University iGEM 2009 team. As their project, they developed improved production and harvesting methods for proteins and other products in multiple organisms using the standardized BioBrick system. In short, they developed a library of fusion-compatible BioBrick parts for targeting compounds for secretion. From their work we learned that recombinant proteins can be targeted to type I and II secretory pathways through genetic fusion with signal peptide targeting sequences. Type I secretion is a simple one step secretion system that can translocate proteins from the cytoplasm to the extracellular medium without protein interaction with the periplasm. Asif Rahman from Utah State was extremely helpful following our contacting him by email. He explained, “It looks like a type I secretion system would be most useful for your group.” The type 1 secretory system (T1SS). The alpha-hemolysin system is one of the best-studied type 1 secretion systems of E. coli. In T1SS the secretion occurs in a single step directly from the cytosol to the extracellular medium. The secretory machinery (translocator) of the alpha-hemolysin system consists of three proteins: C, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein. Proteins with C-terminally fused HlyA signal sequence can be recognized by the HlyB-HlyD-TolC translocator and targeted for secretion. So we fused the HlyA secretion tag to the C terminus end of the coding sequence for RiAFP. The expression of RiAFP fused with HlyA is controlled by one plasmid we call the expression plasmid. The translocator proteins (HlyB-HlyD-TolC) are expressed by a second compatible plasmid, pLG575. So the PlantiFreeze genetic circuit is a dual plasmid system consisting of the expression plasmid and the secretion plasmid, pLG575. These two plasmids had to be compatible (ie, different origins of replication and independent antibiotic selection). Plasmid pLG575 has p15A origin of replication and carries the chloramphenicol resistance marker.Design of the expression plasmid. We decided to engineer a single DNA fragment to serve as the expression genetic circuit in the PlantiFreeze Device. The circuit comprises the following components:BioBrick Standard Protein Coding Region Prefix /T7 Promoter /RBS /RiAFP /HlyA /Double terminator /BioBrick Standard Protein Coding Region SuffixPromoter. Our device uses a T7 system, as did the Yale team, because T7 RNA polymerase is an incredibly fast and powerful enzyme transcribing rapidly and profusely for as long as the T7 RNA polymerase is present. It synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently.The T7 promoter is a BioBrick part BBa_1712074C-terminal His-tag. In our protein expression construct, there is an amino acid motif that consists of six histidine (His) residues fused to the C-terminus of RiAFP. Histidine tags are widely used because they are small and rarely interfere with the function, activity, or structure of target proteins. The polyhistidine-tag is to be used to detect the secreted protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. HlyA-signal peptide. The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target RiAFP for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to RiAFP results in transport of the protein from the cytoplasm to the extracellular medium in a single step.The HlyA signal peptide is BioBrick pat BBa_K208006Terminator. There are several E. coli transcriptional terminators available via the Registry. The most commonly used type of terminator is a forward terminator. When placed downstream of a genetic part that is transcribed, a forward transcriptional terminator will cause transcription to abort. We use part BBa_B0015, a double terminator (B0010-B0012). BBa_B0015. This terminator has an Average Forward Termination Efficiency of 98.4%. Synthesis of the gBlock DNA fragment. The DNA sequences for the components could be found in the Registry. So it was not difficult to design the part. It was 864 base pairs in length. It is fast and simple to order a 864 base pair gBlock™ fragment from Integrated DNA Technologies (IDT) (double-stranded, sequence-verified genomic blocks that ship in only a few working days for affordable and easy gene construction). The first step in ordering a gBlock is automated review by screening tools and expert review by IDT scientists of entered sequences for characteristics that may interfere with synthesis. This review found that there were sequence repetitions longer than 8 base pairs that comprise a combined 91% of the sequences within a window of 70 base pairs beginning at base pair 235. This structure would interfere with synthesis. So the IDT gene product specialist optimized the sequence for expression in E coli and compatible with the gBlock synthesis process. Optimization theoretically just changes codon usage without changing the proteins coded. Below is the optimized sequence for our expression part:gaattcgcgccctttactggtcatccgcttaacgattggaaacacctcctatctcttcttcctgcacacccgcgtgaaagaagttaaaaagatgtatagctgtcgtgcggttggtgtagatgggcgtgccgtcaccgatattcaaggcacctgccacgcgaaggcaacgggggcgggtgcgatggctagcggtacctcggagccggggtcaacgtccaccgcgactgcgaccggccgtggcgctaccgcccggagcacaagtacaggccggggaacagccacgaccacggcaaccgggacagcgagcgccacgtctaacgcaatcggtcagggtaccgcgactaccacagcgactgggtcagcaggcggtcgggccacaggttccgctacgacctccagttctgcatcccagccgacccagacccagactattacggggccgggctttcagaccgccaaatcttttgcacgcaacacggcaactactaccgtgactgcgtctcatcaccatcatcaccatctggcgtatggttcacaaggtgacttaaacccgctgatcaacgaaatttcaaagatcatttcagcggcgggttcctttgatgtgaaagaggaacgcaccgccgccagcttgctccagttatcggggaatgcgagcgattttagttatggccgtaactcgattacgctcaccacgtcagcttaataatattaatcccaggcaagcaataaaactaagggttctgtggagcgccttggtctgtccttttacttactgttcgttggtgagcgctcgttattggagtctcactggctgactttcggttgggcgtttttacgtctgtatacaagctccggccgctgcag Sequence outlined in SeqBuilder (click to enlarge)Importantly, the EcoRI and PstI sites on the 5’ and 3’ ends of this gene were maintained compatible with BioBrick standard 3A assembly. This expression part ordered from IDT would be inserted into a linearized plasmid backbone with antibiotic resistance different from the plasmid controlling secretion (pLG575). We decided to use pSB1A3. This linearized plasmid is supplied by iGEM. We had exhausted our previous supply so ordered a new one.The plan is to insert the expression part into pSB1A3 and do cotransformation of BL21 DE3 cells with the two plasmids. Then we would induce the cotransformants with IPTG to express and secrete RiAFP. Finally, using the Invision In-gel Staining Kit we would analyze the media and BL21 DE3 cell lyzate for RiAFP. This would mean we work up to the very last minute before the Jamboree but we are determined to successfully complete the project.Why use BL21 DE3 cells for the expression platform? Expression can only be achieved in a bacterial strain carrying the gene for the T7 RNA polymerase (see paragraph on T7 promoter above). The most common cell strain to use with a T7 promoter system is BL21 (DE3) (competent cells are chemically competent cells used for high-level protein expression with T7 RNA polymerase-based expression systems). The BL21(DE3) contains the T7 RNA polymerase gene, under the control of the lacUV5 promoter, integrated into the chromosome. IPTG is used to induce the expression of recombinant proteins cloned into vectors downstream of a T7 RNA promoter and transformed into the BL21(DE3) cellspLG575 plasmid. The pLG575 plasmid was generously sent to us by our advisor at Utah State, Asif Rahman. On Nanodrop, the plasmid showed 260 ug/ul. Retrieved from "http://2014hs.igem.org/Team:CSWProteens/projecteam From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailB I O G R A P H I E S       Name: Aiden ‘17Age:16Reasons for Joining iGEM: I have an interest in biology and think that gentically modifiying living things is awesomeInterests: long boarding, programming, biking, fishingOne word to describe: tenaciousCommittees: Fundraising       Name:Ellie "Flopsie" Wolf ‘15Age:16Reasons for Joining iGEM: Need to keep up with the latest sciences and technologies if you wanna control the world, ya know?Interests: Film, comic books and anime, running road races, studies on myths and legends and Cheezit crackers with tea. One word to describe: Cartoonish (is that a word? I'm thinking of something that's so exuberant and animated and exaggerated that it should more likely be in a cartoon than in real life)From: Winchester, MA      Name:Jenny (Jee Youn) Woo ‘16Age:16Reasons for Joining iGEM: Synthetic biology seemed like an interesting topic to engage in.Interests: psychology, psychiatry, biology, and literature. One word to describe: pertinaciousFrom: Seoul, South Korea      Name:Liam Kaufer ‘16Age:15Reasons for Joining iGEM: I like to see science in action!Interests: Physics, biology, Engineering and Aeronautics.One word to describe: AvidCommittees: DesignFrom: Westchester, New York      Name:Yixuan (Mason) Liu ‘16Age:17Reasons for Joining iGEM: Well it's just worth a try, right? I mean, why not?Interests: Mechanics, Music, Physics, History, Philosophy, Programming, Basketball,arts and anything.One word to describe: humanCommittees: DesignFrom: Shanghai, China      Name:Nate Williams ‘16Age:16Reasons for Joining iGEM: I heard about IGEM at CSW, and I thought it would be an incredible experience. I was already interested in biology, and IGEM refined and solidified my interest. Interests: Synthetic Biology, Music, Science, Art. One word to describe: CuriousCommittees: Design, FundraisingFrom: Arlington, MA      Name:Noa Machover ‘15Age:16Reasons for Joining iGEM: I love biology, problem solving, and thinking about practical ways to change and better the world — it felt like a great integration of my passions.Interests: Music, trigonometry, physics, painting, the outdoors, literature, pets, cellular biology, neuroscience, programming, waltzing, knitting, cozy things, good conversation, traveling, small and beautiful thingsOne word to describe: CuriousCommittees: Design, Project Management, CommunicationsFrom: Waltham, MA       Name:Thomas Schaffner ‘15Age:17Reasons for Joining iGEM: I think Synthetic biology will be a crucial industry for my generation. I wanted to gain some first hand experience in the field and I think participating in Igem is a great way to do so. Interests: History, Political Science, Life Sciences One word to describe: indecisive (because I can't decide on a word, I mean the entire notion of describing yourself in a single word is a bit ridiculous) Committees: Design, Team Model (Head of Modeling)From: Concord, MA       Name:Joseph (Joey) Boots-Ebenfield ‘15Age:17Reasons for Joining iGEM: It had been a dream of mine to do iGEM since eighth grade, so I began the journey to create a CSW iGEM team my freshman year.Interests: Synthetic Biology, biotech, healthcare, archaeology, electronics, justice.One word to describe: CaffeinatedTeam Leader/Co-teacher From: Swampscott, MA       Name:Micah Rickles-Young ‘14Age:17Reasons for Joining iGEM: Synthetic biology is the closest thing to being god. God of E. Coli.Interests: Music, Biology, Bioethics, PhysicsCommittees: FundraisingOne word to describe: Human?From: Holliston, MA      Name:Lilly (Claire) Kerper ‘14Age:17Reasons for Joining iGEM: Trying out synthetic biology seemed like a natural extension of my interest in engineering--and it is!Interests: Engineering & physics, languages, visual arts, computer science (wow, it totally sounds like I don't have a life outside of school)One word to describe: DedicatedCommittees: CommunicationsFrom: Lexington, MA      Name:Matt Waters ‘15Age:16Reasons for Joining iGEM: I heard of it and thought that Genetic Engineering sounded interesting.Interests: Biology, engineering, chemistry and animationCommittees: DesignFrom: Concord, MA      Name:Ben Klebe ‘16Age:15Reasons for Joining iGEM: I enjoy engineering and was excited to apply that enjoyment to the field of biology.Interests: Electronics, Programming, Engineering, DesignCommittees: CommunicationOne word to describe: Practical From: Lexington, MA      Name:Summer Ardell ‘15Age:16Reasons for Joining iGEM:I am really interested in both biology and engineering and iGEM is an amazing opportunity to combine my interests.Interests: Robotics, anthropology, world religions, theater, and biology. Committees: FinancesOne word to describe: DiverseFrom: Melrose, MARetrieved from "http://2014hs.igem.org/Team:CSWProteens/teanotebook/protocols From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailP R O T O C O L S1. Stock Solutions2. Competent Cells3. Transformation4. Miniprep5. Bacterial Plates and Liquid Culture6. Running SDS PAGE Gels7. Restriction Digest8. Ligation9. DNA Agarose Gels10. Invision In-gel Staining11. Visualization of stained gelPDF file to be uploaded after Jamboree.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/notebook/protocoadvisors From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailA D V I S O R SWe would like to thank our phenomenal advisors and mentors who helped us along the way. They were patient, readily available to troubleshoot and discuss our project, and we would not have been able to accomplish nearly as much without them.Melodie Knowlton, Ph.DMelodie has just assumed the Chairmanship of the Science Department. Melodie earned her Ph.D. in cell and developmental biology from Harvard University and a B.S. in biology from Ohio State University.Committing to participation in the high school division of iGEM is a big deal for a secondary school. Here is a brief statement from Melodie Knowlton, our current chair of the Science Department:"CSW's Science Department encouraged our students to participate in iGEM because of the cross-discipline nature of the activities. The iGEM competition teaches students how to collaborate and to think critically. It challenges students and helps to deepen their understanding of the sciences. Moreover, synthetic biology provides an engineering context in which to learn molecular biology, genetic engineering and microbiology methods. It also provides a means to explore current and emerging research technologies that are hard to address in most high school science classes. High school iGEM also provides an avenue for students to build relationships with research scientists. Students collaborate with academic faculty in synthetic biology and scientists in biotech companies, extending the teaching of molecular genetic techniques into real world and authentic applications."Howard Goldsweig, MDHoward is a medical oncologist who worked in biotech clinical research and development. As an encore career, he joined the science faculty of the Cambridge School of Weston. Howard is from New Jersey and lives in Boston with his wife and Springer Spaniel, James.Stephanie Hays“Steph” is a PhD student in Systems Biology, Harvard University in Cambridge, Massachusetts. She works on engineering microbial communication in Pam Silver’s Lab. She holds BS/MS degrees in Biochemistry/Biophysics from Rensselaer Polytechnic Institute in Troy, New York. At RPI, she graduated Magna Cum Laude and was induced into the TriBeta Biological Honors Society. Steph is from New Jersey.Asif RahmanAsif is pursuing his PhD in Biological Engineering at Utah State University. His area of study is secretion based recovery of bioplastic from recombinant E.coli and scale-up of the process. Asif is from Auckland, New Zealand. Outside of the lab Asif plays cricket, tennis, swimming, and soccer. He also enjoys traveling.Alyssa HenningAlyssa is a PhD candidate and NSF GRFP Fellow in Jeffrey Catchmark's lab at Penn State. She majored in Bioengineering at Cornell, where she was a founding member of Cornell's iGEM program in 2008. After graduating, she went to Ginkgo Bioworks to further explore synthetic biology. She has been active in many aspects of iGEM since her undergraduate days. She is from Bakersfield, California.  Retrieved from "http://2014hs.igem.org/Team:CSWProteens/advisocontact From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmail Retrieved from "http://2014hs.igem.org/Team:CSWProteens/contacFrom 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailWelcome! We are the CSW Proteens! Scroll down to find more about our project! PlantiFreezeAnnually, about 5% to 15% of agricultural produce is lost due to frost. The formation of ice damages plants by rupturing cells and also through dehydration as water molecules are drawn out of the tissue. Current solutions to this problem - such as using heat or covering crops with protective material - are cumbersome, costly, and not fully preventative. Synthetic antifreeze chemicals have not been proven to work. Even if they did, they would be need to be applied repeatedly, at great cost, and may also leave residues in the environment. Solution The project undertaken by the Cambridge School of Weston (CSW) 2014 iGEM team aims to design a synthetic biology solution to this problem by building upon the work of the 2009 Utah State team that developed a protein secretion mechanism and that of the 2011 Yale team which synthesized an “antifreeze” protein (RiAFP) isolated from a cold-tolerant beetle called Rhagium inquisitor. This is the most potent antifreeze protein so far studied. Our device (Plantifreeze) is designed to mitigate crop damage caused by ice crystal formation on the plant surface. Plantifreeze is a dual plasmid system. Read more about our project. Follow our work in the lab. Human Practices Although it has its roots in the long established discipline of molecular biology, the field of synthetic biology is still in its infancy. Consequently, the overwhelming majority of the public has no awareness of this emerging field. An informal pool of the student body at our own school in the Western suburbs of Boston (ground zero for synthetic biology) confirmed this notion. Consequently, the goal of our Human Practices effort was to enlighten the student body of our secondary school about synthetic biology and to promote interest in synthetic biology and iGEM to younger generations of students.Retrieved from "http://2014hs.igem.org/Team:CSWProteproject/Methods From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailM E T H O D SThis section of our team Wiki gives an overall summary of the various methods we used to construct our PlantiFreeze device. It is important to note that the device is based on a dual plasmid design: one plasmid controlling expression of the recombinant protein, RiAFP, and the other plasmid controlling secretion of the protein. These procedures reflect the revised construction strategy implemented following our discovery that the wrong DNA sequence was originally ordered for synthesis by IDT.Genetic parts were constructed in accordance with the BioBrick technical standard. The DNA sequence comprising the expression plasmid was synthesized as a gBlock™ DNA fragment by Integrated DNA Technologies (IDT, Coralville, IA). Using standard iGEM 3A assembly, the DNA fragment is ligated with the linearized pSB1A3 plasmid (see plasmid sequence and map). pSB1A3 is a BioBrick standard vector for assembly and expression of BioBrick genetic devices. All restriction enzymes and related reagents were donated by New England Biolabs, Ipswich, MA. (click to enlarge) The pSB1A3 plasmid with the DNA fragment insert is used to transform competent NEB 10 beta cells. The cells are plated on agar supplemented with ampicillin. After 16 hours incubation at 37° C, individual colonies are selected and grown up in 5ml of LB broth (with ampicillin) in 15 mL culture tubes.Now that cell cultures of E. coli containing lots of copies of the expression plasmid have been grown up, Quiagen miniprep is used to access the expression plasmid DNA: lyse the cells, separate the DNA from the cellular material, and purify the sample to make sure you only have the plasmid DNA and not the E. coli's own genomic DNA. We used a Qiagen Miniprep Kit (Qiagen, Valencia, CA) and our mini-centrifuge with a max speed of 4400 rpm. Thanks to the generosity of Asif Rahman, our team received the secretion plasmid, pLG575, from the Utah State University. Plasmid pLG575 includes thecoding regions for proteins HlyB and HlyD and carries the gene for chloramphenicol resistance. The next step in the construction of our device is the cotransformation of competent BL21 DE3 cells (New England Biolabs) with the expression plasmid accessed by the miniprep and pLG575. The cells are plated on agar supplemented with ampicillin and chloramphenicol. After 16 hours incubation at 37° C, individual colonies are selected and grown up in 5ml of LB broth (with ampicillin and chloramphenical) in 15 mL culture tubes.Overnight cultures were then used to seed larger 250 ml flasks (50 ml media volume) at an initial optical density (OD600) of 0.05 at time 0 h. 0.1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma Aldrich, Inc. St. Louis, MO) was added to each flask. Flasks were removed at 24, and 48 h and media and lyzed cells were analyzed for RiAFP with the use of InVision™ His-tag In-gel Stain (Life Technologies, Carlsbad, CA), a fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. Electrophoresis of the sample is run on a polyacrylamide gel (1.0-mm thick NuPAGE® Novex Gel, Life Technologies, Carlsbad, CA). The staining is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). A His-tagged Protein Standard (BenchMark™ His-tagged Protein Standard, Life Technologies) is used as a positive control for the InVision™ His-tag In-gel Stain. After staining, His-tagged fusion protein bands are visualized with a UV transilluminator equipped with a camera. Click to enlarge image above.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/project/Methoproject/discussion From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailD I S C U S S I O NThe team felt it was useful to mention some reactions to the overall experience of iGEM at CSW. Here they are:Arguably the single most crucial factor in determining the success of an iGem project, perhaps even more so than the idea itself, is how well the team works as a group. We have certainly had our ups and downs with that aspect of the iGem process. Communication and participation have turned out to be the keys to progress for us. When we met or were in contact and everyone was on the same page, we got work done. On the other hand, when we had limited communication and especially participation, we got little to no work done. Our idea remained mostly unchanged from the beginning, so the only factor controlling our progress towards our goal was teamwork. This day and night difference showed us how effective good teamwork can be.Teamwork and a good project idea are both necessary for success in iGem, but both are useless without guidance from an advisor. The job of the advisor is to guide us through the iGem process from a position of experience while still allowing us to discover and do things for ourselves. One of the most important things we learned about working with our advisor is that although it was his job to guide us, we had to communicate with him to make sure everyone knew what was going on and what was being done. He was overseeing the project’s completion and we had to let him know that we were completing parts of the project, not just expecting him to read our minds.In addition to our advisor, we benefited greatly from the help of external advisors. These were actual professionals working currently in the field of biology. The expertise, facilities, and recourses that our external advisors brought to the table were invaluable. External advisors were also very helpful in troubleshooting the problems that arose during the process due to their high level of expertise.The iGem competition is a very effective teaching tool for several aspects of scientific careers. It taught us about applying for fundraising, about presenting, and about the high level of independence and interdependence in lab work. It was an extremely valuable experience especially for those of us going into lab science as a profession.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/project/discussioproject/results From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailR E S U L T SOur original construction of our expression plasmid did not go according to plan. We received the parts and ligated them, but were confounded when our transformations of this plasmid into competent cells did not work. Thanks to the Silver Lab at Harvard Medical School, we were able to use SeqBuilder software to examine our sequences and the restriction sites to make sure that they were intact. To our dismay, we learned that a deleted base, guanine, in our sequence led to a frame shift. Since this problem was easily pinpointed, we were able to construct a new expression plasmid to be synthesized by IDT. Experiments on this new plasmid will be taking place in the coming week.Because of this delay in our lab experimentation, the actual findings of our work are not on this Wiki, but will be available for presentation at Jamboree 2014. In this Wiki, our experimental methods and expected results will be summarized. The goal of our experimental work is to assay the culture media and the lysate of the cotransformed BK21 DE3 cells in order to detect the presence of RiAFP in these fractions. The analyzes will be run on the fractions before and after induction of the expression of RiAFP with IPTG. A word about the induction of the system by IPTG The lac operator sequence is incorporated into the T7 promoter. The gene for RiAFP is inserted just after the T7 promoter DNA sequence. The lac repressor (LacI) binds to the lac operator sequence and blocks T7 RNA polymerase from binding the promoter sequence. Before the gene for RiAFP is transcribed two things need to happen:1. The lac repressor (LacI) must fall off of the operator DNA sequence in front of the RiAFP gene2. T7 RNA polymerase must be introduced and recognize the T7 promoter in the absence of repressor.When both of these factors are satisfied, transcription of the gene will proceed rapidly. If induction occurs during the mid-log phase of growth (OD600nm between 0.6 and 0.8), the yield of RiAFP protein will be maximized.When lactose binds to LacI it induces a conformational change in the protein structure that renders it incapable of binding to the operator DNA sequence. IPTG is a structural mimic of lactose (it resembles the galactose sugar) that also binds to the lac repressor and induces a similar conformational change that greatly reduces its affinity for DNA. Unlike lactose, IPTG is not part of any metabolic pathways and so will not be broken down or used by the cell. This ensures that the concentration of IPTG added remains constant, making it a more useful inducer of the lac operon than lactose itself. Once the lac repressor can no longer bind the operator, native E. coli RNA polymerase begins transcribing, in high numbers, the T7 RNA polymerase gene engineered into its chromosome. Once the T7 RNA polymerase protein is expressed, it binds to the T7 promoter sequence upstream of the gene for RiAFP on the plasmid insert and transcribes the RiAFP gene. The initiation of transcription of the RiAFP gene culminates in the secretion or RiAFP into the cytoplasm of the BL21 DE3 cells, thence into the periplasm of the cell, and finally into the space outside the cell membrane. We use standard methods to obtain cell-free supernatant from the culture media as well as proteins from the cell lysate. Proteins from these two fractions are separated by electrophoresis on an NuPAGE Bis-Tris gel (4%–12%). The gel is stained with InVision™ His-tag In-gel Stain. This is a fluorescent stain for detection of histidine-tagged recombinant fusion proteins in protein polyacrylamide gels and is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). This technique eliminates membrane transfer and Western blotting steps.One of the lanes of the electrophoresis gel contains BenchMark™ His-tagged Protein Standard, used as a positive control and for molecular weight sizing in his-tagged fusion protein detection. This molecular weight standard produces 10 sharp and clear bands in the range of 10-160 kDa for molecular weight estimation of his-tagged proteins (see figure of benchmark standard). The molecular weight standards are used to measure the relative sizes of the unknown proteins. RiAFP has a molecular weight (MW) of 12.8 kDa. In the lanes with samples of protein from the supernatant and cell lysate before induction with IPTG, there will be no detectable band corresponding to a protein of a MW of 12.8 kDa. However, the lanes with samples of protein from the supernatant and cell lysate after induction there should be detectable tagged protein corresponding to the expected MW of RiAFP Retrieved from "http://2014hs.igem.org/Team:CSWProteens/project/resulproject/safety From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmail S A F E T Y Would any of your project ideas raise safety issues? Regarding organisms, our project raises minimal concerns for safety. We use E. Coli bacteria, which has been classified as biosafety level one by the World Health Organization (WHO) Laboratory Biosafety Manual. E. Coli, “is a non-pathogenic strain that cannot permanently colonize the gut of healthy humans or animals” (WHO Laboratory Biosafety Manual). Our team also uses a DNA sequence from the Rhagium Inquisitor beetle, but we received the part directly and had no contact with the beetle. We have managed to keep our lab safe, and have used reasonable safety precautions. No organisms or materials we handled in our lab were dangerous other than bunsen burners and UV lights, but we nevertheless enacted safety measures. We cleaned all surfaces with 70% ethanol, and sterilized all unsterile materials with an autoclave. We properly disposed of waste in the lab. All team members wore safety gloves and glasses when appropriate. A safety shower-and-eyewash was readily available in the lab, in case of emergency. UV-protection glasses were worn when we used UV lights to detect dye in electrophoresis gels. Heat-gloves were used by team members when handling potentially hot objects. All bunsen burners included safety shutoffs, and team members were instructed how to use these burners safely. Our instructor clearly led us through each lab process and all safety measures necessary before any of said processes were conducted. If our project did not go to plan and the organisms or their byproducts were released, no immediate threat would be posed. As mentioned above, E. Coli is not a harmful organism. Furthermore, it can only live in a lab environment, and would not survive outside. The antifreeze protein that our project will supply also poses no threat. As stated in Yale’s 2011 IGEM team wiki, many antifreeze proteins “are already being used in industry and for consumer products” (http://2011.igem.org/Team:Yale/Safety). Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?None of the devices we made raise any safety issues. As stated above, the antifreeze protein that our device expresses is unharmful and completely safe. Is there a local biosafety group, committee, or review board at your institution? Our high school has no official biosafety review board. However, our science department and our instructor have reviewed all lab procedures we used, and they have ensured the continued safety of anyone in contact with the lab. Our lab also complies to all biology lab standards of the United States, as legally required. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?Many high schools require students to sign a Laboratory Safety Contract. This simple approach to lab safety strengthens student awareness of an appropriate lab safety culture. Are contaminated materials and biohazard waste handled according to current guidelines?Receptacles with biohazard bags are provided for the disposal of contaminated waste. Any material that has been contaminated with bacteria are placed in these buckets. No liquids containing bacteria are ever poured down the sink. No cultures or culture material may be taken from the lab.Non-Disposable contaminated materials are decontaminated and then are autoclaved and reused. Examples are plastic caps, forceps, and glass screw-cap tubes. Designated containers of disinfectant are provided for these items.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/project/safethumanpractices From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailO U T R E A C HAlthough it has its roots in the long established discipline of molecular biology, the field of synthetic biology is still in its infancy. Consequently, the overwhelming majority of the public has no awareness of this emerging field. An informal pool of the student body at our own school in the Western suburbs of Boston (ground zero for synthetic biology) confirmed this notion. Consequently, the goal of our Human Practices effort was to enlighten the student body of our secondary school about synthetic biology and to promote interest in synthetic biology and iGEM to younger generations of students. For the human practices component of our project, we shared our enthusiasm for synthetic biology with our school community. There is an annual festival of light and art at our school called Illuminarium, to celebrate the springtime. At the event, students present their various artworks, such as film, photography or sculptures. We thought that we could show E. coli that had been transformed with pPRL (a purple pigment-producing plasmid) and pGRN (a green pigment-producing. We are grateful to BioBuilder for the kit that allowed us to do the transformations. We explained the process of transformations and the goals of synthetic biology. We also informed the community of our project through posters and conversation. Many students had never heard of iGEM or synthetic biology, and their enthusiasm after speaking with us was apparent. Although Illuminarium is typically a festival for the arts, students and faculty were interested in and receptive of our presentation, demonstrating the interdisciplinary nature of the event and community, as well as the allure of synthetic biology.The team at the Illuminarium table.To better prepare us for the effort, described below, we surveyed the literature and developed a briefing document for distribution among the iGEM team at CSW.The major points made in the briefing document are summarized in bullet points herein:• Providing individuals with a fundamental knowledge of synthetic biology allows each person to form fact-based opinions, hopefully forestalling the development of misconceptions• The level of support for synthetic biology research will receive depends on public opinion.• Government regulations and public policy are driven by the beliefs and viewpoints of the public. If the public are not educated on the benefits and risks of synthetic biology they cannot make informed decisions regarding its support.• Research in this field has enormous potential for the advancement of technology, including medicine, pharmaceuticals, food and agriculture, the environment, and alternative energy.• The potential benefits of the technology must not be overhyped for this risks both creating excessive public anxiety and unrealistic hopes.• It is perfected reasonable and expected for the public to have concern about unforeseen, unintended consequences that might occur from synthetic biology• Emphasize that a variety of government and private organizations study and monitor the potential risks of synthetic biologyOur posters. Click to enlarge.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/humanpracticnotebook/ From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailL A B   N O T E B O O K:2013: November | December | 2014: January | February | April | May | June November 13Our first full-length meeting of the year. We spent much of the time discussing the logistical (financial, organizational, etc.) details of the iGEM Competition, before splitting off to confer with our small, self-assigned subcommittees: Design, Communications, Fundraising, and Project Management.Pictured: Howard, a co-teacher and Faculty Advisor, presenting on the necessary functions of a cohesive iGEM team.November 18Our lecture half of the class consisted mostly of a detailed overview of synthetic biology, its engineering aspects, and our place in the process. During the lab half, we learned how to make gels for electrophoresis.Pictured: Top: Howard draws a diagram of the parts of a biological device.Bottom: a buffer is added to the gel after it sets.November 20We spent the first part of class tossing around ideas for a team name; when it came down to either sticking with the school’s mascot (the gryphon) or choosing something more original, a narrow majority vote decided on the latter. Thus, we are now officially the CSW ProTeens. We then learned more about genetic devices, and specifically their promoters.Pictured: Top: Howard describes the inner workings of the lac operon.November 25Today, Joey was in sole charge of the class. His lecture covered plasmid backbones in detail. We also watched this CrashCourse video on transcription and translation. For the lab, we learned (or reviewed) how to make agarose gels, use parafilm, and operate a centrifuge.Pictured: Left: Joey illustrates how the backbone of a plasmid can be cut to accept new parts.Right: Aiden pours a gel as Liam and Mason look on.December 4Today’s lecture was on translational control, and the workings of DNA polymerase. For the lab, we began to work on making our own ampicillin plates from scratch.Pictured: Top: Howard draws (and vigorously circles) a ribosome binding site.Bottom: Howard demonstrates the process of making amp plates.Decmber 9This class was dedicated to finishing our ampicillin plates. We also broke in the new autoclave.Pictured: The team intently examines the working autoclave.December 11The team split into smaller groups, and each performed a transformation of pPRL, a purple pigment producing plasmid, into E. coli.Pictured: Top: Nate, Aiden and Liam put their plasmids on ice.Bottom: The plasmids are put on our ampicillin plates.December 16For the lecture portion of class, we learned about “RNA thermometers,” and then discussed logic gates and used them to analyze some devices. We then viewed the (successful!) results of our pPRL transformation.Top: Howard explains truth tables.Bottom: Our E. coli is purple!December 18Joey gave a lecture on 3A assembly. Our lab was to transform pGRN, a green pigment producing plasmid, into E. coli.Top: Joey details the standardized DNA sequences that allow plasmids to be cut.Bottom-left: Noa and Jenny work on the transformation lab.January 6Happy New Year! We dedicated today to preparing gels for our next lab.Pictured: a comb is inserted into the not-yet-solidified gel to create wells.January 8We began a restriction digest lab, but due to technical difficulties had to postpone until the next class. We then split into committees for a regular update.Pictured: Joey and Howard divide reagents.January 13We did some more practice with electrophoresis using lambda DNA.Pictured:Top: Noa removes a gel from the electrophoresis chamber.Bottom: Our results were highly visible when the gels were placed on a light box.January 15We dedicated the whole meeting to practicing with a restriction digest.Pictured: Nate, Aiden, and a blurry Liam fill a gel’s wells.January 22In pairs and singles, we researched and presented the team’s top ten project ideas and began to narrow down that list.We dedicated the whole meeting to practicing with a restriction digest.Pictured:Top: Liam and Aiden present their findings.Bottom: Noa and Jenny set up before sharing theirs.January 27After the previous week’s presentations, we worked to more fully develop the most feasible ideas and draft circuits for them.Pictured:Top: Joey and Howard review the process of writing a circuit.Bottom: Lilly begins to draft a circuit on the whiteboard.January 29We gave our final presentations on the most feasible project ideas, which we had been working on for weeks.Pictured:Top: Summer discusses her proposed circuit.Bottom: The final list of ideas to be discussed and voted on. (Voting results coming soon…)February 3The results are in! We, the CSW ProTeens, will pursue plantifreeze for our final project. "Plantifreeze" is our shorthand name for a bacteria that prevents frost from forming on fruits near the end of the fall growing season—in other words, a plant antifreezePictured: Liam reviewing his proposed circuit for the project, just before the team splits into smaller groups to research various parts.March PlanningOur school has a schedule where courses run for sections of 5-weeks. After February, our class section finished and we did not have a structured meeting time. In this interim, we worked on planning out our project, contacting advisors, and ordering parts for our project.April 27This weekend we made stock solutions of antibiotic solutions, prepared sterile LB agar plates supplemented with ampicillin, chloramphenicol, and the combination. We also prepared SOB broth for growing up transformed bacteria. Consequently, we are ready now to begin assembly of our device.April 28We received the pGL575 plasmid from Utah State today by FedEx. Asif suggested that we transform our competent cells with the plasmid ASAP. So we did just that. The plasmid carries the gene for chloramphenicol resistance so we used LB agar with chloramphenicol to select for the transformed cell. We made 5 agar plates so we should have plenty of the plasmid. This plasmid contains the genes for allowing the secretion of proteins made by the bacteria via Type 2 secretion system. Our other plasmid that we are assembling in the lab, regulates secretion of the RiAFP protein. That's why our device is a dual plasmid system: one plasmid controls expression of the protein and the other plasmid regulates secretionApril 29We just checked the selection plates for transformed NEB 10 beta cells. Four of the five plates showed colonies so we were successful with the transformation. The plates are in the frig ready to be grown up to supply the pGL575 plasmid for the device. Next step is to assemble the gene for expression of RiAFP with tag for 6xHis and peptide signal for secretion. We need to put that fusion gene into a plasmidMay 1Kudos to Aiden who did the majority of the lab work. We took the gBlocks of parts A and B of the expression plasmid and the linearized plasmid pSB3A1 and digested the prefix and suffix of each with restriction enzymes: EchoRI, SpeI, Xbal, PstI, PslI. These enzymes recognize restriction sites and cut them in ways that allow part A to fuse with part B and for the fusion of A-B to fit into pSB3A1. The next step is to permanently ligate the parts so that we end up with a circular plasmid carrying the fusion part. The ligation will be performed on Thursday morning between 10 am and 1 pm. Drop by.May 6The transformation failed. We later discovered that we used 0.2 micrograms of plasmid instead of the suggested 1 microgram while constructing the expression plasmid from parts A and B. This altered the volume of the reaction, and was likely the cause of failure.May 8In absence of gBlocks, we can not do anything with the expression plasmid. We took individual colonies of NEB 10 cells transformed with pLG575 plasmids and put them in LB broth cultures to incubate at room temp for 24 hours. Growing up these cells will afford us a large supply of the pLG575 plasmids.We ordered more gBlocks from IDT. We will carefully rewrite the protocol for digestion and ligation of the parts that go into the linearized pSB3A1 so transformation can be successful with a future attempt. Also will run concurrent positive control in the future to avoid our inability to analyze any future failures.May 14After very careful preparation, the parts for the expression plasmid were digested, ligated and the resulting ligation product was used to transform NEB 10 cells today with the capable assistance of Aiden, Nate, and Noa. Many things were changed from the unsuccessful transformation from last week. The transformed cells are in the 37 degree C incubator so please everyone hold your breathMay 15Transformation is successful. Next step is to grow out colonies of transformed cell in liquid culture so we have good supply. Then we mini prep both plasmids and cotransform DB21 DE3 cells and look for recombinant protein in supernatant and lysate.So we have the expression plasmid in NEB 10 beta cells on agar supplemented with amp. The next step is to grow up colonies form the agar plate in LB broth. This will give us a large supply of plasmids. We will miniprep the liquid culture getting bare plasmids for contranformation of DB21 DE3 competent cells with both plasmids. By using Western blot we confirm presence of RiAFP in supernatant and lysate. What did we do right this time. In the ligation step, we were very careful to use the correct proportion of DNA from the inserts (Parts A and B) and the vector (linearized pSB1A3). We used 50 ng of vector DNA and made use of a calculator on the NEB web site to calculate the amt of insert DNA to use. There should be a 3 to 1 molar excess of insert DNA to vector DNA to ensure that all the linearized vector is fully occupied by insert DNA. The calculation used the nucleotide lengths of the inserts (about 450 bp for each part) and the vector (2155 bp) to make the calculation. Also in an incubation in ligation we incorrectly used 22 degrees C instead of room temperature originally. This was corrected in the lates transformation.May 18After we transformed the NEB 10 beta cells with the newly assembled plasmid (T7 RBS RiAFP 6xHis HlyA term term), Ellie selected a few individual colonies from the agar plate supplemented with amp and put them in 50 mL of LB broth supplemented with amp in sterile 250 ml flasks with a sterile magnetic stirrer and grew up the cells over 16 hours at room temperature with gentle stirring. Our newly acquired rotating shaker has a broken belt so it is on the bench. After the 16 hour incubation, the broth had turned from clear to opaque. We transferred 10 ml of the broth to sterile falcon tubes and stored them at 4 degrees C. We do minipreps on the cells to isloate and pruify the plasmids. We also transformed NEB 10 cells wit the pLG575 plasmids and stored them in10 ml broth at 4 degrees C. So now we have ample supplies of both plasmids.The next step is to miniprep the transformed cells. Then we cotransform the two plasmids into DB21 DE3 competent cells. Our device includes an expression vector containing RiAFP cloned downstream of the T7 promoter. DB21 DE3 competent cells carry a chromosomal copy of the phage T7 RNA Polymerase gene, which is controlled by a lac promoter. When inducer (IPTG) is added, T7 RNA Polymerase is expressed and becomes dedicated to transcription of RiAFP.Plasmid pLG575 includes the coding regions for proteins HlyB and HlyD.May 20We are ordering the DB21 DE3 competent cells from NEB very shortly in preparation of cotransformation of these cells with the expression and secretion plasmids.May 22We took individual colonies growing on agar supplemented with chloramphenicol and put them in SOB culture broth. SOB is a slightly modified LB broth. The cells, NEB 10 beta cells, transformed with pLG575 plasmid will grow in the broth and provide a source of the plasmid. We also transformed NEB 10 cells with the expression plasmid.Currently we have liquid cultures of NEB cells with the expression plasmid and liquid cultures of NEB cells with the secretion plasmid. At this point we are poised to do minipreps on both types of cultures. The minipreps will give us bare plasmids that in turn will be used to transform BL21 DE3 E coli cells. These are chemically competent E. coli cells suitable for transformation and T7 promoter driven expression of RiAFP expression. The two plasmids will be used to cotransform BL21 DE3 cells. Following cotransformation, expression of RiAFP will be induced by IPTG.May 24On Tuesday we will do miniprep on the cells containing the expression plasmid. At that point we will be in a position to cotransform the BL21 DE3 cells that constitute the PlantiFreeze device. Competent BL21 DE3 cells will be shipped to the lab on Wednesday on dry ice. We will transform the cells on Wednesday. On Thursday the 16 hour incubation of the transformed cells will be ready. On Thursday, we will take individual colonies from the agar plate and put them into SOC media. On Friday of next week if all goes well we will have an ample supply of the BL21 DE3 cells transformed with both the expression and secretion plasmids. At this point the moment of true has arrived! By introducing IPTG into the culture we should be able to induce the cells to express RiAFP and secrete the recombinant protein into the medium in which the cells are growing. Here is where Steph and the Silver lab come into action. We need to assay the medium and the lyzed cells to confirm the RiAFP was expressed by the E coli and secreted. We will do this using Western blot.There will be other analyzes that we'll also do:• Effect of each plasmid and both plasmids on the growth rate of BL21 DE3 cells (by turbidity analysis using spectrophotmometer• Gel electrophoresis of some of the intermediate constructsMay 24Rather than using Western blot we are thinking about the use of Invision In-gel stain from Life Technologies. Visualization of the fluorescent bands uses:UV transilluminator (302 nm) equipped with a camera capable of integrationTo view and photograph a gel on the UV transilluminator, use a video camera, CCD (Charged Couple Device) camera, or a cooled CCD camera with ethidium bromide filter or band pass filter encompassing the emission maxima(590 nm) of the stain. May 27DL21 BE3 competent cells from NEB arrive tomorrow. Today we will do miniprep on the cells containing the expression plasmid. So how 'bout we order:InVision™ His-Tag In-Gel Staining KitInVision™ His-tag In-gel Stain is supplied as a 1X ready-to-use staining reagent (500 mL) [enough to run 20 mini gels]BenchMark™ His-tagged Protein Standard (125 µL) is also included in the kitNuPAGE® Novex® 10% Bis-Tris Gels (pre-cast polyacrylamide gels)May 28Thanks to Joey and Thomas for doing the miniprep on the expression plasmid. We now have pure DNA from both plasmids and are poised to cotransform BL21 DE3 competent cells. This is the last step in the assembly of the PlantiFreeze device. After that step we do functional assays to confirm that the device works to express and secret RiAFP. As a side venture we'll be exploring the effect of transformation of the BL23 DE 3 cells with each plasmid alone and both together. We'll be constructing growth curves based on optical density of the culture media.We have ordered from LifeTechnologies Invision in-gel stain, a latter, and precast gels for confirming the expression and secretion of the protein. These experiments will be done with Steph Hayes at Pam Silver's lab at Harvard Medical School.Keep your fingers crossed that everything goes right and we'll be in the home stretch. Still need to do data analysis, the Wiki, our poster and the presentation so we need everybody at battle stations a while longer.Finally, we need to do more minipreps more growing up liquid cultures, more transformations so please avail yourself of the opportunity to get your hands wet in the lab. Its fun, its instructional, and you help is really needeMay 29Lab today 1 to 5:• cotransformation of Bl21 De 3 cells• miniprep of plasmids• making agar plates• making liquid cultures• planning measurement of oD of culture for growth curvesMay 31Yesterday was a banner day for lab work. Thanks to Joey, Micah, Aiden, and Thomas. We did a miniprep of the NEB 10 cells transformed with the secretion plasmid pLG575. Did the milestone cotransformation of BL21 DE3 cells with the expression and secretion plasmids. On Saturday after 16 hours incubation at 37 degrees C we should know if it worked. This time we ran a positive control of pUC16 DNA so if something goes wrong will have the control to help us unravel the problem.If the cotransformation works our next step is to grow up the cotransformed cells in LB broth and then induce them with IPTG to express and secrete RiAFP.Remember we plan on determining the influence on, the other and both plasmids on the growth of BL21 DE3 cells using the optical density of the liquid culture on the spectrophotometer. If time before the close of the Wiki permits we might also do digestions of the plasmids and look at their electrophoresis patterns.June 1No growth on either the plates with contransformation or the positive control. It is obviously useful to have a positive control because now we can conclude that we are doing something wrong with the transformation procedure or the competent cells are no longer competent. In fact, in our hands, tansformation has been very unreliable. But this is the first time we ran a positive control.June 2The NanoDrop is a specialized spectrophotometer that measures the amount of DNA in a sample as small as 1 microliter. We are having trouble with the cotransformation and we are worried that the plasmid DNA we are getting from the minipreps was too little. As a benchmark, it is good to have more than 50 nanograms/microliter of plasmid DNA.We have two plasmids: pLG575 and the expression plasmid. We have 5 nanograms/microliter of this plasmid. Obviously very low. The other plasmid is the expression plasmid. We have 47 nanograms/microliter of this plasmid. Good.We can adjust the miniprep to increase the concentration of pLG575 DNA,The puzzle is that we should have gotten transformation of the expression plasmid. So we still have to look at the transformation procedure.June 6This has been a frustrating and puzzling week. The cotransformation performed on Sunday failed, leading us to wonder if we got plasmid DNA from the minipreps of the expression and secretion plasmids. Steph generously allowed us to use the Nanodrop analyzer in her lab. We found that the DNA concentration was low but acceptable for pLG575 and in a good range for the expression plasmid. We made some alterations to the miniprep procedure. We repeated the transformations in a wide ranging way and were amazed that the only plate with growth was the one with agar only. This observation directed us to think about a problem with the antibiotics so we made new stock solutions and were especially careful about antibiotic concentrations in the most recent transformations. On Thursday, Joey, Micah, and Thomas were in the lab. We used BL21 DE competent cells and did the following:• cotransformation with the original pLG575 plasmid from Utah State and the expression plasmid• transformation of pUC19 positive control plasmid• transformation with pLG575 plasmid obtained by miniprep using modifications to increase concentration• transformation of the pPURP plasmid from the BioBuilder lab exerciseAll in all, maddeningly frustrating, but we will persevere.June 7Puzzling, frustrating, and humiliating! We used commercial BL21 DE3 competent cells in the transformation reactions. pLG575 (orig) refers to the plasmids shipped to use by Utah State; pLG575 (miniprep) refers to the plasmid DNA we got by miniprep of the transformed NEB 10 beta cells transformed with the pLG575 plasmids.pPURP is the plasmid from BioBuilders and pUC19 is the positive control plasmid sent with the competent cells by NEB. We mixed new stock solution of ampicillin and chloramphenicol and carefully checked the concentrations in the LB agar, so I am really bewildered by the discrepancies between the expected and observed results.June 10Thank you Aiden, Noa, Ben, and Joey for working in Steph's lab at Harvard Medical School. In an attempt at troubleshooting our problem with transformation we did transformations with several different strains of competent cells. The plasmids we used included: pLG575, the expression plasmid, pUC19.June 11Yesterday, we used five different strains of competent E coli and five separate plasmids for transformation of the strains of competent cells. Here are the results of the growth of the plates with the appropriate antibiotics:Results from Steph’s LabDisregarding one or two anomalies, the analysis indicates two things:The BL21 DE3 competent cells don't appear to be competent. Keeping them on dry ice may not have been able to maintain a stable -80 degree centigrade temp allowing them to become incapable of transformation (see column pUC19). Please note that the prior batch of competent cells from NEB (NEB 10 beta cells) were transformed by the pLG575 plasmids we recieved from Utah State and the plasmid we constructed from the gBlocks form IDT. I did not observe thawing of the BL23 DE3 cells. The plasmid DNA we obtained by miniprep were not able to transform any competent cells (see last three columns). The cause of the failure of the miniprep remains uncertain.June 12Hi, all. Let’s summarize where we are at the moment.We have plasmids with the expression cassette (amp resistant) and the secretion cassette (chloramphenicol resistant), respectively in transformed NEB 10 beta cells. This is where we reached a stone wall. Using minipreps, we were unable to cotransform BL21 DE3 cells and induce those cells with IPTG to express and secrete RiAFP. We have an in-gel stain that will enable us to detect RiAFP in cytoplasm and culture media standing by and ready to use (takes about 3 hours to do).Under the guidance of Steph, team members performed transformations using several E coli strains and different plasmids (the results summarized in an email message sent to all yesterday). The analysis of those results suggest that the commercial competent cells from NEB (BL21 D3) were degrade by unstable maintenance of -80 C temp and that our miniprep procedure was flawed. It is easy to overcome the competent cell flaw. Not so easy is pin pointing what's wrong with the miniprep. Since we have amp and chloramphenicol resistant NEB 10 cells in the Bio 3 frig, we can analysis the plasmids in those cells and figure out a strategy to go forward. We have in the frig in Bio 3 agar plates and LB broth cultures of the NEB 10 beta cells which are amp resistant cells andchloramphenicol resistant cells meaning they were transformed by the expression and secretion plasmids, respectively. I will bring them to the AlbertsLab tomorrow for analysis confirming the presence of the appropriate plasmids in the cells. Needless to say, the clock is ticking and it will be a challenge to finish all the lab work. The Wiki closes on 20 June, but the lab work can go on beyond that date and any new data can be incorporated in the Poster and the PowerPoint slide deck. Even then time may run out despite the best efforts.June 13Noa's review of the Parts synthesized by IDT prompted me to do a deep dive into the parts sequence synthesized by IDT. Oh boy, did we mess up and compounding that problem we didn't check the parts sequence when they came back from IDT. But since they have a 3-5 business day turn around we can recover but we want to order just one DNA fragment which will be inserted into a linearized plasmid with an antibiotic resistance different from the secretion plasmid. That plasmid will then be cotransformed with our secretion plasmid into BL21 DE3 competent cells. Uncovering the incorrect sequence in the expression plasmid solves our current problem of not being able to cotransform in that we'll have a brand new plasmid and we will use new competent cells. Here is new revised approach:we need a plasmid which contains these components and BioBrick standard prefix and suffix (not the RFC 23):• T7 promoter• RBS• RiAFP coding sequence• HlyA coding sequence• 6x His marker• double terminatorWe insert (using digestion and ligation) the above fragment into a linearized plasmid with a resistance marker other than chloramphenicol (the secretion plasmid is chloramphenicol resistant). We then use Invision in gel stain to detect presence of RiAFP in cytoplasm and media.We'll be working up to the last minute: here's the timeline:• Receive new part from IDT: 19 June• Insert new part into linearized plasmid: 19 June• Transform competent cells to grow up stock of plasmid: 20 June• Grow up liquid culture of transformant: 21 June• Miniprep of plasmid DNA: 22 June• Transform BL21 DE3 cells: 22-23 June• Grow up transformed BL21 DE3 cells and induce expression and secretion of RiAFP: 23-24 June• Use Invision system to detect RiAFP in fractions: 25 June• Analyze results: 25-26 JuneJune 14After the disheartening discovery that Part A had been incorrectly designed, we decided to engineer a single DNA fragment to serve as the expression genetic circuit in thePlantiFreeze Device. The circuit would comprise the following components:BioBrick Standard Protein Coding Region Prefix /T7 Promoter /RBS /RiAFP/HlyA /Double terminator /BioBrick Standard Protein Coding Region Suffix The DNA sequences for the components could be found in the Registry. So it was not difficult to design the part. It was 864 base pairs in length. It is fast and simple to order a 864 base pair gBlock fragment form IDT (double-stranded, sequence-verified genomic blocks that ship in only a few working days for affordable and easy gene construction). The first step in ordering a gBlock is automated review by screening tools and expert review by IDT scientists of entered sequences for characteristics that may interfere with synthesis. This review found that there were sequence repetitions longer than 8 base pairs that comprise a combined 91% of the sequences within a window of 70 base pairs beginning at base pair 235. This structure would interfere with synthesis. So the IDT gene product specialist optimized the sequence for expression in E coli and compatible with the gBlock synthesis process. Optimization theoretically just changes codon usage without changing the proteins coded. Importantly, the EcoRI and PstI sites on the 5’ and 3’ ends of this gene were maintained compatible with BioBrick standard 3A assembly. However, Noa at Steph's lab ran the optimized new sequence thru the seqbuilder program and identified that the optimization process put in another Pst1 restriction site at position 637 in addition to the appropriate one at position 863 (the one compatible with the BioBrick standard). Having the secondPstI restriction site breaks the BioBrick standard and makes correct assembly impossible. Following the finding of this glitch, the fragment was reoptimizedremoving the PstI site at 836. The part was order for synthesis and delivery as soon as possible. New sequence outlined in SeqBuilder (click to enlarge)This new part just ordered from IDT would be inserted into a linearized plasmid backbone with antibiotic resistance different from the plasmid controlling secretion (pLG575). We decided to use again pSB1A3. This linearized plasmid is supplied by iGEM. We had exhausted our previous supply so ordered a new one.The plan is to insert the new part into pSB1A3 and again do cotransformation of BL21 DE3 cells with the two plasmids. Then we would induce thecotransformants with IPTG to express and secrete RiAFP. Finally, using theInvision In-gel Staining Kit we would analyze the media and BL21 DE3 cell lyzatefor RiAFP. This would mean we work up to the very last minute before the Jamboree but we are determined to successfully complete the project.One last problem is a reliable source of pLG575. Because our mini-centrifuge has a max speed of 4000 rpm (as compared to the recommended of 12000 rpm) our yield of pLG575 from Qiagen miniprep is abysmal (on Nanodrop: 5 ug/uL) The pLG575 generously sent to us by our advisor at Utah State, Asif Rahman, on Nanodrop showed 260 ug/ul. I'll ask Asif to send us another tube of pLG575 from his lab. Also, we will do the miniprep with Steph using a centrifuge of appropriate speed.So that's our plan! The team is up to the challenge. In the lull while we wait for the new part, we will work on the Wiki due 20 June. and work on the Poster and our 20 minute Presentation.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/notebookproject/frostprotection From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailA NOVEL APPROACH TO FROST PROTECTIONExtent of the problem. The success of the agricultural production is heavily dependent on the weather. For example, in April 2014, the American Southeastern states suffered one of the worst frosts in recent years. Blasts of arctic air brought prolonged record-breaking low temperatures. In Florida, citrus, strawberries, tomatoes, beans and other crops were damaged. In anticipation of compromised supplies, prices for many affected fruits and vegetables shot up. Frost damage to crops is not unusual; it causes American farmers to lose billions of dollars annually.Frost forms when an outside surface cools past the dew point. The dew point is the point where the air gets so cold, the water vapor in the atmosphere turns into liquid. If it gets cold enough, little bits of ice, or frost, form. The ice is arranged in the form of ice crystals. In general, -2 to -3° C frost over a period of at least an hour can be expected to cause damage to crops, -1° C for an extended period such as 3 to 4 hours can also cause similar damage. Current solutions. Currently farmers’ attempts to prevent frost damage are decidedly low-tech. Methods include burning smudge pots to produce warm smoke; running wind machines to move the frigid air; and spraying water on the plants to form an insulating coat of ice. It is a surprising experimental observation that frost protection can be achieved by preventing ice crystal formation on the plant surface. Indeed, this approach may be more important than inhibiting ice formation in the interior of the plant cell. Evidence suggests that ice crystals formed on the surface of plants must physically grow into the interior of the plant in order to initiate freezing in the plant. This can occur through stomates (very small openings in the epidermis of a leaf or stem through which gases and water vapor pass — singular = “stoma”) or cracks in the cuticle. Based on this data showing how freezing occurs, one method of frost protection might be by prevention of the ice nucleation of leaves by delaying the penetration of ice from a frozen droplet on the leaf surface. The efficiency of this protection was such that ice penetration was delayed on average by up to 2 h and in a short freezing test this was sufficient to enable a higher proportion of plants to supercool and avoid freezing and thus remain undamaged. Several new approaches to frost protection are under investigation. Exogenously applied cryoprotectants may provide a barrier that can prevent external ice from inducing plants to freeze. Many compounds have been screened, such as sorbitol or polyethylene glycol. However, most appear to either inhibit plant growth at active concentrations or only marginally effective in controlling ice nucleation in vegetative field crops. Some acrylic compounds have been marketed on the basis of giving frost protection by covering the leaf surfaces with an inert layer. Hydrophilic formulations of kaolin dust have been reported to protect tomato plants from frost damage.Biologic solution. Animals and plants living in cold climates produce natural antifreeze proteins (AFP) that serve as a survival mechanism and prevent organic fluids from crystallizing and forming ice. The production of antifreeze proteins in living things is one of the major evolutionary routes taken by a variety of organisms. First described in fish, they have also been reported in insects. Antifreeze protein activity has also been identified in many plants, but with low activity.A synthetic biology solution to the frost damage problem is a spray-on film of a nonpathogenic strain of E. coli that covers the crop and contains a secreted antifreeze protein (AFP). AFP's possess the ability to bind to the ice crystals surface, inhibiting the formation of ice. The growing ice surface becomes energetically unfavorable for further absorption of water molecules as the surface curvature increases, leading to the stopping of ice growth.RiAFP refers to an antifreeze protein (AFP) produced by the Rhagium inquisitor longhorned beetle. It is a type V antifreeze protein with a molecular weight of 12.8 kDa; this type of AFP is noted for its hyperactivity. R. Inquisitor is a freeze-avoidant species, meaning that, due to its AFP, R. inquisitor prevents its body fluids from freezing altogether. This contrasts with freeze-tolerant species, who’s AFPs simply depress levels of ice crystal formation in low temperatures. The antifreeze protein from the Rhagium inquisitor beetle is the most active antifreeze protein discovered to date — hundreds of times more potent than salt in inhibiting the formation of ice.Rhagium InquisitorThe story of a biotechnology solution (Frostban™) to frost damage frozen out by federal regulators. An ice-minus bacterium is a common name given to a mutant of the common bacterium Pseudomonas syringae (P. syringae). This strain of P. syringae lacks the ability to produce a certain surface protein, usually found on wild-type P. syringae. This lack of surface protein provides a less favorable environment for ice formation when the bacteria cover plant surfaces.Both the wild-type and mutant strains of P. syringae occur naturally. However, the ice-minus bacteria to be used for spraying crops are made on a large scale using recombinant DNA technology. In the mid 1980’s, Advanced Genetic Sciences, a pioneering agricultural biotechnology company headquartered in Oakland, California, developed Frostban, a bacterial-based treatment capable of reducing frost damage to fruit and nut crops. As the first U.S. field trial, Frostban became a lightening rod for opponents of the emerging biotechnology industry. In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment when a strawberry field in California was sprayed with the ice-minus strain of bacteria. The results were promising, showing lowered frost damage to the treated plants. The testing was very controversial and drove the formation of US biotechnology policy. Frostban was never marketed.Broader anti-icing applications. Anti-icing’s primary function is to prevent the bond of snow and ice freezing to aircraft and pavement surfaces. The composition of anti-icing fluids varies considerably depending upon the specific application and may include propylene glycol or ethylene glycol, sugar beet by-product added to salt brine, and potassium acetate. However, a common concern is the toxicity of these different formulations including corrosion to vehicles, impact on infrastructure, and, of course, damage to environment. A synthetic biology solution in the mold of PlantiFreeze may be an approach to these issues.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/project/frostprotectionotebook/parts From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailP A R T S Part: BBa_K1251000• The project undertaken by the Cambridge School of Weston (CSW) 2014 iGEM team builds upon the work of the 2009 Utah State team that developed a protein secretion mechanism and that of the 2011 Yale team which synthesized an “antifreeze” protein (RiAFP) isolated from a cold-tolerant beetle called Rhagium inquisitor. • To save time and simplify construction, the part was made by direct synthesis and ordered as gBlocks™ from IDT. gBlocks Gene Fragments are custom, double-stranded, sequence-verified fragments of DNA up to 700 bp. • We directly synthesized a composite part based on parts from other teams• There are no illegal restriction sites (EcoR1, Xba1, Spe1, Pst1) that would interfere with BioBrick standard assembly. The part is flanked by the BioBrick prefix and suffix.• The gBlock fragment inserted into a Registry supported plasmid backbone. • The part controls RiAFP expression which is tagged with a 6x His motif and a HlyA secretion signal. Initiation of transcription of RiAFP/6xHis/HlyA is controlled by a phage promoter, T7. A double terminator stops transcriptionPromoter. Our device uses a T7 system containing a lac operator, as did the Yale team, because T7 RNA polymerase is an incredibly fast and powerful enzyme transcribing rapidly and profusely for as long as the T7 RNA polymerase is present. It synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently. Expression can only be achieved in a bacterial strain carrying the gene for the T7 RNA polymerase. The most common cell strain to use with a T7 promoter system is BL21(DE3) (competent cells are chemically competent cells used for high-level protein expression with T7 RNA polymerase-based expression systems). The BL21(DE3) contains the T7 RNA polymerase gene, under the control of the lacUV5 promoter, integrated into the chromosome. IPTG is used to induce the expression of recombinant proteins cloned into vectors downstream of a T7 RNA promoter and transformed into the BL21(DE3) cells.The T7 promoter is a BioBrick part BBa_1712074C-terminal His-tag. In our protein expression construct, there is an amino acid motif that consists of six histidine (His) residues fused to the C-terminus of RiAFP. Histidine tags are widely used because they are small and rarely interfere with the function, activity, or structure of target proteins. The polyhistidine-tag is to be used to detect the secreted protein via anti-polyhistidine-tag antibodies or alternatively by in-gel staining (SDS-PAGE) with fluorescent probes bearing metal ions. HlyA-signal peptide. The HlyA is a signal peptide found in the C-terminal signal sequence of alpha-hemolysin (HlyA). It is used to target RiAFP for secretion via the Type I secretion pathway of gram-negative bacteria. Fusion of the HlyA signal peptide to RiAFP results in transport of the protein from the cytoplasm to the extracellular medium in a single step.The HlyA signal peptide is BioBrick pat BBa_K208006Terminator. There are several E. coli transcriptional terminators available via the Registry. The most commonly used type of terminator is a forward terminator. When placed downstream of a genetic part that is transcribed, a forward transcriptional terminator will cause transcription to abort. We use part BBa_B0015, a double terminator (B0010-B0012). BBa_B0015. This terminator has an Average Forward Termination Efficiency of 98.4%.Our PlantiFreeze device is a dual plasmid system in BL21 DE3 E coli. Sequence outlined in SeqBuilder (click to enlarge). The image on the left is the single-strand DNA sequence of the part. The image on the right is the plasmid map. Retrieved from "http://2014hs.igem.org/Team:CSWProteens/notebook/parnotebook/references From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmail R E F E R E N C E S1. Albiniak AM, Matos CF, Branston SD, Freedman RB, Keshavarz-Moore E, Robinson C. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli. The FEBS journal 2013;280:3810-21.2. Branston SD, Matos CF, Freedman RB, Robinson C, Keshavarz-Moore E. Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery. Biotechnology and bioengineering 2012;109:983-91.3. Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied microbiology and biotechnology 2004;64:625-35.4. Gentschev I, Dietrich G, Mollenkopf HJ, et al. The Escherichia coli hemolysin secretion apparatus--a versatile antigen delivery system in attenuated Salmonella. Behring Institute Mitteilungen 1997:103-13.5. Gusta LV, Wisniewski M, Nesbitt NT, Gusta ML. The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves. Plant physiology 2004;135:1642-53.6. Hacker J, Neuner G. Ice propagation in plants visualized at the tissue level by infrared differential thermal analysis (IDTA). Tree physiology 2007;27:1661-70.7. Hakim A, Nguyen JB, Basu K, et al. Crystal structure of an insect antifreeze protein and its implications for ice binding. The Journal of biological chemistry 2013;288:12295-304.8. Hakim A, Thakral D, Zhu DF, Nguyen JB. Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein. Acta crystallographica Section F, Structural biology and crystallization communications 2012;68:547-50.9. Hamed F, Fuller MP, Telli G. The pattern of freezing of grapevine shoots during early bud growth. Cryo letters 2000;21:255-60.10. Jong WS, Sauri A, Luirink J. Extracellular production of recombinant proteins using bacterial autotransporters. Current opinion in biotechnology 2010;21:646-52.11. Kotzsch A, Vernet E, Hammarstrom M, et al. A secretory system for bacterial production of high-profile protein targets. Protein science : a publication of the Protein Society 2011;20:597-609.12. Kotzsch A, Vernet E, Hammarström M, et al. A secretory system for bacterial production of high-profile protein targets. Protein Science 2011;20:597-609.13. Kristiansen E, Ramlov H, Hagen L, Pedersen SA, Andersen RA, Zachariassen KE. Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L.). Comparative biochemistry and physiology Part B, Biochemistry & molecular biology 2005;142:90-7.14. Linton E, Walsh MK, Sims RC, Miller CD. Translocation of green fluorescent protein by comparative analysis with multiple signal peptides. Biotechnology Journal 2012;7:667-76.15. Low K, Muhammad Mahadi N, Md. Illias R. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts. Applied microbiology and biotechnology 2013;97:3811-26.16. Lv J, Song Y, Jiang L, Wang J. Bio-inspired strategies for anti-icing. ACS nano 2014;8:3152-69.17. Matos CF, Branston SD, Albiniak A, et al. High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli. Biotechnology and bioengineering 2012;109:2533-42.18. Mergulhão FJ, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli. Biotechnology advances 2005;23:177-202.19. Moeller L, Gan Q, Wang K. A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway. Journal of experimental botany 2009;60:3337-52.20. Ni Y, Chen R. Extracellular recombinant protein production from Escherichia coli. Biotechnology letters 2009;31:1661-70.21. Pearce RS, Fuller MP. Freezing of barley studied by infrared video thermography. Plant physiology 2001;125:227-40.22. Perez-Perez J, Marquez G, Barbero JL, Gutierrez J. Increasing the efficiency of protein export in Escherichia coli. Bio/technology (Nature Publishing Company) 1994;12:178-80.23. Su L, Chen S, Yi L, Woodard R, Chen J, Wu J. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microbial Cell Factories 2012;11:8.24. Sugamata Y, Shiba T. Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli. Applied and environmental microbiology 2005;71:656-62.25. Taschler D, Beikircher B, Neuner G. Frost resistance and ice nucleation in leaves of five woody timberline species measured in situ during shoot expansion. Tree physiology 2004;24:331-7.26. Thomas JD, Daniel RA, Errington J, Robinson C. Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Molecular microbiology 2001;39:47-53.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/notebook/referencsponsors From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmail Thank you to Integrated DNA Technologies for giving us a deep discount on synthesis of the DNA fragments we used to construct the expression plasmid. Thank you to New England Biolabs for giving us many items from the catalog for free. The project would not have been possible without their support. Thanks to the Silver Lab at Harvard Medical School/Wyss Institute for advising us and allowing us to visit and work in the lab.Thanks to Utah State for supplying us with the secretion plasmid pLG575 at no cost. Retrieved from "http://2014hs.igem.org/Team:CSWProteens/sponsonotebook From 2014hs.igem.org Home Team MembersThe Cambridge School of WestonAdvisorsSponsorsAttributions Project Frost ProtectionProject DescriptionMethodsResultsDiscussionSafety Notebook Lab JournalProtocolsPartsReferences Outreach Contact Us TumblrFacebookEmailL A B   N O T E B O O K:2013: November | December | 2014: January | February | April | May | June November 13Our first full-length meeting of the year. We spent much of the time discussing the logistical (financial, organizational, etc.) details of the iGEM Competition, before splitting off to confer with our small, self-assigned subcommittees: Design, Communications, Fundraising, and Project Management.Pictured: Howard, a co-teacher and Faculty Advisor, presenting on the necessary functions of a cohesive iGEM team.November 18Our lecture half of the class consisted mostly of a detailed overview of synthetic biology, its engineering aspects, and our place in the process. During the lab half, we learned how to make gels for electrophoresis.Pictured: Top: Howard draws a diagram of the parts of a biological device.Bottom: a buffer is added to the gel after it sets.November 20We spent the first part of class tossing around ideas for a team name; when it came down to either sticking with the school’s mascot (the gryphon) or choosing something more original, a narrow majority vote decided on the latter. Thus, we are now officially the CSW ProTeens. We then learned more about genetic devices, and specifically their promoters.Pictured: Top: Howard describes the inner workings of the lac operon.November 25Today, Joey was in sole charge of the class. His lecture covered plasmid backbones in detail. We also watched this CrashCourse video on transcription and translation. For the lab, we learned (or reviewed) how to make agarose gels, use parafilm, and operate a centrifuge.Pictured: Left: Joey illustrates how the backbone of a plasmid can be cut to accept new parts.Right: Aiden pours a gel as Liam and Mason look on.December 4Today’s lecture was on translational control, and the workings of DNA polymerase. For the lab, we began to work on making our own ampicillin plates from scratch.Pictured: Top: Howard draws (and vigorously circles) a ribosome binding site.Bottom: Howard demonstrates the process of making amp plates.Decmber 9This class was dedicated to finishing our ampicillin plates. We also broke in the new autoclave.Pictured: The team intently examines the working autoclave.December 11The team split into smaller groups, and each performed a transformation of pPRL, a purple pigment producing plasmid, into E. coli.Pictured: Top: Nate, Aiden and Liam put their plasmids on ice.Bottom: The plasmids are put on our ampicillin plates.December 16For the lecture portion of class, we learned about “RNA thermometers,” and then discussed logic gates and used them to analyze some devices. We then viewed the (successful!) results of our pPRL transformation.Top: Howard explains truth tables.Bottom: Our E. coli is purple!December 18Joey gave a lecture on 3A assembly. Our lab was to transform pGRN, a green pigment producing plasmid, into E. coli.Top: Joey details the standardized DNA sequences that allow plasmids to be cut.Bottom-left: Noa and Jenny work on the transformation lab.January 6Happy New Year! We dedicated today to preparing gels for our next lab.Pictured: a comb is inserted into the not-yet-solidified gel to create wells.January 8We began a restriction digest lab, but due to technical difficulties had to postpone until the next class. We then split into committees for a regular update.Pictured: Joey and Howard divide reagents.January 13We did some more practice with electrophoresis using lambda DNA.Pictured:Top: Noa removes a gel from the electrophoresis chamber.Bottom: Our results were highly visible when the gels were placed on a light box.January 15We dedicated the whole meeting to practicing with a restriction digest.Pictured: Nate, Aiden, and a blurry Liam fill a gel’s wells.January 22In pairs and singles, we researched and presented the team’s top ten project ideas and began to narrow down that list.We dedicated the whole meeting to practicing with a restriction digest.Pictured:Top: Liam and Aiden present their findings.Bottom: Noa and Jenny set up before sharing theirs.January 27After the previous week’s presentations, we worked to more fully develop the most feasible ideas and draft circuits for them.Pictured:Top: Joey and Howard review the process of writing a circuit.Bottom: Lilly begins to draft a circuit on the whiteboard.January 29We gave our final presentations on the most feasible project ideas, which we had been working on for weeks.Pictured:Top: Summer discusses her proposed circuit.Bottom: The final list of ideas to be discussed and voted on. (Voting results coming soon…)February 3The results are in! We, the CSW ProTeens, will pursue plantifreeze for our final project. "Plantifreeze" is our shorthand name for a bacteria that prevents frost from forming on fruits near the end of the fall growing season—in other words, a plant antifreezePictured: Liam reviewing his proposed circuit for the project, just before the team splits into smaller groups to research various parts.March PlanningOur school has a schedule where courses run for sections of 5-weeks. After February, our class section finished and we did not have a structured meeting time. In this interim, we worked on planning out our project, contacting advisors, and ordering parts for our project.April 27This weekend we made stock solutions of antibiotic solutions, prepared sterile LB agar plates supplemented with ampicillin, chloramphenicol, and the combination. We also prepared SOB broth for growing up transformed bacteria. Consequently, we are ready now to begin assembly of our device.April 28We received the pGL575 plasmid from Utah State today by FedEx. Asif suggested that we transform our competent cells with the plasmid ASAP. So we did just that. The plasmid carries the gene for chloramphenicol resistance so we used LB agar with chloramphenicol to select for the transformed cell. We made 5 agar plates so we should have plenty of the plasmid. This plasmid contains the genes for allowing the secretion of proteins made by the bacteria via Type 2 secretion system. Our other plasmid that we are assembling in the lab, regulates secretion of the RiAFP protein. That's why our device is a dual plasmid system: one plasmid controls expression of the protein and the other plasmid regulates secretionApril 29We just checked the selection plates for transformed NEB 10 beta cells. Four of the five plates showed colonies so we were successful with the transformation. The plates are in the frig ready to be grown up to supply the pGL575 plasmid for the device. Next step is to assemble the gene for expression of RiAFP with tag for 6xHis and peptide signal for secretion. We need to put that fusion gene into a plasmidMay 1Kudos to Aiden who did the majority of the lab work. We took the gBlocks of parts A and B of the expression plasmid and the linearized plasmid pSB3A1 and digested the prefix and suffix of each with restriction enzymes: EchoRI, SpeI, Xbal, PstI, PslI. These enzymes recognize restriction sites and cut them in ways that allow part A to fuse with part B and for the fusion of A-B to fit into pSB3A1. The next step is to permanently ligate the parts so that we end up with a circular plasmid carrying the fusion part. The ligation will be performed on Thursday morning between 10 am and 1 pm. Drop by.May 6The transformation failed. We later discovered that we used 0.2 micrograms of plasmid instead of the suggested 1 microgram while constructing the expression plasmid from parts A and B. This altered the volume of the reaction, and was likely the cause of failure.May 8In absence of gBlocks, we can not do anything with the expression plasmid. We took individual colonies of NEB 10 cells transformed with pLG575 plasmids and put them in LB broth cultures to incubate at room temp for 24 hours. Growing up these cells will afford us a large supply of the pLG575 plasmids.We ordered more gBlocks from IDT. We will carefully rewrite the protocol for digestion and ligation of the parts that go into the linearized pSB3A1 so transformation can be successful with a future attempt. Also will run concurrent positive control in the future to avoid our inability to analyze any future failures.May 14After very careful preparation, the parts for the expression plasmid were digested, ligated and the resulting ligation product was used to transform NEB 10 cells today with the capable assistance of Aiden, Nate, and Noa. Many things were changed from the unsuccessful transformation from last week. The transformed cells are in the 37 degree C incubator so please everyone hold your breathMay 15Transformation is successful. Next step is to grow out colonies of transformed cell in liquid culture so we have good supply. Then we mini prep both plasmids and cotransform DB21 DE3 cells and look for recombinant protein in supernatant and lysate.So we have the expression plasmid in NEB 10 beta cells on agar supplemented with amp. The next step is to grow up colonies form the agar plate in LB broth. This will give us a large supply of plasmids. We will miniprep the liquid culture getting bare plasmids for contranformation of DB21 DE3 competent cells with both plasmids. By using Western blot we confirm presence of RiAFP in supernatant and lysate. What did we do right this time. In the ligation step, we were very careful to use the correct proportion of DNA from the inserts (Parts A and B) and the vector (linearized pSB1A3). We used 50 ng of vector DNA and made use of a calculator on the NEB web site to calculate the amt of insert DNA to use. There should be a 3 to 1 molar excess of insert DNA to vector DNA to ensure that all the linearized vector is fully occupied by insert DNA. The calculation used the nucleotide lengths of the inserts (about 450 bp for each part) and the vector (2155 bp) to make the calculation. Also in an incubation in ligation we incorrectly used 22 degrees C instead of room temperature originally. This was corrected in the lates transformation.May 18After we transformed the NEB 10 beta cells with the newly assembled plasmid (T7 RBS RiAFP 6xHis HlyA term term), Ellie selected a few individual colonies from the agar plate supplemented with amp and put them in 50 mL of LB broth supplemented with amp in sterile 250 ml flasks with a sterile magnetic stirrer and grew up the cells over 16 hours at room temperature with gentle stirring. Our newly acquired rotating shaker has a broken belt so it is on the bench. After the 16 hour incubation, the broth had turned from clear to opaque. We transferred 10 ml of the broth to sterile falcon tubes and stored them at 4 degrees C. We do minipreps on the cells to isloate and pruify the plasmids. We also transformed NEB 10 cells wit the pLG575 plasmids and stored them in10 ml broth at 4 degrees C. So now we have ample supplies of both plasmids.The next step is to miniprep the transformed cells. Then we cotransform the two plasmids into DB21 DE3 competent cells. Our device includes an expression vector containing RiAFP cloned downstream of the T7 promoter. DB21 DE3 competent cells carry a chromosomal copy of the phage T7 RNA Polymerase gene, which is controlled by a lac promoter. When inducer (IPTG) is added, T7 RNA Polymerase is expressed and becomes dedicated to transcription of RiAFP.Plasmid pLG575 includes the coding regions for proteins HlyB and HlyD.May 20We are ordering the DB21 DE3 competent cells from NEB very shortly in preparation of cotransformation of these cells with the expression and secretion plasmids.May 22We took individual colonies growing on agar supplemented with chloramphenicol and put them in SOB culture broth. SOB is a slightly modified LB broth. The cells, NEB 10 beta cells, transformed with pLG575 plasmid will grow in the broth and provide a source of the plasmid. We also transformed NEB 10 cells with the expression plasmid.Currently we have liquid cultures of NEB cells with the expression plasmid and liquid cultures of NEB cells with the secretion plasmid. At this point we are poised to do minipreps on both types of cultures. The minipreps will give us bare plasmids that in turn will be used to transform BL21 DE3 E coli cells. These are chemically competent E. coli cells suitable for transformation and T7 promoter driven expression of RiAFP expression. The two plasmids will be used to cotransform BL21 DE3 cells. Following cotransformation, expression of RiAFP will be induced by IPTG.May 24On Tuesday we will do miniprep on the cells containing the expression plasmid. At that point we will be in a position to cotransform the BL21 DE3 cells that constitute the PlantiFreeze device. Competent BL21 DE3 cells will be shipped to the lab on Wednesday on dry ice. We will transform the cells on Wednesday. On Thursday the 16 hour incubation of the transformed cells will be ready. On Thursday, we will take individual colonies from the agar plate and put them into SOC media. On Friday of next week if all goes well we will have an ample supply of the BL21 DE3 cells transformed with both the expression and secretion plasmids. At this point the moment of true has arrived! By introducing IPTG into the culture we should be able to induce the cells to express RiAFP and secrete the recombinant protein into the medium in which the cells are growing. Here is where Steph and the Silver lab come into action. We need to assay the medium and the lyzed cells to confirm the RiAFP was expressed by the E coli and secreted. We will do this using Western blot.There will be other analyzes that we'll also do:• Effect of each plasmid and both plasmids on the growth rate of BL21 DE3 cells (by turbidity analysis using spectrophotmometer• Gel electrophoresis of some of the intermediate constructsMay 24Rather than using Western blot we are thinking about the use of Invision In-gel stain from Life Technologies. Visualization of the fluorescent bands uses:UV transilluminator (302 nm) equipped with a camera capable of integrationTo view and photograph a gel on the UV transilluminator, use a video camera, CCD (Charged Couple Device) camera, or a cooled CCD camera with ethidium bromide filter or band pass filter encompassing the emission maxima(590 nm) of the stain. May 27DL21 BE3 competent cells from NEB arrive tomorrow. Today we will do miniprep on the cells containing the expression plasmid. So how 'bout we order:InVision™ His-Tag In-Gel Staining KitInVision™ His-tag In-gel Stain is supplied as a 1X ready-to-use staining reagent (500 mL) [enough to run 20 mini gels]BenchMark™ His-tagged Protein Standard (125 µL) is also included in the kitNuPAGE® Novex® 10% Bis-Tris Gels (pre-cast polyacrylamide gels)May 28Thanks to Joey and Thomas for doing the miniprep on the expression plasmid. We now have pure DNA from both plasmids and are poised to cotransform BL21 DE3 competent cells. This is the last step in the assembly of the PlantiFreeze device. After that step we do functional assays to confirm that the device works to express and secret RiAFP. As a side venture we'll be exploring the effect of transformation of the BL23 DE 3 cells with each plasmid alone and both together. We'll be constructing growth curves based on optical density of the culture media.We have ordered from LifeTechnologies Invision in-gel stain, a latter, and precast gels for confirming the expression and secretion of the protein. These experiments will be done with Steph Hayes at Pam Silver's lab at Harvard Medical School.Keep your fingers crossed that everything goes right and we'll be in the home stretch. Still need to do data analysis, the Wiki, our poster and the presentation so we need everybody at battle stations a while longer.Finally, we need to do more minipreps more growing up liquid cultures, more transformations so please avail yourself of the opportunity to get your hands wet in the lab. Its fun, its instructional, and you help is really needeMay 29Lab today 1 to 5:• cotransformation of Bl21 De 3 cells• miniprep of plasmids• making agar plates• making liquid cultures• planning measurement of oD of culture for growth curvesMay 31Yesterday was a banner day for lab work. Thanks to Joey, Micah, Aiden, and Thomas. We did a miniprep of the NEB 10 cells transformed with the secretion plasmid pLG575. Did the milestone cotransformation of BL21 DE3 cells with the expression and secretion plasmids. On Saturday after 16 hours incubation at 37 degrees C we should know if it worked. This time we ran a positive control of pUC16 DNA so if something goes wrong will have the control to help us unravel the problem.If the cotransformation works our next step is to grow up the cotransformed cells in LB broth and then induce them with IPTG to express and secrete RiAFP.Remember we plan on determining the influence on, the other and both plasmids on the growth of BL21 DE3 cells using the optical density of the liquid culture on the spectrophotometer. If time before the close of the Wiki permits we might also do digestions of the plasmids and look at their electrophoresis patterns.June 1No growth on either the plates with contransformation or the positive control. It is obviously useful to have a positive control because now we can conclude that we are doing something wrong with the transformation procedure or the competent cells are no longer competent. In fact, in our hands, tansformation has been very unreliable. But this is the first time we ran a positive control.June 2The NanoDrop is a specialized spectrophotometer that measures the amount of DNA in a sample as small as 1 microliter. We are having trouble with the cotransformation and we are worried that the plasmid DNA we are getting from the minipreps was too little. As a benchmark, it is good to have more than 50 nanograms/microliter of plasmid DNA.We have two plasmids: pLG575 and the expression plasmid. We have 5 nanograms/microliter of this plasmid. Obviously very low. The other plasmid is the expression plasmid. We have 47 nanograms/microliter of this plasmid. Good.We can adjust the miniprep to increase the concentration of pLG575 DNA,The puzzle is that we should have gotten transformation of the expression plasmid. So we still have to look at the transformation procedure.June 6This has been a frustrating and puzzling week. The cotransformation performed on Sunday failed, leading us to wonder if we got plasmid DNA from the minipreps of the expression and secretion plasmids. Steph generously allowed us to use the Nanodrop analyzer in her lab. We found that the DNA concentration was low but acceptable for pLG575 and in a good range for the expression plasmid. We made some alterations to the miniprep procedure. We repeated the transformations in a wide ranging way and were amazed that the only plate with growth was the one with agar only. This observation directed us to think about a problem with the antibiotics so we made new stock solutions and were especially careful about antibiotic concentrations in the most recent transformations. On Thursday, Joey, Micah, and Thomas were in the lab. We used BL21 DE competent cells and did the following:• cotransformation with the original pLG575 plasmid from Utah State and the expression plasmid• transformation of pUC19 positive control plasmid• transformation with pLG575 plasmid obtained by miniprep using modifications to increase concentration• transformation of the pPURP plasmid from the BioBuilder lab exerciseAll in all, maddeningly frustrating, but we will persevere.June 7Puzzling, frustrating, and humiliating! We used commercial BL21 DE3 competent cells in the transformation reactions. pLG575 (orig) refers to the plasmids shipped to use by Utah State; pLG575 (miniprep) refers to the plasmid DNA we got by miniprep of the transformed NEB 10 beta cells transformed with the pLG575 plasmids.pPURP is the plasmid from BioBuilders and pUC19 is the positive control plasmid sent with the competent cells by NEB. We mixed new stock solution of ampicillin and chloramphenicol and carefully checked the concentrations in the LB agar, so I am really bewildered by the discrepancies between the expected and observed results.June 10Thank you Aiden, Noa, Ben, and Joey for working in Steph's lab at Harvard Medical School. In an attempt at troubleshooting our problem with transformation we did transformations with several different strains of competent cells. The plasmids we used included: pLG575, the expression plasmid, pUC19.June 11Yesterday, we used five different strains of competent E coli and five separate plasmids for transformation of the strains of competent cells. Here are the results of the growth of the plates with the appropriate antibiotics:Results from Steph’s LabDisregarding one or two anomalies, the analysis indicates two things:The BL21 DE3 competent cells don't appear to be competent. Keeping them on dry ice may not have been able to maintain a stable -80 degree centigrade temp allowing them to become incapable of transformation (see column pUC19). Please note that the prior batch of competent cells from NEB (NEB 10 beta cells) were transformed by the pLG575 plasmids we recieved from Utah State and the plasmid we constructed from the gBlocks form IDT. I did not observe thawing of the BL23 DE3 cells. The plasmid DNA we obtained by miniprep were not able to transform any competent cells (see last three columns). The cause of the failure of the miniprep remains uncertain.June 12Hi, all. Let’s summarize where we are at the moment.We have plasmids with the expression cassette (amp resistant) and the secretion cassette (chloramphenicol resistant), respectively in transformed NEB 10 beta cells. This is where we reached a stone wall. Using minipreps, we were unable to cotransform BL21 DE3 cells and induce those cells with IPTG to express and secrete RiAFP. We have an in-gel stain that will enable us to detect RiAFP in cytoplasm and culture media standing by and ready to use (takes about 3 hours to do).Under the guidance of Steph, team members performed transformations using several E coli strains and different plasmids (the results summarized in an email message sent to all yesterday). The analysis of those results suggest that the commercial competent cells from NEB (BL21 D3) were degrade by unstable maintenance of -80 C temp and that our miniprep procedure was flawed. It is easy to overcome the competent cell flaw. Not so easy is pin pointing what's wrong with the miniprep. Since we have amp and chloramphenicol resistant NEB 10 cells in the Bio 3 frig, we can analysis the plasmids in those cells and figure out a strategy to go forward. We have in the frig in Bio 3 agar plates and LB broth cultures of the NEB 10 beta cells which are amp resistant cells andchloramphenicol resistant cells meaning they were transformed by the expression and secretion plasmids, respectively. I will bring them to the AlbertsLab tomorrow for analysis confirming the presence of the appropriate plasmids in the cells. Needless to say, the clock is ticking and it will be a challenge to finish all the lab work. The Wiki closes on 20 June, but the lab work can go on beyond that date and any new data can be incorporated in the Poster and the PowerPoint slide deck. Even then time may run out despite the best efforts.June 13Noa's review of the Parts synthesized by IDT prompted me to do a deep dive into the parts sequence synthesized by IDT. Oh boy, did we mess up and compounding that problem we didn't check the parts sequence when they came back from IDT. But since they have a 3-5 business day turn around we can recover but we want to order just one DNA fragment which will be inserted into a linearized plasmid with an antibiotic resistance different from the secretion plasmid. That plasmid will then be cotransformed with our secretion plasmid into BL21 DE3 competent cells. Uncovering the incorrect sequence in the expression plasmid solves our current problem of not being able to cotransform in that we'll have a brand new plasmid and we will use new competent cells. Here is new revised approach:we need a plasmid which contains these components and BioBrick standard prefix and suffix (not the RFC 23):• T7 promoter• RBS• RiAFP coding sequence• HlyA coding sequence• 6x His marker• double terminatorWe insert (using digestion and ligation) the above fragment into a linearized plasmid with a resistance marker other than chloramphenicol (the secretion plasmid is chloramphenicol resistant). We then use Invision in gel stain to detect presence of RiAFP in cytoplasm and media.We'll be working up to the last minute: here's the timeline:• Receive new part from IDT: 19 June• Insert new part into linearized plasmid: 19 June• Transform competent cells to grow up stock of plasmid: 20 June• Grow up liquid culture of transformant: 21 June• Miniprep of plasmid DNA: 22 June• Transform BL21 DE3 cells: 22-23 June• Grow up transformed BL21 DE3 cells and induce expression and secretion of RiAFP: 23-24 June• Use Invision system to detect RiAFP in fractions: 25 June• Analyze results: 25-26 JuneJune 14After the disheartening discovery that Part A had been incorrectly designed, we decided to engineer a single DNA fragment to serve as the expression genetic circuit in thePlantiFreeze Device. The circuit would comprise the following components:BioBrick Standard Protein Coding Region Prefix /T7 Promoter /RBS /RiAFP/HlyA /Double terminator /BioBrick Standard Protein Coding Region Suffix The DNA sequences for the components could be found in the Registry. So it was not difficult to design the part. It was 864 base pairs in length. It is fast and simple to order a 864 base pair gBlock fragment form IDT (double-stranded, sequence-verified genomic blocks that ship in only a few working days for affordable and easy gene construction). The first step in ordering a gBlock is automated review by screening tools and expert review by IDT scientists of entered sequences for characteristics that may interfere with synthesis. This review found that there were sequence repetitions longer than 8 base pairs that comprise a combined 91% of the sequences within a window of 70 base pairs beginning at base pair 235. This structure would interfere with synthesis. So the IDT gene product specialist optimized the sequence for expression in E coli and compatible with the gBlock synthesis process. Optimization theoretically just changes codon usage without changing the proteins coded. Importantly, the EcoRI and PstI sites on the 5’ and 3’ ends of this gene were maintained compatible with BioBrick standard 3A assembly. However, Noa at Steph's lab ran the optimized new sequence thru the seqbuilder program and identified that the optimization process put in another Pst1 restriction site at position 637 in addition to the appropriate one at position 863 (the one compatible with the BioBrick standard). Having the secondPstI restriction site breaks the BioBrick standard and makes correct assembly impossible. Following the finding of this glitch, the fragment was reoptimizedremoving the PstI site at 836. The part was order for synthesis and delivery as soon as possible. New sequence outlined in SeqBuilder (click to enlarge)This new part just ordered from IDT would be inserted into a linearized plasmid backbone with antibiotic resistance different from the plasmid controlling secretion (pLG575). We decided to use again pSB1A3. This linearized plasmid is supplied by iGEM. We had exhausted our previous supply so ordered a new one.The plan is to insert the new part into pSB1A3 and again do cotransformation of BL21 DE3 cells with the two plasmids. Then we would induce thecotransformants with IPTG to express and secrete RiAFP. Finally, using theInvision In-gel Staining Kit we would analyze the media and BL21 DE3 cell lyzatefor RiAFP. This would mean we work up to the very last minute before the Jamboree but we are determined to successfully complete the project.One last problem is a reliable source of pLG575. Because our mini-centrifuge has a max speed of 4000 rpm (as compared to the recommended of 12000 rpm) our yield of pLG575 from Qiagen miniprep is abysmal (on Nanodrop: 5 ug/uL) The pLG575 generously sent to us by our advisor at Utah State, Asif Rahman, on Nanodrop showed 260 ug/ul. I'll ask Asif to send us another tube of pLG575 from his lab. Also, we will do the miniprep with Steph using a centrifuge of appropriate speed.So that's our plan! The team is up to the challenge. In the lull while we wait for the new part, we will work on the Wiki due 20 June. and work on the Poster and our 20 minute Presentation.Retrieved from "http://2014hs.igem.org/Team:CSWProteens/noteboo