Team:Danzi Kesh 8/Notebook

Process

We think outside of the box!

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Notebook

The notebook describes two processes which occur simultaneously: The experiment in the laboratory and social process. Each month we have described the activity for these two areas separately.

February 

The decision on participation in the competition 
The decision in participating in the competition was brought forward to the class. After a long and thorough discussion of the pros and cons we decided to enter the contest as a team, despite the difficulty in managing a large group.
One of the main difficulties was: lack of complete knowledge of genetic engineering, that's why we started to study with Maggie the subject of genetic engineering as part of classroom lessons.

Crash course in genetic engineering
Despite the enthusiasm and excitement of the experience before us we began to understand the knowledge gap we need to overcome before entering the laboratory. The topic itself is fascinating to us as students, we suddenly realized just how limitless the world and science has become an almost unlimited field.

Activity beginning
Meeting with Associate Professor Roy Amit (synthetic biology assistant professor, Faculty of Biotechnology and Food Engineering and Russell Berrie Nanotechnology Institute) from the Technion.
Received an explanation of the IGEM competition.

LAB : No activity 

march

Forming the Team
forming the Team began in March, already then we began to talk of bonding the major and turning into a Team competing in an international competition called Igem held in Boston.
Excitement was in the air, a new product development that does not exist in the market and the flight abroad with the major of
biotechnology would sound like a fantasy. In those moments we did not know and did not understand the meaning of the IGEM entry experience.

Exposing the idea to the Team
All members of the Team gathered to be exposed to the idea upon which we were going to work over the coming months, at the meeting we met the Advisor Dr. Yadid and we started to discuss the development of the product the best we could, students suggestions were accepted and we were able to combine our ideas with Dr. Yadid's.

Meeting:       

  1. Meeting at Migal (Upper Galilee Research Institute, Institute specializes in food biotechnology research) Professor Segula Mutzafi, Search Advisor for the project. (Dr. Itamar Yadid selected)
  2. Meeting with the mayor (Nissim Malka) and director of the city's education, experience in finding funding.
  3. Meetings with other donors, Tel Aviv.

LAB : No activity 

april

Parents meeting
After we met with Dr. Friend and after exposure to the long road ahead it was important for us to share with our parents and to commit ourselves to the process. We told the parents about the competition and what it requires of us to do, at first, some parents thought it was far-fetched and unrealistic, when they arrived for a meeting with the teachers and the school principal and were exposed to the process from the mouths of the leaders of the process, they internalized the idea, understood it was possible and gave us their blessings.

Brainstorming, meeting with a group of IGEM looking for project ideas

  1. General idea was found - A kit to identify food allergens. Meeting with students' parents, exposure to IGEM Group's and the general idea.
  2. Search for allergen proteins in food.
  3. A decision was taken – gluten allergen.
  4. Reading articles on gluten, Search the gluten protein component,   Search amino acids that make gluten.
  5. The amino acid glutamine was found to make up the protein allergen.

Meeting:       

  1. Team heads meeting with Dr. Itamar Yadid (checking the idea).
  2. Meeting with Dr. Itamar Yadid at the school (introductory meeting with students and searched for different allergen proteins (a decision was made to work the gluten allergen).
  3. Presentation of the project idea at Tel Hai College and a national conference for biotechnology teachers.

Dividing into groups
Since we are a large Team (22 students) and time is short and a lot has to be done, we divided the Team into groups in accordance with the tasks. In fact, all five teams worked hard and were dependent on each other to get the complete product.
Set up the team site and divided into organized work groups: writing, documentation, logistics, laboratory and presentation:

  1. Lab Team: This team worked on the scientific aspects in product development from the idea stage. Their work was great and they invested a lot of time and effort, without all their work the work of the other groups was useless. The lab team was responsible for lab work and the writing of work process into a daily notebook.
  2. Documentation team: The team was responsible for documenting and marketing the team in any way possible. The documentation team was responsible for preserving significant and awkward moments to be able to describe the
    process from the beginning. The documentation team was responsible for producing the group's videos, short documentation and ongoing blogs, photos, videos, articles and more .. The documentation team has been active in all
    social networks: Google +, Facebook, Twitter and even Instagram. Their aim is to spread the idea and to maintain the process.
  3. Site and writing team: in charge of the wiki site construction. Their work was very important for the transfer of information passed on to them from all the other groups, the translation from Hebrew into English, and looking at website design that attracts the eye. The writing team documented in words and images the events that took place in the classroom ever since the decision to participate in the competition was made.
  4. Presentation Team: The face of our Team. Members of this team will represent us in any event related to the spreading of the project and the competition itself. Team members were interviewed by the local press and met with donors. They meet frequently with Dave and work on their English language accent and way of speaking, yet ultimately; they are the ones who are supposed to represent us in the competition in Boston and speak professionally and proficiently and to
    represent us with dignity.
  5. Logistics Team: running and working overtime behind the scenes to take care and make sure everything is, that no group is not missing anything everyone is working consistently and nobody gets stuck in the back, they push the team forward and take care of all things great and small alike. This team has produced all the classroom activities, for example, coordinating meetings, writing and distributing surveys and more.

It is important to note that all groups are necessary to carry out the process and think equally, without the contribution of each
member the entire project would have been in vain, and so each and every member has to give his 100%. Most students took part in two teams.
After the exposure of the project to financers, scientists and academics from across the country the Team travelled to represent us at several conferences: The Annual Manufacturers Conference, International Science Day at Tel Hai College and the annual scientific meeting at Tel Hai College.

LAB : No activity 

may

Synthetic Biology Conference – May 21
When we started off we established, in cooperation with K-grade graduates in biotechnology, the synthetic biology conference in the country's first youth in the field of synthetic biology. The conference was held at the school.
The conference was attended by approximately 400 students who study science and live in the northern periphery of the state of Israel. The Mayor of Kiryat Shmona, professors, journalists, members of the Knesset and scientists from Tel Hai College attended the conference as observers as well as a few lecturers.
At first we started to expose our project and its objectives, we received a lot of compliments and the participants were impressed by our initiative and devotion towards the process.
The conference was very successful and our name began to spread across the country.

LAB : Process Planning 

june

  1. A meeting with Igem team leaders from Israel. The meeting was held with scientists and with start-up companies in order to introduce them to the ideas of the Israeli teams.
  2. A decision on how to design the biological denominator of the product using bio brick.
  3. Promoting awareness of celiac disease in a primary school in the city, a public opinion survey among school students, and random visitors in the mall.
  4. Activity Day at "Meginim" Primary School 8/6/2015.

LAB- Week 1:

We performed a preliminary test to our series of experiments:
Insert of RFP gene to the pSB1C3 plasmid and then we performed a transformation to E.coli.

Stage 2:

  1. Preparation of growth platforms (substrates) for  the bacteria -  The first platform with two ampicillin and the second one with chloramphenicol. Inc.
  2. Cutting RFP gene by two restriction enzymes - Ecor1, pst1. 
  3. Running the RFP gene in electrophoresis gel.
  4. Split the Gel together with the gene and cleaning the gene.
  5. Split the plasmid with restriction enzymes Ecor1, pst1.
  6. Ligation – joining the plasmid and the gene.
  7. Performing transformation using the Heat Shock method to the E.coli bacteria competents - Type DH5α.
  8. Planting the increase in antibiotic-resistant substrates chloramphenicol and ampicillin prepared in the first stage. 

Results:

Figure 1: seperation of the gene using   electrophoresis gel.

Picture 1: Electrophoresis results: Psb1c3 plasmid- 2000bp, Rfp gene- 1100bp

Cleaning the DNA from the gel: 

Eppendorf test tube - 1 gram
Electrophoresis gel + test tube - 1.19 g
Calculation of how much the gel cube alone weighs - 0.19 grams

Nanodrop calculation:

Convert the DNA from concentrated units to molar units:
A rule of thumb - the molar ratio between the plasmid and the gene 1: 3
bp insert*ratio*ng vector=ng insert
bp vector 

Plate 1:  
PSB1C3 + RFP, substrate LB + chloramphenicol, 1ml concentration, transformation worked, the growth of bacteria with resistance to chloramphenicol and with RFP Gene conferring the color red, with low concentration.

Plate 2:
PSB1C3 + RFP, substrate LB + chloramphenicol, 100ml concentration, transformation worked, the growth of bacteria with resistance to chloramphenicol and RFP Gene conferring with red color, high concentration.

Plate 3:
PSB1C3 + RFP, LB + ampicillin substrate, concentration of 100ml, transformation worked, Growth of bacteria with resistance to ampicillin and with RFP Gene conferring with red color, high concentration. 

LAB Week 2: No activity 

LAB Week 3:

Preparation of the PET plasmid towards ligation with the Gln_H gene:
This plasmid contains a strong promoter – 7T, Activated with the lactose operon mechanism, Gene resistance to ampicillin, MCs, and six amino acids His – which will be used by us as a "glue" for the colona and will contribute to cleaning the proteins. 

We will insert this plasmid by transformation to bacteria bl21(de3), that is a bacteria we erased the proteases, and thus its expression of proteins is stronger.

  1. We split the plasmid .
  2. We added phosphatase (so the plasmid will not close on itself before the insert entering).
  3. We confirmed presence by running in electrophoresis gel.
  4. We measured the concentration by nanadrop.

Results:

Picture 2: Electrophoresis in gel: you can see the split PET plasmid.
There are 2 bands because we ran 2 identical samples in order to increase the chances to getting the plasmid.

Testing concentration of the plasmid:

Clean Eppendorf weight: 1 gram
Eppendorf with gel weight:
Vial 1 – 1.52 grams
Vial 2 – 1.6 grams
Concentration of molar plasmid using nanodrop test:
A rule of thumb - the molar ratio between the plasmid and the gene 1: 3
bp insert*ratio*ng vector=ng insert
bp vector
Vial 1 –  9 ng
Vial 2 – 12.5 ng

LAB Week 4: No activity 

july

Week 1:
After publication of the judges’ competition booklet we decided to compete in the social area as well. As part of this decision we have executed a number of actions: part of the team developed the area with the cooperation of the community in Kiryat Shmona, celiac patients from around the country and unions for celiac patients from Canada and the United States.

Week 2:
Exposure to children ages 9-12 through summer camp activities that deals with the subject of celiac disease, promoting legislation in the Israeli parliament. A meeting with members of parliament who are members Children’s Rights in the Knesset. MP Yifat Sasha Biton, Committee Chairman understood the economic and social significance of the legislation that we  proposed. Another MP turned out to be a celiac patient, sided with the arguments that we brought and decided to launch the move of legislation in favor of celiac patients. Lowering prices for gluten-free foods, ingestion of gluten-free menus in the educational system, etc .. Finally, we met with the Minister of Education himself, the Minister of Education Mr. Bennett, Team representatives listened long, and finally he said that the legislation is possible but complicated. The Minister of Education
promised to investigate the matter and take action to the extent possible in order to change the conduct in the framework of his ministry, the Ministry of Education.

Interview Dr. Maayan Gal and Dr. Irena.

Public Opinion Survey opened at the Internet. 

Week 3:
A poll was transferred on all the social networks, the second survey specifically designated for celiac sufferers. Its aim was to examine if there is compliance with our product among the population affected by Celiac Disease. 170 celiac patients responded to the survey.

Scouts Summer camp Activity.

Week 4:
Visiting Minister of Science laboratory Rami Danon.

LAB Week 1:

We synthesized the Gln-H gene from six different sources and a combination of different bricks:
EC HAD –e.coli Synthesis  sours with had molecule
EC GFP-  e.coli Synthesis  sours with gfp molecule
GBS HAD-  gbs Synthesis  sours with had molecule
GBS GFP- gbs  Synthesis sours with gfp molecule
TM HAD-  TERMATOGA MARITIMA  Synthesis sours with had molecule
TM GFP-   TERMATOGA MARITIMA   Synthesis sours with gfp molecule

These actions we performed on every one of the synthetic bricks we created:

  1. Cut the Gln-H gene from inside the PUC57 plasmid, cleaned the gene and transferred the gene to the PETtr plasmid we prepared at the previous meeting.
  2. Placed the gene and the plasmid on electrophoresis gel.
  3. Identified the gene and its split together with the gel
  4. Cleaned the gene from the gel.
  5. Measured the concentration of the gene.
  6. Ligation.
  7. Transformation to bacteria e.coli DH5α components.
  8. Seeding on lb+amp plate.

Results:

Picture 3:
Upper bend – puc57 plasmid 2700bp
Lower bend – Gln-H gene 1500bp
Gene No. 6 – did not show expression, need to repeat the action.  
Gene No. 3 – a spread bend is visible

Picture 4:
Results of running electrophoresis gel to genes 3,6                   
Upper bend – puc57 plasmid 2700bp                    Lower bend – Gln-H gene 1500bp 

LAB Week 2:

Ligation of Gln-H into plasmid expression PETtr

Performed transformation of PETtr to DH5a  bacteria       
Purified the plasmids from the bacteria using Plasmid Prep process
Performed transformation to bacteria BI21 (DE3) – expression bacteria and seeded on Petri plates, with heat shock and electroporation method. The growing platform contains lb agar+amp+1% glu

Transformation results: 

Transformation succeeded! We chose the transformation plates of the bacteria that underwent electrophoresis ( they produced better results in relation to others)

Example of 3 seeded systems (test + experiment) genes 1,2,3:

Picture 1:  
On the left control plate Ib+amp – no colony growth, as expected. 
On the right side Ib+amp gene No. 1 that underwent transformation using electrophoresis method, you can see colony growth.

Picture 2:  
On the left of the control plate Ib+amp – no colony growth, as expected On the right gene Ib+amp gene No. 2 that underwent transformation using electrophoresis, you can see colony growth. 

Picture 3:  
On the left of the control plate Ib+amp – no colony growth , as expected On the right gene Ib+amp gene No. 3 that underwent transformation using electrophoresis, you can see colony growth.

LAB Week 3:

Performing colony PCR for verification of the presence the gene in the plasmid expression.  
Performing 4 repeats for each gene
Positive control – a whole plasmid is closed
Negative control – no plasmid

Results:

Picture 5:
after colony pcr Genes 1,2,4 – present in the expression plasmid PETtr – next stage – protein purification.  
Genes 3-5, 4-5 did not show luminescence when we ran on electrophoresis there was no presence of the gene.
The remaining genes (1-3, 2-3, 3-3, 1-5, 2-5) showed low luminescence which proves a small presence gene in the plasmid expression. We will perform a repeat process with genes 3, 5, 6.

LAB Week 4:

Genes 3, 5, 6
An additional attempt to increase the gene by performoing fusion pcr.
The difference between the colony pcr methods is performed by splitting using enzyme reaction before increasing by PCR.
In pcr fusion, at first we perform PCR and only after that we perform splitting, ligation and transformation.

Picture 6:
running on electrophoresis gel to genes 3, 5, 6 after fusion pcr, to test for gene presence.
We can see a weak expression of the genes however a clear luminescence in the 1500bp area. It was performed by 2 repeats of each gene.

Repeat experiment to genes 3, 5, 6.
We will perform:

  1. repeat ligation.
  2. Transformation to bl21(de3).
  3. Colony pcr
  4. Running on electrophoresis gel.

Results:

A repeat ligation and transformation to bacteria bl21(de3) and genes 3, 5, 6 and another performance of genes 3, 5, 6 colony pcr

Picture 7
colony pcr repeat to genes 3, 5, 6 we can see according to the big marker that the ligation was a success, the genes are in the PETtr plasmid, the transformation also succeeded, the next stage- extracting protein from the bacteria expression bl21(de3) (gene size 1500bp). Repeats to all the clones.

august

  1. Online Skype collaboration meetings. We conducted two online meetings – with Team Sydney and Team Paris. The conversations were very important for us especially to understand similar experiences in another place.
  2. Meeting with the Technion Youth team. The meeting contributed equally to both teams on how to give a presentation and poster. In addition, we will collaborate in the areas of wiki and modeling between the teams.
  3. The lab team made considerable progress to expressing the protein and checking the system.
  4. The documentation team doesn’t cease to run around all the groups taking more and more pictures, as well as the wiki team who building a site for the first time.
  5. The animation is on air after many arguments and doubt. The documentation team took a brave decision, you are invited to judge for yourselves.
  6. In the middle of August we felt that everyone was falling apart from the pressure so we decided to put everything on hold and take a week off in order to gather strength and return refreshed and full of energy.
  7. We have begun working on the presentation, not easy however, in full force; simultaneously the lab is finishing off their part gradually. Te results were not up to our expectations due to lack of time we will not be able to solve the riddle that has formed, it is possible we will leave the task for other students.
  8. Processing results and the writing stage are increasing – the team documents all the processes that have been done up until now together with the documentation team which contributes fascinating pictures of the entire process.

LAB Week 1:

Clone 1,2,4 Expression and purification of Allerglns
Cleaning the Gln-H protein from bacteria bl21(de3) by kolona with nickel strips, we added an extension of six histadines to the protein, therefore, the protein will bind to the nickel kolona. We will release it at the end of the process with the help of a buffer
constraint.
Perform a dialysis overnight, check activity by spectrophotometer.

  1. Fluidize the bacteria with a lysis buffer.
  2. Wash the protein with a wash buffer.
  3. Release the protein with an elution buffer.
  4. Run samples in protein gel.
  5. Dialysis overnight.
  6. Check activity.

LAB Week 2:

Genes 1
Vial 1 = substrate + protein – should not work (not laminate)
Vial 2 = protein + substrate + gluten (should work)
Vial 3 = gluten + substrate – should not work 

Genes 2 
Vial 1 = substrate + protein – should not work (not laminate)
Vial 2 = protein + substrate + gluten – should work
Vial 3 = gluten + substrate – should not work 

Genes 3
Vial 1 = protein (should not work – not laminate)
Vial 2 = gluten + protein (should work – laminate)
Vial 3 = gluten ( should not work – not laminate) 

Results:

Pictures 13-15- Running protein gel SDS 1,2,4 respectively

Picture 13
gene No. 1 EC_HAD is expressed in the system, probably was split into two sub units, we presume the reason is in the separation process, a change in the electrical charge due to a change in PH may cause protein catabolism to sub units. Another possibility is the existence of other proteins that were separated in the elution process to release the Gln-H protein. Nevertheless, one can see that the protein is expressed well in the system. 

Picture 14
gene No. 2 EC-GFP we can see the expression of the Gln-H protein in channels 7-14. Expression of the protein in the gene is lower; the intensity of the mark is very low compared to the protein extracted from gene No. 1.

Picture 15
gene No. 15 GBS-GFP is expressed in channels 8-14, expression of the Gln-H protein is lower than the protein expression from gene No.1 however greater than the protein extracted from gene No.2. 

Biobrick: preparing the plasmids:
The following actions we did to each of the synthetic bricks we created:

  1. Splitting the Gln-H gene from the plasmid PUC57
  2. Electrical charging the gene and plasmid on electrophoresis gel
  3. Identifying the gene and it’s split together on the gel
  4. Cleaning the gene from the gel
  5. Measuring the concentration of the gene
  6. Check activity.
  7. Ligation into the PSB1C3 plasmid – transporter plasmid
  8. Preparing a kit – 8 tubes
  9. Drying
  10. Filling the biobrick data on the Igem site
  11. Send the biobrick

LAB Week 3:

Expression and purification of Allerglns - genes 3, 5, 6  
Cleaning the Gln-H genes from the bacteria bl21(de3) by Kolona with nickel strips, we added an extension of six histadines to the protein, therefore, the protein will bind to the nickel kolona. We will release it at the end of the process with the help of a buffer constraint. Perform a dialysis overnight, check activity by spectrophotometer.

  1. Fluidize the bacteria with a lysis buffer
  2. Wash the protein with a wash buffer 
  3. Release the protein with an elution buffer
  4. Run samples in protein gel
  5. Dialysis overnight
  6. Check activity.

Genes 3
Vial 1 = substrate + protein – should not work (not laminate)
Vial 2 = protein + substrate + gluten (should work)
Vial 3 = gluten + substrate – should not work 

Genes 5
Vial 1 = substrate + protein – should not work (not laminate)
Vial 2 = protein + substrate + gluten (should work)
Vial 3 = gluten + substrate – should not work 

Genes 6
Vial 1 = protein (should not work – no luminescence)
Vial 2 = gluten + protein (should work (luminescence)
Vial 3 = gluten (should not work – no luminescence)

Results:

Pictures 16-18 gel running proteins SDS 3, 5, 6 respectively. We can see by the mark the size of the Gln-H protein at 55kd. In clone 5 we can see the clean protein notably.

Picture 16
gene No.3 EC-HAD is expressed in channels 5-14, expression of the Gln-H protein in the gene in considerably lower than protein expression from gene No. 5, howeve,r higher than expression of the protein extracted from gene No.6

Picture 17
gene No.5 TM-HAD is expressed from channel 5 in all channels, 5-14 expression of the Gln-H protein in the gene is the highest observed.

Picture 18
gene No. 6 TM-GFP is expressed in channels 5-15, expression of Gln-H protein in the gene is lower than the protein in other genes.

LAB Week 4: No activity 

september

Week 1:

We started practicing our presentation in different forums as well as practice our conversational English. We have a series of invites to come and talk about the idea of the project from start to finish. We were at a mini-Igem at the Technion, Tel-Aviv Port, and Tel Hai College and even at school. We received much “food for thought” and a lot of encouragement from our spectators.

The pressure is on the rise and we have begun working on the uniforms, and the small details.

Week 2:

Meeting with Erel margalit in his house.

Week 3:

Participation in the conference:" Cities Summit Tel Aviv ".
      
Presentation of the Alergln kit to our students and teachers in science majors.

Mini IGEM- Technion.

Week 4:

Coming soon...

LAB Week 1: modeling