Difference between revisions of "Team:Goettingen/Notebook"

 
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<h2>Notebook</h2>
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<p>
 
<p>
−
    <h1><strong>Plasmid Extraction - using peqGOLD Plasmid Miniprep Kit I,
+
<h1> Notebook </h1>
−
PEQLAB Technologies</strong></h1>
+
 
 
</p>
 
</p>
  
 +
    <a href="" onClick=" $('#menu1').slideToggle(300, function callback() {  }); return false;"><h2>15.12.14</h2></a>
 +
 +
<div id="menu1">
 
<p>
 
<p>
−
     Select few TOP10/BL21 <em>E.coli</em> transformed colonies from the LB Amp plates containing the propagated transformed colonies and inoculate them into
+
     <strong>1.) Finding a topic</strong>
−
    liquid LB medium (5 ml) containing preferred antibiotic of desired concentration in each tube.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Incubate the LB tubes with the transformed colonies at 37ᵒC in a shaker for about 12 – 15 hours (overnight) with agitation (150 rpm).
+
     After establishing who is interested in being a member of this year’s Team, everyone went out to find possible supervisors and projects. Prof. Rolf Daniel
 +
    together with Dr. Elzbieta Brzuszkiewicz and Dr. Silja Brady, whom some of us know from the first microbiology lab course, have a project for us:
 
</p>
 
</p>
 
<p>
 
<p>
−
     Extract the plasmids from the incubated LB cultures using the peqGOLD Plasmid Miniprep Kit I.
+
     <strong>2.) Project Proposal</strong>
 
</p>
 
</p>
 
<p>
 
<p>
−
     Centrifuge the culture at 10000 x g for 2 min to obtain the pellet and repeat the process until the culture is completely centrifuged. Store the pellet of
+
     We would create a cellulosome like multi enzyme scaffold as well as several linker regions with which
−
    1 ml of the culture at -20ᵒC for future use.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Resuspend the pellet in 250 µl of Solution I of the Kit (which is normally kept at 4ᵒC because of the RNase) and vortex until the pellet is resuspended.
+
     enzymes of interest could be fused. This way, if for example a company wants to degrade different
 
</p>
 
</p>
 
<p>
 
<p>
−
     Add 250 µl of Solution II to the resuspended mixture and gently mix by inverting and rotating the tubes 6 -10 times to obtain a cleared lysate. Incubate
+
     materials in a solution or wants a specific enzyme complex they would then list the needed enzymes
−
    the mixture for 2 min to obtain optimum results.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Add 350 µl of Solution III to the cleared lysate and gently mix by inverting the tubes 6 -10 times until a flocculent white precipitate is formed.
+
     and order them from us. We would then transform the enzymes before / after the linker regions, the
−
    Centrifuge at 10000 x g for 10 min at room temperature.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Transfer the clear supernatant to a fresh PerfectBind DNA Column in a 2 ml Collection Tube. Centrifuge the Column with the Collection Tube for 1 min at
+
     enzymes would be assembled in the cell on the scaffold and then exported by the model organism.
−
    10000 x g at room temperature. Discard the flow-through liquid.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Add 500 µl of PW Plasmid buffer to the PerfectBind DNA Column in the Collection Tube and centrifuge for 1 min at 10000 x g. Discard the flow-through.
+
     As the scaffold itself could be easily tagged, i.e. by a strep tag, we could then purify the multi enz
 
</p>
 
</p>
 
<p>
 
<p>
−
     Add 750 µl of DNA Wash buffer to the PerfectBind DNA Column in the Collection tube and centrifuge for 1 min at 10000 x g. Discard the flow-through. Repeat
+
     yme complex and deliver it to the company. As there are enzymes for thousands of different reactions, the applicability would be broad. As Dr.
−
     this step to obtain optimum results.
+
     Brzuszkiewicz and Dr. Brady weren't sure whe
 
</p>
 
</p>
 
<p>
 
<p>
−
     Place the PerfectBind DNA Column in the Collection tube and centrifuge for 2 min at 10000 x g to dry the column matrix. This step is essential to remove
+
     ther something like this has already been done, we will still have to do some research into the topic itself.
−
    ethanol from the column.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Place the PerfectBind DNA Column into a fresh 1.5 ml Eppendorf tube. Add 50 µl of pre-warmed sterile deionized water directly to the binding matrix in the
+
     <strong>2.) Deciding which topic to choose</strong>
−
    PerfectBind DNA Column and centrifuge for 1 min at 5000 x g to elute the DNA.
+
 
</p>
 
</p>
 
<p>
 
<p>
−
     Discard the PerfectBind DNA Column and store the eluted plasmid DNA at -20ᵒC.
+
     As we currently have a grand total of one project, the question is whether we wait for other topics,
 
</p>
 
</p>
 
<p>
 
<p>
−
     Check the concentration of the plasmids by using a NanoDrop and note down the values for future experiments.
+
     as not all lecturers have answered yet or, if there are no objections, go ahead with the project of
 
</p>
 
</p>
 +
<p>
 +
    Prof. Daniel. For this, please sent me a mail with your thoughts until Sunday, as we need to decide fast.
 +
</p>
 +
<p>
 +
    <strong>3.) Distribution of tasks</strong>
 +
</p>
 +
<p>
 +
    Everybody should look into Prof. Daniels topic, to see whether something similar has been done and
 +
</p>
 +
<p>
 +
    what the current status on this research area is. In the coming days Verena will invite you to the
 +
</p>
 +
<p>
 +
    iGEM 2015 Dropbox folder, in which you can upload interesting papers.
 +
</p>
 +
<p>
 +
    <strong>Booklet:</strong>
 +
    Dennis agreed to do the first draft over the holidays, Avril mentioned that she could proof
 +
</p>
 +
<p>
 +
    read the english version, Stefani would help with the design and further improvement.
 +
</p>
 +
<p>
 +
    <strong>Fiscal management</strong>
 +
    : David and Joshy have agreed to try and set up a preliminary budget plan.
 +
</p>
 +
<p>
 +
    Logo: Stefani will design our first logo.
 +
</p>
 +
<p>
 +
    <strong>Wikipedia:</strong>
 +
    Joshy agreed to handle our wikipedia for the time being.
 +
</p>
 +
<p>
 +
    <strong>Music video:</strong>
 +
    Verena has big plans for a laboratory-themed song and corresponding music video.
 +
</p>
 +
<p>
 +
    <strong>Website:</strong>
 +
    we still need someone who either knows a bit about website design or wants to learn how
 +
</p>
 +
<p>
 +
    to set up a web site. We can use the old site as template, but we still need to convert/cannibalise it
 +
</p>
 +
<p>
 +
    into our own. Please mail Verena or me if you would like to try your hand as a web designer.
 +
</p>
 +
<p>
 +
    <strong>4.) Dismissing members</strong>
 +
</p>
 +
<p>
 +
    We are a comparatively small group with an ambitious project and a limited number of members. The coming months are very important, since this decides
 +
    pretty much how easy we will have it later in the year. We currently have registered everybody who either mailed Verena or Julian or came to one of our
 +
    meetings and are currently at 10 members total, 12 being the limit. If we get to the limit,we have to put people on the waiting list. Since we are a small
 +
    group with an ambitious project, having someone in the group who doesn't bother to cancel to agreed appointments or, worse, won't do work she/he agreed on
 +
    doing, would not only unnecessary aggravate the rest of the group but would also slow us down.
 +
</p>
 +
<p>
 +
    Therefore, with the coming meeting, we will introduce a three strike system. If you can't come to a meeting, that's no problem - just tell someone who will
 +
    attend in advance or post it on facebook. If you don't do so three times, you will be excluded from the iGEM group.
 +
</p>
 +
<p>
 +
    If someone keeps making trouble within the group, the organisation team reserves the right to vote on excluding them.
 +
</p>
 +
<br clear="all"/>
 +
    </div>
  
 +
    <a href="" onClick=" $('#menu2').slideToggle(300, function callback() {  }); return false;"><h2>08.01.15</h2></a>
  
 +
<div id="menu2">
 +
<p>
 +
    <strong>1.) Topic: </strong>
 +
</p>
 +
<p>
 +
    We intend to utilise a scaffold protein in order to assemble enzymes with different functions into one complex, which will hopefully increase their
 +
    efficiency as well as being customisable.
 +
</p>
 +
<p>
 +
    As model organisms <em>B.subtilis</em> or <em>E.coli</em> seem to be to most viable at this point
 +
</p>
 +
<p>
 +
    Issues may arise from the connection of linker regions to the enzymes and the subsequent loss of function.
 +
</p>
 +
<p>
 +
    <strong>2.) Booklet: </strong>
 +
</p>
 +
<p>
 +
    Most parts of the booklet have been written by Dennis. Avril will proofread the sections and then pass them on to Stefani for layout and design.
 +
</p>
 +
<p>
 +
    Further information needs to be added about the research projec, budget and sponsoring.
 +
</p>
 +
<p>
 +
    <strong>3.) Website/Wiki:</strong>
 +
</p>
 +
<p>
 +
    Udhaya and Joschy will take care of the Wiki and Webpage together. Bing Yao from a previous Igem Team has offered to help and teach.
 +
</p>
 +
<p>
 +
    <strong>4.) Locations:</strong>
 +
</p>
 +
<p>
 +
    Julian has asked Prof. Daniel to ask if someone has a space for us to have an office in their lab. Other options are the Schwann-Schleiden Research Centre,
 +
    experimental medicine or the physics department.
 +
</p>
 +
<p>
 +
    <u>Update:</u>
 +
    Verena has found out, that there is an iGem Computer in the Microbiology library.
 +
</p>
 +
<p>
 +
    Usage of the practical room as labspace is in an agreement, and should therefore be possible.
 +
</p>
 +
<p>
 +
    Equipment from the previous team will be assessed by Verena.
 +
</p>
 +
<p>
 +
    <strong>5.) Organisation Team</strong>
 +
    was confirmed in its current setup of: Julian, Verena, Udhaya and Avril. If desired or the need should arise, the team can be extended further along the
 +
    line.
 +
</p>
 +
<p>
 +
    <strong>6.) Finances:</strong>
 +
</p>
 +
<p>
 +
    Budget for 3 different situations can be found in the dropbox (by Daniel). The booking prices are from the 5th of January and are subject to change.
 +
</p>
 +
<p>
 +
    Visa prices need to be determined and passports updated.
 +
</p>
 +
<p>
 +
    Julian will take over the Finances including the management of a bank account, bookings and the overview of our spending and financial support.
 +
</p>
 +
<p>
 +
    <u>Update:</u>
 +
    200€ are left over from the previous Igem Team to be used by this Igem Team.
 +
</p>
 +
<p>
 +
    <strong>7.) Timetable and Deadlines:</strong>
 +
</p>
 +
<p>
 +
    Julian has made a preliminary timetable (Dropbox). In addition to this, the deadline for the booklet will be the <strong>14th of January</strong> and the
 +
    deadline for the first round of Sponsor contacting will be the <strong>18th of January</strong>. Unresponsive sponsors will be contacted weekly. The
 +
    sponsor list will be split up between the team members at the next meeting (13th of January).
 +
</p>
 +
<p>
 +
    The best weekdays for team meetings are Mondays and Thursdays .
 +
</p>
 +
<p>
 +
    <strong>8.) Roles:</strong>
 +
</p>
 +
<p>
 +
    Press and Publicity: Steffi and Dennis
 +
</p>
 +
<p>
 +
    Finances: Julian
 +
</p>
 +
<p>
 +
    Sponsoring managers: Sindhu and Nida
 +
</p>
 +
<br clear="all"/>
 +
    </div>
  
 +
    <a href="" onClick=" $('#menu3').slideToggle(300, function callback() {  }); return false;"><h2>13.01.15</h2></a>
  
 +
<div id="menu3">
 +
<p>
 +
    <strong>1.) Logo and Booklet:</strong>
 +
</p>
 +
<p>
 +
    Stefani presented two ideas for a possible logo and asked for opinions. She will flesh them out a bit and we can decide on our next meeting which one we
 +
    will choose as our main logo.
 +
</p>
 +
<p>
 +
    Stefani also presented a possible design for our booklet.
 +
</p>
 +
<p>
 +
    Udhaya had an idea for a possible slogan we could use, fitting one of our possible logos: “Placing enzymes where they should be”.
 +
</p>
 +
<p>
 +
    <strong>2. ) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    Sindhu will assign the companies we have so far, so everyone will have to contact 4 to 5.
 +
</p>
 +
<p>
 +
    <strong>3.) Equipment:</strong>
 +
</p>
 +
<p>
 +
    Verena and Udhaya went through the inherited things from last year’s iGEM-Group. They are now listed in our inventory.
 +
</p>
 +
<p>
 +
    <strong>4.) Meetings:</strong>
 +
</p>
 +
<p>
 +
    We decided to only hold meetings every two weeks, as every week seems a bit excessive, especially when we meet alternative on Mondays and Tuesdays. The
 +
    schedule is in our timetable.
 +
</p>
 +
<p>
 +
    <strong>5.) Task for next week:</strong>
 +
    <br/>
 +
    Everybody should think about a fitting project title until our next meeting.
 +
</p>
 +
<br clear="all"/>
 +
    </div>
  
 +
    <a href="" onClick=" $('#menu4').slideToggle(300, function callback() {  }); return false;"><h2> 15.01.15 </h2></a>
  
 +
<div id="menu4">
 +
<p>
 +
    <strong>Present:</strong>
 +
    Verena, Alaa, Udhaya, Julian, Daniel, Avril, Silja, Ela, Prof. Daniel, Heiko Liesegang
 +
</p>
 +
<p>
 +
    Supervisors will be met for monthly update
 +
</p>
 +
<p>
 +
    <strong>1.) Useful contacts:</strong>
 +
</p>
 +
<p>
 +
    Supervisors Ela (Sequencing) and Silja (Biotech), Head of Unit Prof. Daniel, Bioinformatics Heiko Liesegang, Secretary Daniela, Lab Technicians: Ute &amp;
 +
    Gabriele, Synthetic Bio Jun.-Prof. Neumann.
 +
</p>
 +
<p>
 +
    Prof. Daniel will meet with Prof. Braus to discuss grants, which will probably require writing a formal letter
 +
</p>
 +
<p>
 +
    Petra from Prof. Daniel's lab used to work as a webdesigner and can be asked for support on the webpage
 +
</p>
 +
<p>
 +
    Supervisors will check the current sponsor list for additional contacts
 +
</p>
 +
<p>
 +
    Silja will do the <strong>lab safety talk</strong> before we start the labwork
 +
</p>
 +
<p>
 +
    Heiko Liesegang is happy to support the team with Bioinformatics and Industrial appliance of the project (he is situated in the cellar).
 +
</p>
 +
<p>
 +
    Verena will add the supervisors to the dropbox, so they can have access to the list of sponsors, inventory, etc.<a name="_GoBack"></a>
 +
</p>
 +
<p>
 +
    <strong>2.) Project:</strong>
 +
</p>
 +
<p>
 +
    The scaffold protein will be taken from <em>Clostridium thermocellum</em>, which is very well described and has been fully sequenced, as well as a known
 +
    structure. The host will be codon optimised <em>E.coli</em>, which can be used with a vector system and can have the structure expressed intra- or
 +
    extracellularly. -&gt; Proof of principle on <em>E.coli</em> before large scale adaptation with <em>B.subtilis</em>
 +
</p>
 +
<p>
 +
    <strong>3.) Research to be done by team:</strong>
 +
</p>
 +
<p>
 +
    - Which enzymes: ideally well known
 +
</p>
 +
<p>
 +
    - How many "dockerins" on the scaffold
 +
</p>
 +
<p>
 +
    - Which biotechnical processes offer themselves -&gt; e.g. waste water, cow udders
 +
</p>
 +
<p>
 +
    - How can the enzymes be linked to the scaffold: e.g multiple enzymes with the same dockerin for exchangeability
 +
</p>
 +
<p>
 +
    - Which Tags can be used
 +
</p>
 +
<p>
 +
    - Which cloning techniques
 +
</p>
 +
<p>
 +
    - Will the bacteria survive and work continuously or die after their application
 +
</p>
 +
<p>
 +
    - How will they be removed from the product
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    Ideally we would use a vector system to form a toolkit, which can be modified by the customers. Therefore a modular design and simplicity would be of
 +
    advantage.
 +
</p>
 +
<p>
 +
    <strong>4.) Planning:</strong>
 +
    Times expected time by 2 or split group into teams, which work on separate goals: e.g. Scaffold group, lipolytic enzymes group, cellulolytic enzymes group,
 +
    etc.
 +
</p>
 +
<p>
 +
    Each group should work with multiple enzymes, to guarantee that they create at least one functional tool.
 +
</p>
 +
<p>
 +
    <strong>5.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Can start at any time as the Daniel-group will provide us with the resources until we have raised enough money. This can then be paid back at a later
 +
    state. Kits and essential chemicals, etc. are also available through the Lab. -&gt; Start on the Scaffold can begin. Julian, Udhaya and Verena will meet
 +
    Silja on Tuesday to create the theoretical basis/Clone manager plans.
 +
</p>
 +
<p>
 +
    <strong>6.) Meetings</strong>
 +
    will have to take place at least weekly as soon as the labwork has started. Next monthd need to be planned and scheduled with everyone's availability and
 +
    working ability, exams, etc.
 +
</p>
 +
<p>
 +
    <strong>7.) Logo</strong>
 +
    will have added science things, e.g. helix, magnifying glass. The theme should be used with the university colours (dark and light blue). The booklet can
 +
    have a different title layout to the main part, which can also be designed in a modular, toolbox manner.
 +
</p>
 +
<p>
 +
    Think of a name for the cellulosome &amp; make it cool: e.g.: superosome, flexosome, legosome
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    <strong>8.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    Possible other sponsors: IVA (for kits), Illumina, Roche
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<br clear="all"/>
 +
    </div>
  
 +
    <a href="" onClick=" $('#menu5').slideToggle(300, function callback() {  }); return false;"><h2> 19.01.15</h2></a>
  
 +
<div id="menu5">
 +
<p>
 +
    Present: Silja, Ela, Julian, Verena, Avril, Stefani, Joschi, Sindhu, Nida, Alaa, Dennis, Daniel
 +
</p>
 +
<p>
 +
    Student Card numbers were collected for easier access to the labs.
 +
</p>
 +
<p>
 +
    <strong>1.) Project:</strong>
 +
</p>
 +
<p>
 +
    We need to decide whether we want the enzymes to degrade or synthesis and focus on one application to start with.
 +
</p>
 +
<p>
 +
    Possible enzymes/applications: nitrilases, university waste water/water works/ environmental, contaminated soils, TNT degrading, rubber degrading, Vitamin
 +
    production from intermediates (can be easily proven if it works), bacterial indigo synthesis/other colours
 +
</p>
 +
<p>
 +
    The easiest is to take a lipase, esterase and/or phosphatase and see if it can degrade different substrates.
 +
</p>
 +
<p>
 +
    Ela emphasized that the labbooks need to be written with particular care in order to keep the overview and find small mistakes.
 +
</p>
 +
<p>
 +
    A time plan and team division should be formed in the near future.
 +
</p>
 +
<p>
 +
    <strong>The safety lecture will be given on Monday the 26.1.2015 at 10am. Everyone is required to attend this lecture!</strong>
 +
</p>
 +
<p>
 +
    <strong>2.) Booklet:</strong>
 +
</p>
 +
<p>
 +
    Projectname was decided: <strong>Flexosome</strong> (with 10/12 votes), runners up were: Custosome and Supersome
 +
</p>
 +
<p>
 +
    IGEM logo should be within the text to minimise white space
 +
</p>
 +
<p>
 +
    A group picture should be added to make the booklet more personal.
 +
</p>
 +
<p>
 +
    Boxes on page 1 need to be filled/removed/changed
 +
</p>
 +
<p>
 +
    <strong>3.) Sponsoring packages:</strong>
 +
</p>
 +
<p>
 +
    Bronze package: &gt;500€: Logo will be displayed on the website
 +
</p>
 +
<p>
 +
    Silver: &gt;2500€: Logo on Website and T-Shirt
 +
</p>
 +
<p>
 +
    Gold: &gt;5000€: Logo will be displayed everywhere
 +
</p>
 +
<p>
 +
    <strong>4.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    Contacts exist to Henkel, Bayer &amp; Illumina. Silja and Ela will introduce us to these and then we can send our booklet/proposal for funding.
 +
</p>
 +
<p>
 +
    Other possible sponsors are: SI clone manager or VWR
 +
</p>
 +
    </div>
  
 +
    <a href="" onClick=" $('#menu6').slideToggle(300, function callback() {  }); return false;"><h2> 02.02.15 </h2></a>
  
 +
<div id="menu6">
 +
<p>
 +
    <strong>Attendance </strong>
 +
</p>
 +
<p>
 +
    Avi, Udhaya, Nida, Verena and Julian
 +
</p>
 +
<p>
 +
    <strong>1.) Booklet:</strong>
 +
</p>
 +
<p>
 +
    Avi and Stefani went to the printer center, they test-printed a booklet and gave some options and prices. They showed the booklet to Ela and she also
 +
    talked to Prof. Daniel. He wants the logo and booklet to have more biology related design on it and he wants it to be printed on better paper. His working
 +
    group can pay for it. So Stefani is still working on the booklet. The printing can be done on Din A3 sized paper which will reduce costs (30 € for 80
 +
    booklets!?) For a professional binding we have to give it to a Buchbinder, Avi had contacted the Buchbinder and the guy said the costs will be 20 € for
 +
    half an hour and 40 € if it takes a full hour.
 +
</p>
 +
<p>
 +
    <strong>2.) Sponsoring:</strong>
 +
</p>
 +
<p>
 +
    Sindhu and Nida divided our company list. Each person has to contact 6 companies. The updated list is in the Dropbox.
 +
</p>
 +
<p>
 +
    <strong>3.) iGEM GWDG account and email address:</strong>
 +
</p>
 +
<p>
 +
    Verena contacted the GWDG and filled in the form to create an iGEM 2015 user account incl. email-address. As soon as it is done, the GWDG will send a
 +
    letter to Prof. Daniels department.
 +
</p>
 +
<p>
 +
    <strong>4.) Bachelor students from other faculties:</strong>
 +
</p>
 +
<p>
 +
    Debbie asked a friend who is studying B. Sc. Informatics here in Göttingen and he is interested in participating to help with programming and stuff for the
 +
    website. Since he is focussing on a different area but does not seem to be completely uneducated in websites and webdesign and is 24 years old, we decided
 +
    that if we cannot find another skilled (younger) person, we can try it.
 +
</p>
 +
<p>
 +
    <strong>5.) Group photo</strong>
 +
</p>
 +
<p>
 +
    A preliminary group photo was taken last week and will be included in the booklet as long as we don’t have another one.
 +
</p>
 +
<p>
 +
    <strong>6.) Scaffoldin</strong>
 +
</p>
 +
<p>
 +
    Udhaya, Julian and Verena met up with Ela and Silja to further design the Scaffoldin. On the first meeting some interesting organisms for putative
 +
    cohesins/dockerins could be found. Now Ela compared a variety of cohesin structures (sequences) from those organisms by alignment and bioinformatical
 +
    reconstruction of phylogenetic trees. Ela gave all organisms a letter code. For example: “CTHE” for <em>Clostridium thermocellum.</em> Due to the fact that
 +
    we want to use (only) 4 different cohesins, we need to cut off scaffoldin proteins that naturally offer more than 4 cohesins in the protein structure. To
 +
    ensure that there will be no unexpected interactions with our desired cohesins (and to have a back-up if the first construct doesn’t work), we finally
 +
    decided to go with a naturally long scaffoldin protein and a shorter one which only harbors 4 cohesin places:
 +
</p>
 +
<p>
 +
    <u>1) Main construct</u>
 +
    : <em>Clostridium thermocellum</em>
 +
</p>
 +
<p>
 +
    Scaffolding protein <u>CipA</u> (cohesin type I) with dockerin type II. CipA contains in sum <strong>9 cohesins.</strong> The first two are somehow
 +
    disconnected by a CBM (cellulose binding motif). A deletion could lead to problems, so we decided to cut off cohesion 1-4 (incl. the CBM). The resulting
 +
    protein offers space for 5 cohesins. The first of these 5 will be the natural cohesin of <em>C. thermocellum</em> so that we have 4 free cohesin places for
 +
    our desired cohesins.
 +
</p>
 +
<p>
 +
    Desired Scaffolding protein structure:
 +
</p>
 +
<p>
 +
    SP EC – linker 6 CTHE – coh6 (CTHE) – cohX – cohY – cohZ – DOC (CTHE)
 +
</p>
 +
<p>
 +
    <u>2) Back-Up:</u>
 +
    <em>Acetivibrio cellulolyticus</em>
 +
</p>
 +
<p>
 +
    Scaffolding protein <u>ScaB</u> (cohesin type II) with dockerin type I. ScaB contains in sum <strong>4 cohesins.</strong> The first of these 4 will be
 +
    (again) the natural cohesin of <em>A. cellulolyticus</em> so that we have 3 free cohesin places for our desired cohesins. It is not clear at the moment
 +
    which dockerin will be used at the end of the structure.
 +
</p>
 +
<p>
 +
    Desired Scaffolding protein structure:
 +
</p>
 +
<p>
 +
    SP EC – coh type II – cohX – cohY – cohZ – DOC (ACEL) / DOC (CTHE)
 +
</p>
 +
<p>
 +
    For further information on the scaffoldin please look in the “Scaffoldin” Dropbox-folder: PowerPoint “scaffoldin_overview”
 +
</p>
 +
<p>
 +
    <u>3)Websites/tools</u>
 +
    : NCBI (BLAST etc.), InterPro Scan for sequences, alignments, selection and branding of domains in Artemis.
 +
</p>
 +
<p>
 +
    Calculated 3D structures of CipA (<em>Clostridium thermocellum</em>) by LOOPP and Phyre<sup>2</sup>
 +
</p>
 +
<p>
 +
    What has to be done now is to find the sequences for the fitting dockerin-binding proteins. This will be discussed on the next scaffolding-meeting tomorrow
 +
    (03.2.15, 2 p.m.) with Ela, Silja, Udhaya, Julian and Verena.
 +
</p>
 +
<p>
 +
    Moreover it is time for us to contact companies doing gene synthesis. Possible companies can be found at “Gene synthesis consortium”
 +
</p>
 +
<p>
 +
    (<a href="http://www.genesynthesisconsortium.org/members.php">http://www.genesynthesisconsortium.org/members.php</a>)
 +
</p>
 +
<p>
 +
    A template message how to contact these companies was sent by you per email from Julian last week.
 +
</p>
 +
<br clear="all"/>
 +
    </div>
  
 +
    <a href="" onClick=" $('#menu7').slideToggle(300, function callback() {  }); return false;"><h2> 16.02.15 </h2></a>
  
 +
<div id="menu7">
 +
<p>
 +
    Ela and Silja have ordered the organisms from which we will extract the dockerins from the DSMZ. To work with those, we need to start preparing media.
 +
    Udhaya has agreed to find a company until the 7<sup>th</sup> which will then produce our scaffoldin constructs.
 +
</p>
 +
    </div>
  
−
<p> Document the dates you worked on your project.</p>
+
    <a href="" onClick=" $('#menu8').slideToggle(300, function callback() {  }); return false;"><h2>  18.02.15</h2></a>
  
−
<h5>What should this page have?</h5>
+
<div id="menu8">
−
<ul>
+
<p>
−
<li>Chronological notes of what your team is doing.</li>
+
    <strong>1.) Labwork:</strong>
−
<li> Brief descriptions of daily important events.</li>
+
</p>
−
<li>Pictures of your progress. </li>
+
<p>
−
<li>Mention who participated in what task.</li>
+
    We have formed our first groups for the two scaffoldins as well as the three enzymes. Debby and Alaa will ask around in the Biochemistry Department for
−
</ul>
+
    concrete ideas for a more complex enzyme systems.
 +
</p>
 +
<p>
 +
    Coming next week, Silja and Elzbieta will show at least one person from each enzyme group, how to extract the dockerines from the organisms. The groups
 +
    will then each extract a dockerin and prepare the enzymes.
 +
</p>
 +
<p>
 +
    <strong>2.) Sponsors: </strong>
 +
</p>
 +
<p>
 +
    Sindhu will prepare a letter for Prof. Daniel, which will then also be sent out with each booklet.
 +
</p>
 +
<p>
 +
    The booklets are currently in print; as soon as Avril hears from the printers she will tell us. We will then start sending them out.
 +
</p>
 +
<p>
 +
    On our next meeting, we will finalise our working groups as well as set up minimum working hours for the lab.
 +
</p>
  
 +
    </div>
 +
<a href="" onClick=" $('#menu9').slideToggle(300, function callback() {  }); return false;"><h2> 23.02.15</h2></a>
  
−
<h4>Inspiration</h4>
+
    <div id="menu9">
−
<p>You can see what others teams have done to organize their notes:</p>
+
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    We agreed on a minimum work hour per month set at 30 h. Each participant can divide their own time as they wish, as long as at least two persons are
 +
    present in the lab as well as an additional PhD / Postdoc on the ground floor in case of accidents.
 +
</p>
 +
<p>
 +
    If the minimum work hours aren't kept, the person may be kicked from the team. The hours may be transferred to another person, if both parties agree.
 +
</p>
 +
<p>
 +
    The work groups have been put together.
 +
</p>
 +
<p>
 +
    Each work group will loan their own equipment. The loaning person is responsible for any damages. Any damages will have to be paid by the person who loaned
 +
    the equipment.
 +
</p>
 +
<div>
 +
    <p>
 +
        Avril has also proposed to create a time table of sorts in which everybody can reserve lab space. If we do work all at the same time in the lab it’ll
 +
        get crowded soon as we only have alloted work space for up to 6 people.
 +
    </p>
 +
    <p>
 +
        First ordered organisms have arrived.
 +
    </p>
 +
    <p>
 +
        Julian and Dennis will sort out Media on Tuesday
 +
    </p>
 +
    <p>
 +
        <strong>2.) Booklet:</strong>
 +
    </p>
 +
    <p>
 +
        Avril could not reach Herr Nolte from printing last week, and will go there on Tuesday to get it all sorted.
 +
    </p>
 +
</div>
 +
<p>
 +
    <u>Update:</u>
 +
    Booklets are printed and will be cut and put into letters on Wednesday.
 +
</p>
 +
<p>
 +
    <strong>3.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    Sindhu/Nida have updated Sponsors, however everyone needs to check the addresses and whether they are correct. Sindhu has also started writing a general
 +
    letter to send to sponsors.
 +
</p>
 +
<p>
 +
    <strong>4.) Office:</strong>
 +
</p>
 +
<p>
 +
    Julian: Sorted out an <strong>office</strong>, which is big and most likely free for the entire time of iGem. It is on the    <strong>2nd floor of the Geography</strong> building. There are two keys, which a system is needed so everyone who needs the office has access.
 +
</p>
 +
</div>
  
−
<ul>
+
<a href="" onClick=" $('#menu10').slideToggle(300, function callback() {  }); return false;"><h2> 16.03.15</h2></a>
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+
−
</ul>
+
  
 +
    <div id="menu10">
 +
 +
<p>
 +
    <strong>Attendance </strong>
 +
</p>
 +
<p>
 +
    Alaa, Dennis, Julian, Sindhu, Udhaya, Verena and Ela
 +
</p>
 +
<p>
 +
    <strong>1.) Money - GZMB</strong>
 +
</p>
 +
<p>
 +
    Julian has managed to write a letter for the GZMB to ask for money and let Prof. Dr. Rolf Daniel sign it. Prof. Daniel will send it this afternoon and we
 +
    should have a response within 2-3 days.
 +
</p>
 +
<p>
 +
    <strong>2.) Schedule – overview</strong>
 +
</p>
 +
<p>
 +
    <strong>Scaffoldins:</strong>
 +
</p>
 +
<p>
 +
    Finances needed: Maybe Prof. Daniel will pay for one of the constructs, ordering (2-3 weeks), vector cloning (4-6 weeks). Labwork could therefore start in
 +
    4-6 weeks
 +
</p>
 +
<p>
 +
    <strong>Dockerins:</strong>
 +
</p>
 +
<p>
 +
    Strains have to be ground and DNA isolated, PCR and cloning into pBAD, extraction and purification when fused to enzymes. Lab work can start now.
 +
</p>
 +
<p>
 +
    <strong>Enzymes:</strong>
 +
</p>
 +
<p>
 +
    Chosen enzymes need to be cloned into pJET (no activity) and then into pBAD with Dockerins. Expression and purification follow afterwards. Labwork can
 +
    start now.
 +
</p>
 +
<p>
 +
    <strong>3.) Working groups</strong>
 +
</p>
 +
<p>
 +
    Due to started lab work, exams and practical courses we had to rethink our groups for the moment.
 +
</p>
 +
<p>
 +
    Ela gave us the advice that it would be the best to have (at the moment) 2 groups working on the enzymes (5 enzymes plus GFP), 1 group working on the
 +
    dockerins and 1 group pushing the sponsors. Without money we cannot register and go on with the project!
 +
</p>
 +
<p>
 +
    <u>Enzyme groups:</u>
 +
</p>
 +
<p>
 +
    1) Avi, Stefani, Joschy
 +
</p>
 +
<p>
 +
    2) Debby, Alaa, Udhaya
 +
</p>
 +
<p>
 +
    <u>Dockerin group:</u>
 +
</p>
 +
<p>
 +
    Julian, Dennis
 +
</p>
 +
<p>
 +
    <u>Sponsor group:</u>
 +
</p>
 +
<p>
 +
    Sindhu, Verena (until the 24<sup>th</sup> march, afterwards also free for lab work)
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    <strong>4.) Lab times</strong>
 +
</p>
 +
<p>
 +
    We agreed on a minimum work hour per month set at 20 h per person. Due to organisation this month does not count.
 +
</p>
 +
<p>
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
    <strong>5.) Keys</strong>
 +
</p>
 +
<p>
 +
    All lab keys and our office key can be found in our own locker. Ask Julian for the numbers of the combination lock!
 +
</p>
 +
<p>
 +
    <strong>6.) Time table</strong>
 +
</p>
 +
<p>
 +
    We agreed on creating an overall time table that will be uploaded into the Dropbox and updated so that everybody knows exactly what has been done so far in
 +
    the lab and has to be done in the next days!
 +
</p>
 +
<p>
 +
    <strong>7.) Lab equipment lending</strong>
 +
</p>
 +
<p>
 +
    At least one person of each group has to come <strong>tomorrow 17<sup>th</sup> March at 10:30 p.m.</strong> to lend lab equipment the groups will need the
 +
    next weeks.
 +
</p>
 
</div>
 
</div>
 +
 +
<a href="" onClick=" $('#menu11').slideToggle(300, function callback() {  }); return false;"><h2> 23.03.15</h2></a>
 +
 +
    <div id="menu11">
 +
   
 +
 +
<p>
 +
    <br/>
 +
    Missing: Sindhu
 +
    <br/>
 +
    <br/>
 +
    <strong>1.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    We need to wait 2 weeks until we hear from the GZMB about funding. This means we will have to pay the late fee for iGEM. Any sponsor bills for sponsors we
 +
    find until then will be held back until we have a thumbs up from the GZMB funding.
 +
</p>
 +
<p>
 +
    Avi will put a new file with sponsors into the Dropbox, to see if everyone can edit it. The list will be updated with additional contacts
 +
    <br/>
 +
    Debbie has received two no answers and no other replies.
 +
    <br/>
 +
    Julian: No news from Sparkasse, another contact will be updated once he can reach Prof. Stülke
 +
    <br/>
 +
    Add more local sponsors
 +
    <br/>
 +
    Joschy will call his own sponsors, who haven't replied
 +
    <br/>
 +
    Will ask Verena to contact VW again, but without calling it sponsoring (differences in taxation)
 +
    <br/>
 +
    <br/>
 +
    <strong>2.) Lab updates:</strong>
 +
</p>
 +
<p>
 +
    <strong>D:</strong>
 +
    growing bacteria is tricky; two of 4 cultures may be growing but are very slow. They were plated on solid medium in order to see if they will grow there.
 +
    The first media and trace elements didn't work. But have been redone now. The two cultures that have grown so far will also be extracted (tomorrow).
 +
    <br/>
 +
    <strong>E1:</strong>
 +
    designed primers for esterase and phosphatase in order to fuse it with dockerin and plasmid. The plasmid has a His-tag. Tomorrow they will do the first PCR
 +
    with the primers for the phosphatase. Will ask Gabrielle about Cycler.
 +
    <br/>
 +
    <strong>E2:</strong>
 +
    nothing so far.
 +
    <br/>
 +
    <br/>
 +
    <strong>3.) Website:</strong>
 +
</p>
 +
<p>
 +
    Alex, who studies economical informatics, has joined our team and will work on the website and help with the Wiki. Julian will contact Cedric about login
 +
    for the website. All company logos shall be added to the Dropbox when they are received.
 +
    <br/>
 +
    Joschy has been thinking about the webpage: maybe have a picture in the background instead of a plain one.
 +
    <br/>
 +
    <br/>
 +
</p>
 +
<p>
 +
    <strong>4.) Other stuff:</strong>
 +
</p>
 +
<p>
 +
    Joschy will have a look at the structure or a possible picture for our flexosome once he has the sequences. Binding simulation on pymol.
 +
    <br/>
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu12').slideToggle(300, function callback() {  }); return false;"><h2> 07.04.15</h2></a>
 +
 +
    <div id="menu12">
 +
 +
<p>
 +
    <strong>1.) Lab:</strong>
 +
</p>
 +
<p>
 +
    Dockerin1: One plate shows some growth but a PCR has shown nothing so far.
 +
</p>
 +
<p>
 +
    Liquid cultures had a pellet in 3 cases, from which J. and D. will try to extract the DNA.
 +
</p>
 +
<p>
 +
    Enzyme 1: have got 2 enzymes ready for insertion (phosphatase and esterase)
 +
</p>
 +
<p>
 +
    Enzyme2: will start working in the evenings during the practical.
 +
</p>
 +
<p>
 +
    Avril can show people how to work clone manager to design primers.
 +
</p>
 +
<p>
 +
    Scaffolding order via Ela or get her to tell someone how to do it.
 +
</p>
 +
<p>
 +
    People who are not in the plant module should see if they can work alongside each other. Who is taking which course next semester?
 +
</p>
 +
<p>
 +
    <strong>2.) Website</strong>
 +
    : Alex should be put in touch with Bing Yao from a previous iGEM Team.
 +
</p>
 +
<p>
 +
    <strong>3.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    Sparkasse letter needs to be resent to the correct person.
 +
</p>
 +
<p>
 +
    New list of sponsors from biotech booklet needs to be contacted.
 +
</p>
 +
<p>
 +
    <strong>4. Boston:</strong>
 +
</p>
 +
<p>
 +
    Prices for flight and accommodation in Boston will be checked by Monday.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu23').slideToggle(300, function callback() {  }); return false;"><h2>13.04.15</h2></a>
 +
 +
<div id="menu23">
 +
<p>
 +
<p>
 +
    <strong>1.) Website:</strong>
 +
</p>
 +
<p>
 +
    Individual skills need to be worked out to find out who can do which part. Joschy is currently learning how to use HTML. He has made a navigation bar. It
 +
    may be helpful to have some external input.
 +
</p>
 +
<p>
 +
    The colour scheme will be our iGEM colours (blues)
 +
</p>
 +
<p>
 +
    The content must be updateable by every group, can be taken from the booklet to start with
 +
</p>
 +
<p>
 +
    Every team member needs to write a max. 150 word paragraph about themselves and submit a photograph to the dropbox.
 +
</p>
 +
<p>
 +
    <strong>2.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Dockerins: DNA has been extracted and we may get 3-4 Dockerins. Dockerins need to be finished in order to have the enzyme groups continue.
 +
</p>
 +
<p>
 +
    Scaffoldin: Transformation into E.coli worked and the plasmids can now be extracted and verified. First expression tests can follow. Alaa and Sindhu will
 +
    continue with the Scaffoldin from tomorrow.
 +
</p>
 +
<p>
 +
    E2: Primers will be designed on Thursday
 +
</p>
 +
<p>
 +
    E1: nothing to report due to a Practical, no work can be done atm.
 +
</p>
 +
<p>
 +
    Clone Manager: Julian will try to see if we can get access to it somehow.
 +
</p>
 +
<p>
 +
    <strong>3.) Sponsors:</strong>
 +
</p>
 +
<p>
 +
    GZMB is meeting soon and will decide on funding. We have so far got 4 Sponsors.
 +
</p>
 +
<p>
 +
    <strong>4.) Flights:</strong>
 +
</p>
 +
<p>
 +
    Alaa has looked up flights if we book now:
 +
</p>
 +
<p>
 +
    700-870€, Hotel: 45-60€ p.P /pN. We need to book fast as most hotels and hostels will be booked by fellow igemers.
 +
</p>
 +
<p>
 +
    <strong>5.) Costs:</strong>
 +
</p>
 +
<p>
 +
    Anticipated Labcosts: 18000€
 +
</p>
 +
<p>
 +
    Flights: 800€ pP
 +
</p>
 +
<p>
 +
    Hotel: 600€ pP
 +
</p>
 +
<p>
 +
    We need a minimum of 6000€ from the GZMB if we want to continue with the project.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu24').slideToggle(300, function callback() {  }); return false;"><h2>21.05.15 </h2></a>
 +
 +
<div id="menu24">
 +
<p>
 +
<p>
 +
    About the supervisors needs to be written for our Webpage
 +
</p>
 +
<p>
 +
    <strong>Labwork:</strong>
 +
</p>
 +
<p>
 +
    Dockerins: Attempt to put them in pBAD has failed so far. Retransformation control worked however. ACEL needs to be reinserted into pJET, BCEL needs to be
 +
    restarted from PCR, CTHE ligation needs to be redone. Change elution steps to gain higher concentrations (30µl instead of 50)
 +
</p>
 +
</div>
 +
 +
 +
<a href="" onClick=" $('#menu13').slideToggle(300, function callback() {  }); return false;"><h2> 19.06.15</h2></a>
 +
 +
    <div id="menu13">
 +
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Heiko will have an iGEM meeting slot everyday from 9-9.30am in his office.
 +
</p>
 +
<p>
 +
    E1:
 +
</p>
 +
<p>
 +
    -skipping BL21 step, because Esterase showed activity in TOP10 activity plates.
 +
</p>
 +
<p>
 +
    - Another option is to add a T7 Plasmid to induce the gene
 +
</p>
 +
<p>
 +
    - Phosphatase is expressing nicely (blue)
 +
</p>
 +
<p>
 +
    Phosphatase will be sent for sequencing next week.
 +
</p>
 +
<p>
 +
    E3:
 +
</p>
 +
<p>
 +
    Ready for expression plates with Cellulase in BL21
 +
</p>
 +
<p>
 +
    Scaffoldin:
 +
</p>
 +
<p>
 +
    Has not worked yet, probably due to the unspecific binding of the primers (good binding conditions are hard to find). Using a ligase free ligation in
 +
    pet101 which is sensitive to temperature and ions
 +
</p>
 +
<p>
 +
    -&gt;do a microdialysis to clean ligation from salts and ions before doing transformation.
 +
</p>
 +
<p>
 +
    The second scaffoldin was ordered and should arrive in the next couple of weeks.
 +
</p>
 +
<p>
 +
    RFP:
 +
</p>
 +
<p>
 +
    Cells are not pink and don’t fluoresce under the fluorescence microscope. Sequences are fine but will be done again in order to make sure.
 +
</p>
 +
 +
</div>
 +
 +
<a href="" onClick=" $('#menu14').slideToggle(300, function callback() {  }); return false;"><h2> 25.06.15</h2></a>
 +
 +
    <div id="menu14">
 +
 +
<p>
 +
    Present: Heiko, Ela, Julian, Dennis, Steffi, Avi, Verena, Alaa, Debbie, Nida
 +
</p>
 +
<p>
 +
    <strong>1.) Strain documentation: </strong>
 +
</p>
 +
<p>
 +
    The team has now got a box in the -80 ̊C freezer to start a strain collection. The Box is on the ground floor, in the middle freezer, top compartment, top
 +
    shelf marked: Rack 1 Robert at the back, 2<sup>nd</sup> from the bottom (blue box)
 +
</p>
 +
<p>
 +
    - To register the strain an information sheet needs to be filled in and submitted to Jaqueline
 +
</p>
 +
<p>
 +
    - Strains should be frozen from overnight culture in 30% glycerine (e.g. 600ul liquid culture + 900ul Glycerine (from 50% stock solution))
 +
</p>
 +
<p>
 +
    <strong>2.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Cellulase:
 +
</p>
 +
<p>
 +
    pJET_cellulase transformation and restriction worked fine in TOP10 and was then transformed into BL21 cells. Restriction showed a fragment of the correct
 +
    size, however sequencing failed. Activity plates showed no activity so far, but will be incubated over a longer period of time and in addition singularised
 +
    to avoid contamination of other colonies.
 +
</p>
 +
<p>
 +
    Scaffoldin:
 +
</p>
 +
<p>
 +
    Trafo in Top10 and BL21 did not work, which is why the TOPO cloning may be problematic.
 +
</p>
 +
<p>
 +
    - A control with an empty vector should be run.
 +
</p>
 +
<p>
 +
    - If the annealing is successful the bands should change in a reaction, which can be verified to exclude those issues.
 +
</p>
 +
<p>
 +
    - In addition Primers should be designed from within the sequence to verify the ends of the scaffoldin. Primers should be around 18bp long and 200bp within
 +
    the construct, which also has TOPO overlaps
 +
</p>
 +
<p>
 +
    - In addition the scaffoldin band of the PCR could be correct, but with an impurity alongside -&gt; transformation should however still work to some extent
 +
    (second problem?)
 +
</p>
 +
<p>
 +
    - For the covalent binding stick to the protocol an do not reduce salt concentrations
 +
</p>
 +
<p>
 +
    - For electroporation the TOPO cloning mixture needs to be desalinated with microdialysis, otherwise the trafo won't work
 +
</p>
 +
<p>
 +
    Recommendation: Check salts, positive control, annealing conditions and sequencing after PCR purification.
 +
</p>
 +
<p>
 +
    RFP:
 +
</p>
 +
<p>
 +
    Sequences are 100%match, but colonies show no fluorescence. Therefore, restricted RFP sequences will be ligated into pBAD in order to verify its function
 +
    in the target plasmid.
 +
</p>
 +
<p>
 +
    GFP sequences for Primer design and corresponding plasmids/colonies can be acquired from Jaqueline.
 +
</p>
 +
<p>
 +
    E1&amp;2:
 +
</p>
 +
<p>
 +
    Sequencing and restriction control were ok. Strains are frozen and documentation is being prepared.
 +
</p>
 +
<p>
 +
    Ask Silja for origin of the enzymes for better documentation. One is from Heiko Nacke, the other from Genis. In addition the enzyme sequences can be
 +
    Blasted for identification.
 +
</p>
 +
<p>
 +
    Phosphatase contains PstI, which is why currently it is not compatible with biobricks. In the future the restriction sites will have to be changed, if
 +
    submission should occur (PCR modification).
 +
</p>
 +
<p>
 +
    Dockerins:
 +
</p>
 +
<p>
 +
    Heat shock transformation did not work for any of the 4 dockerins, but positive control worked fine.
 +
</p>
 +
<p>
 +
    Vector was cut with EcoRI and KpnI as a test and KpnI was not very efficient. Due to this the restrictions were done separately.
 +
</p>
 +
<p>
 +
    Next step is a ligation and a gel to verify it immediately after to find reasons for failure:
 +
</p>
 +
<p>
 +
    - T4 ligase working?
 +
</p>
 +
<p>
 +
    - Phosphorylation issues -&gt; skip this step
 +
</p>
 +
<p>
 +
    - Find Plasmid formula to calculate ideal ligation ratio (depends on insert length and is usually 3:1) -&gt; overloading the vector may cause different
 +
    ligation variants
 +
</p>
 +
<p>
 +
    - Can E.coli express the dockerins?
 +
</p>
 +
<p>
 +
    Oder dockerins as codon optimised gBlocks from IDT, as a backup.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu17').slideToggle(300, function callback() {  }); return false;"><h2>24.07.15</h2></a>
 +
 +
<div id="menu17">
 +
<p>
 +
    Present: Debbie, Avi, Julian, DEnnis
 +
</p>
 +
<p>
 +
    Excused: Verena, Steffi, Nida, Sindhu
 +
</p>
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Enzyme 3: Transformations didn't work, so the project will be restarted from PCR stage and a new ligation.
 +
</p>
 +
<p>
 +
    The sequencing is also not working, possibly due to wrong primers or wrong primer concentrations.
 +
</p>
 +
<p>
 +
    Dockerin/Scaffoldin: both scaffoldins are at the expression stage
 +
</p>
 +
<p>
 +
    Dockerins in gBlocks have arrived.
 +
</p>
 +
<p>
 +
    Old Dockerins may not be restrictable due to missing bands around the restriction sites.
 +
</p>
 +
<p>
 +
    New Plasmid isolation is on the way and work will continue on the dockerins as before.
 +
</p>
 +
<p>
 +
    <strong>2.) 3D Model:</strong>
 +
</p>
 +
<p>
 +
    Debbie has checked the sequences and has the Amino Acid sequences for all enzymes, but cannot find the scaffoldin sequences. When used they result in short
 +
    fragments. Dockerins do not work either due to too many stopcodons.
 +
</p>
 +
<p>
 +
    Double check the files and give her new files.
 +
</p>
 +
<p>
 +
    <strong>3.) Human Practices:</strong>
 +
</p>
 +
<p>
 +
    Julian will contact people at the Xlab.
 +
</p>
 +
<p>
 +
    A stop motion movie is being made that simply explains our project
 +
</p>
 +
<p>
 +
    A talk needs to be organised around our project and synthetic biology -&gt; Beer and Pretzels? Julian will contact some people and organise it.
 +
</p>
 +
<p>
 +
    Once returned from iGEM, the presenters will also present their project to the faculty at a faculty talk on the 27.10.2015
 +
</p>
 +
<p>
 +
    <strong>4.) Meetup:</strong>
 +
</p>
 +
<p>
 +
    The group that will travel to Marburg for the German Meetup will prepare short presentation
 +
</p>
 +
<p>
 +
    <strong>5.) Poster:</strong>
 +
</p>
 +
<p>
 +
    Dennis is happy to help with the organisation of the poster
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu18').slideToggle(300, function callback() {  }); return false;"><h2>18.08.15</h2></a>
 +
 +
<div id="menu18">
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Ligation and transformation of enzymes and dockerins with extra bases worked all around there are positive restriction results (but for 2). Plasmids have
 +
    been sent for sequencing.
 +
</p>
 +
<p>
 +
    - enzymes are being induced in fat medium for next day protein purification. Induction must be done with L-arabinose due to the pBAD vectore.
 +
</p>
 +
<p>
 +
    Scaffoldin: ACEL purification yielded 1mg/ml of Protein, but it still needs to be verified on an SDS gel.
 +
</p>
 +
<p>
 +
    Cellulase_CCEL construct is being sequenced.
 +
</p>
 +
<p>
 +
    <strong>2.) BioBricks:</strong>
 +
</p>
 +
<p>
 +
    Avril will design Primers with the proper restriction sites for the BioBrick shipping vector. EcoRI and PstI will be used.
 +
</p>
 +
</div>
 +
 +
<a href="" onClick=" $('#menu20').slideToggle(300, function callback() {  }); return false;"><h2>24.08.15</h2></a>
 +
 +
<div id="menu20">
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Each section of labwork needs to be reassigned in order to optimize our work
 +
</p>
 +
<p>
 +
    Dennis &amp; Julian will work on Gelfiltrations and Protein extractions. They may need some help with all the inductions.
 +
</p>
 +
<p>
 +
    Alaa will make sure all protocols are written for the Wiki in time for the Wiki Freeze. Every group needs to write up their protocols and results.
 +
</p>
 +
<p>
 +
    Avril will make Primers for BioBricks of Dockerins with and without Enzymes and ask the Aachen Team for some Enzymes (design Primers for these as well)
 +
</p>
 +
<p>
 +
    Stefi will work on Poster and Presentation
 +
</p>
 +
<p>
 +
    Enzyme Dockerin constructs and BioBricks need to be continued and brought to work. See if we can find an enzyme cascade that we could attach to the
 +
    Flexosome.
 +
</p>
 +
<p>
 +
    <strong>2.) Finances:</strong>
 +
</p>
 +
<p>
 +
    An overview over our current finances has to be made to know what we have for Tshirts and labcosts.
 +
</p>
 +
<p>
 +
    <strong>3.) Safety Form:</strong>
 +
</p>
 +
<p>
 +
    The safety form deadline is this week. Avril will work on it and double check it with Heiko.
 +
</p>
 +
</div>
 +
<a href="" onClick=" $('#menu21').slideToggle(300, function callback() {  }); return false;"><h2>27.08.15</h2></a>
 +
 +
<div id="menu21">
 +
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Enzymes: Phosphatase activity test in different Buffer is working
 +
</p>
 +
<p>
 +
    Cellulase: Primers have not come yet
 +
</p>
 +
<p>
 +
    Induction: Pho_CTHE and rfp_ACEL are in fat medium and the spare amount will be used with the FPLC purification.
 +
</p>
 +
<p>
 +
    Primers have been designed for most constructs
 +
</p>
 +
<p>
 +
    Verena, Udhaya and Sindhu will continue the work on Dockerins, Est_BCEl and cjblue for BioBrick creation.
 +
</p>
 +
<p>
 +
    <strong>2.) Wiki:</strong>
 +
</p>
 +
<p>
 +
    Alaa and Udhaya have been working on the Method Collection for the Wiki
 +
</p>
 +
<p>
 +
    Ask Alex to make it pretty and insert the Aix-Marseille Gold Medal for collaboration
 +
</p>
 +
<p>
 +
    <strong>3.) ETC:</strong>
 +
</p>
 +
<p>
 +
    Steffi is working on Poster and Presentation. She has designed Tshirts with Verena and both have looked for enzyme pathways within the biobricks.
 +
</p>
 +
 +
</div>
 +
<a href="" onClick=" $('#menu22').slideToggle(300, function callback() {  }); return false;"><h2>03.09.15</h2></a>
 +
 +
<div id="menu22">
 +
<p>
 +
<p>
 +
    <strong>1.) Labwork:</strong>
 +
</p>
 +
<p>
 +
    Protein purification of CTHE has been successful. Other constructs are not working. Not sure whether due to pBAD vector, therefore sequences need to be
 +
    checked.
 +
</p>
 +
<p>
 +
    Aachen Enzymes have arrived and the first BioBricks made (still need to be sequenced). BioBricks with colours amilCP, cjBlue and efforRed are being used as
 +
    backup.
 +
</p>
 +
<p>
 +
    Purification: Could the enzymes be deactivated by the purification?
 +
</p>
 +
<p>
 +
    Can we check scaffoldin interactions, even though enzymes may not function? So far it hasnt been possible to purify any of the enzymes or RFP and not to
 +
    show a 100% positive activity test.
 +
</p>
 +
<p>
 +
    - Restart expression tests
 +
</p>
 +
<p>
 +
    - start cellulase induction
 +
</p>
 +
<p>
 +
    - check RFP again with a control
 +
</p>
 +
<p>
 +
    - could it be possible to use the scaffoldin to purify the enzyme-dockerins?
 +
</p>
 +
<p>
 +
    - check for pBAD promoter error and ask Robert for other expression vectors.
 +
</p>
 +
<p>
 +
    - try and get some sort of interaction even if it is crude
 +
</p>
 +
<p>
 +
    - is there a BioBrick expression vector?
 +
</p>
 +
<p>
 +
    <strong>2.) Presentation:</strong>
 +
</p>
 +
<p>
 +
    First presentation meeting today
 +
</p>
 +
</div>
 +
<a href="" onClick=" $('#menu25').slideToggle(300, function callback() {  }); return false;"><h2>10.09.15</h2></a>
 +
 +
<div id="menu25">
 +
<p>
 +
<p>
 +
    <strong>1.) BioBricks:</strong>
 +
</p>
 +
<p>
 +
    Est-BCEL plasmid needs to be re-inoculated to get more plasmid. ACEL, BCEL and CCEL plasmids in the BioBrick vector look good and just need sequencing.
 +
</p>
 +
<p>
 +
    <strong>2.) Aachen:</strong>
 +
</p>
 +
<p>
 +
    HPS and PHI have been succesfully inserted into pBAD with sequencing being fine. Primers for expression in pET100 have arrived.
 +
</p>
 +
<p>
 +
    <strong>3.) Colours:</strong>
 +
</p>
 +
<p>
 +
    amilCP and eforRed have been sequenced but only amilCP is good, cjBlue is still in the BB vectore´
 +
</p>
 +
<p>
 +
    <strong>4.) Cellulase:</strong>
 +
</p>
 +
<p>
 +
    Restriction control in pBAD is good. Insertion into pET100 can be tried.
 +
</p>
 +
<p>
 +
    <strong>5.) pET100:</strong>
 +
</p>
 +
<p>
 +
    Esterase and Phosphatase have been inserted into pET100 with the dockerins. Need to wait if ligation and transformation has worked or if it needs to be
 +
    redone.
 +
</p>
 +
</p>
 +
</div>
 +
 +
 +
</html>
 +
 +
[[File:TeamGoettingen_notebook.jpg|center|250px]]
 +
<html>
 +
</div></div> <!--These are the closing tags for div id="mainContainer" and div id="contentContainer". The corresponding opening tags appear in the template that is {{included}} at the top of this page.-->
 
</html>
 
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Latest revision as of 11:19, 18 September 2015



Notebook

15.12.14

08.01.15

13.01.15

15.01.15

19.01.15

02.02.15

16.02.15

18.02.15

23.02.15

16.03.15

23.03.15

07.04.15

13.04.15

21.05.15

19.06.15

25.06.15

24.07.15

18.08.15

24.08.15

27.08.15

03.09.15

10.09.15

TeamGoettingen notebook.jpg