Team:Grenoble/Notebook Rawhide

From 2011.igem.org

(Difference between revisions)
 
(6 intermediate revisions not shown)
Line 6: Line 6:
   <h1>July 28<SUP>th</SUP> to 3<SUP>st</SUP></h1>
   <h1>July 28<SUP>th</SUP> to 3<SUP>st</SUP></h1>
 +
<div class="blocbackground">
 +
    <img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
 +
      <h2 >Team Marmottes<span>Marion</span></h2>
 +
      <p>According to the results from the sequencing order will have to start over constructions since the 1st-steps. A Purification step will be add for all 1st-step construction in order to  get round our resistance issues.</p>
 +
 +
<ul>
 +
  <li>Cloning session :<br/>
 +
    -> 1st-step construction: RBS-CinI<br/>
 +
    -> 2nd-step constructions:<br/>
 +
        TetR-CinR<br/>
 +
        MerR-CinR<br/>
 +
        MerR-LuxR<br/>
 +
        LacI-LuxI<br/>
 +
    -> 3rd-step construction<br/>
 +
        pLac + TetR-LuxR</li>
 +
    <p>Digestions: Because we are having issues to get 2nd-step constructions. We tried both 3A Assembly and Standard protocols. </p>
 +
    <p>Cheking and purification gels: We add a cheking step, before performing ligations on Standard Protocol. To maximise our chance to get 2nd-step constructions, we tried to purified the inserts.</p>
 +
    <p>On purification gel, plasmids seem to be digested but inserts is missing. On checking gel, cinI wasn't digested correctly. </p>
 +
  <li>Sequencing Order<br/>
 +
    ->RESULTS</li>
 +
  <p>RBS-TetR RBS-LuxI RBS-CinR RBS-LacI pLac-GFP pCin-GFP pMerT-GFP pLux-Lyco</p>
 +
  <p>6 out of 8 sequence alignments failed.<br/>
 +
  To perform this constructions, we used standard protocol: we kept the plasmid of the shortest biobrick which we inserted the longest biobrick. <br/>
 +
  The problem was identified as coming from a wrong identification of antibiotic resistants. In most constructions below, vector and inserts had the same antibiotic resistance. So, even wrong constructions were selected on Petri Dishes.</p>
 +
  <li>Enzyme checking</li><p>Because we had no results with the latest cloning session, we tested our enzymes.</p>
 +
  <p>We tried to digest each site individually. We obtained linearized plasmid. So, enzymes are well working.</p>
 +
  <li>Cloning session:<br/>
 +
    -> Biobricks which require an RBS and have the same antibiotic resistance as RBS plasmid, were transfered on pSB1AC3 plasmid with chloramphenicol resistance:<br/>
 +
    TetR<br/>
 +
    LuxR<br/>
 +
    LuxI<br/>
 +
    LacI<br/>
 +
    GFP<br/>
 +
    Lycopen<br/>
 +
      -> Fha1 was also transfered on pSB1AT3 plasmid to simplify selection of the right constructions.</li>
 +
    <p>Digestions: a gel checking of the restriction results were performed.</p>
 +
    <p>Spreading over Petri dish</p>
 +
   
 +
    <p>On the Restriction Gel, We obtain the right size for all inserts.<br/>
 +
    Because pSB1AC3 is a double resistance plasmid (ampicillin and chloramphenicol) and RBS plasmid is on ampicillin, as previously the right constructions won't be selected.</p>
 +
   
 +
    <li>Cloning session:<br/>
 +
      -> 3rd-step construction:<br/>
 +
      pLac + MerR-CinR<br/>
 +
      pConst + MerR-CinR<br/>
 +
      -> 2nd-step construction:<br/>
 +
      RBS-CinI</li>
 +
    <p>Preculture: To perform this new cloning session, we first selected 5 colonies from the Petri dish of MerR-CinR construction.</p>
 +
    <p>Digestions: a gel checking of the restriction results were performed.</p>
 +
    <p>Purification: In order to check and extract the wright insert.</p>
 +
    <p>Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids.</p>
 +
    <p>Spreading over Petri dish</p>
 +
   
 +
    <li>Cloning Session:<br/>
 +
      -> 1st-step construction START OVER:<br/>
 +
      RBS-LacI
 +
      RBS-TetR
 +
      RBS-LuxI
 +
      Fha-LacI
 +
      Fha-LuxI
 +
      Fha-LuxR
 +
      Fha-CinI
 +
      Fha-CinR
 +
      Fha-TetR
 +
      pLux-Lycopene
 +
      pCin-Lycopene
 +
      <br/>
 +
        -> Construction Test<br/>
 +
      pCin-GFP
 +
      pLux-GFP
 +
      pTet-GFP
 +
      pConst-GFP
 +
      pLac-GFP
 +
      pConst-RBS-CinR
 +
      pConst-RBS-LuxR</li>
 +
     
 +
      <p>Digestions: a gel checking of the restriction results were performed.</p>
 +
      <p>Purification: In order to check and extract the wright insert.</p>
 +
      <p>Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids.</p>
 +
      <p>Spreading over Petri dish</p>
 +
     
 +
      <p>On Puritication gel, 4 biobricks were not correctly digested: CinI, LuxI, LuxR, RBS-LuxR. All the other were apparently well purified.<br/>
 +
      Ligations were performed as usual.<br/>
 +
      Results on Petri Dishes were unsatifying: we obtained an agglomerate of colonies. And this in all Petri Dishes. Does the issue come from too old Petri Dishes? Or LB pre culture?<br/>
 +
      So, we tried a 2nd-spreding on Petri Dishes and the result was the same.<br/>
 +
      We also checked by PCR if we could work anyway with those colonies. But the results were not conclusive.</p>
 +
     
 +
      <li>New stock on Petri Dishes</li><p>We spread again -80°C stock in order to start new miniprep of the troubling biobricks:<br/>
 +
  Lycopene<br/>
 +
  RBS<br/>
 +
  Fha<br/>
 +
  TetR<br/>
 +
  LuxR<br/>
 +
  LuxI<br/>
 +
  CinI<br/>
 +
  CinR<br/>
 +
  pTet<br/>
 +
  pMerT<br/>
 +
  pCin<br/>
 +
  pConst<br/>
 +
  pLux</p>
 +
 
 +
      <li>Cloning Session:<br/>
 +
  We started over constructions which couldn't be performed during the previous cloning because of wrong digestion.<br/>
 +
  Fha-LuxR<br/>
 +
  Fha-LuxI<br/>
 +
  Fha-CinR<br/>
 +
  Fha-CinI<br/>
 +
  pCin-Lycopene<br/>
 +
  pLux-Lycopene<br/>
 +
  pConst-RBS-LuxR<br/>
 +
  RBS-LuxI<br/>
 +
  RBS-LuxR</li>
 +
  <p>Digestions: a gel checking of the restriction results were performed.</p>
 +
  <p>Purification: In order to check and extract the wright insert.</p>
 +
  <p>Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids. We add a control for the ligations: plasmids without its inserts (pLux, pCin, Fha, RBS).</p>
 +
  <p>Spreading over Petri dish</p>
 +
 +
 +
</ul>
 +
  </div>
   <div class="blocbackground">
   <div class="blocbackground">
Line 21: Line 142:
   <h1>July 22<SUP>nd</SUP> to 27<SUP>th</SUP></h1>
   <h1>July 22<SUP>nd</SUP> to 27<SUP>th</SUP></h1>
 +
 +
Stock of electro competent cells
   <div class="blocbackground">
   <div class="blocbackground">
Line 36: Line 159:
</ul>
</ul>
   </div>
   </div>
-
  <h1>July 14<SUP>th</SUP> to 21<SUP>st</SUP></h1>
+
 
   <div class="blocbackground">
   <div class="blocbackground">
     <img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
     <img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
Line 79: Line 202:
       </div>
       </div>
 +
 +
  <h1 id="week2">July 14<SUP>th</SUP> to 21<SUP>st</SUP></h1>
 +
 +
  <div class="blocbackground">
 +
    <img src="https://static.igem.org/mediawiki/2011/5/5c/Morgane.png" class="icon"/>
 +
      <h2 >Team Marmottes<span>Morgane</span></h2>
 +
      <p>Better clonings rate: Clonings performed with recommended enzymes by iGEM. </p>
 +
 +
<ul>
 +
 +
  <li>Stock of electro competent cells </li><p>RESULTS : Great Transformation Rate</p>
 +
  <li>New stock of Biobrick Miniprep </li><p>DO checking PCR cheking</p>
 +
  <li>Cloning of every first steps of our construction: (Standard Assembly Method)<br/>
 +
    -> 11 clonings</li>
 +
  <p>We changed our strategy: clonings were performed with enzymes recommended by iGEM (New England Biolabs). </p>
 +
  <p>One Petri Dish was free of colony. We performed PCRs on 5 colonies from each Petri Dish. According to PCR results, 5 constructions out of 11 seem to have the right size.</p>
 +
  <li>Cloning session :<br/>
 +
    -> every 1st-step constructions that failed last time, so 6 constructions.<br/>
 +
    -> 2 2nd-step constructions:<br/>
 +
        MerR-LuxR<br/>
 +
        TetR-LuxR</li>
 +
  <p>Only digestions were performed because of a lack of equipments.<br/>
 +
  We decided to use the Standard Protocol for the first step constructions because the assembly combinates a short and a long gene. The long gene is inserted in the plasmid of the small gene.<br/>
 +
  But the Second step constructions were performed with the 3A Assembly Protocol.</p>
 +
</ul>
 +
      </div>
   <div class="blocbackground">
   <div class="blocbackground">
Line 93: Line 242:
   </div>
   </div>
-
   <h1>July 7<SUP>th</SUP> to 13<SUP>th</SUP></h1>
+
   <h1 id="week1">July 7<SUP>th</SUP> to 13<SUP>th</SUP></h1>
   <div class="blocbackground">
   <div class="blocbackground">
Line 124: Line 273:
   </div>
   </div>
-
  <h1>June 29<SUP>th</SUP> to July 6<SUP>th</SUP></h1>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/8/80/Maxime.png" class="icon"/>
 
-
      <h2 >Team Godlike<span>Maxime</span></h2>
 
-
      <p>Working on parameters</p>
 
-
 
-
<ul>
 
-
 
-
  <li>Models</li><p>Our deterministic model is now finished. The whole system is properly simulated and we simplificated it. We will now work on the stochastic approach. Stochastic is very important for our system as it will define the precision of the whole measure.</p>
 
-
  <li>Parameters</li><p>This is now the main part of our work. We have to look for the best set of parameters for our system to properly modelize it. We dissect litterature as well as other and former teams results to get a whole set. For the time being we do not have proper set.<br/>The parameters we obtained are often contradictory from one set to another. We will try to characterize some of the parameters ourselves. We are working with biologists on experiments to characterize those parameters.</p>
 
-
</ul>
 
-
  </div>     
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/5/5c/Morgane.png" class="icon"/>
 
-
      <h2 >Team Marmottes<span>Morgane</span></h2>
 
-
      <p>Thanks to the delivery of new MerR material, we will finally be able to include MerR to our System.<br/>Because former clonings gave no results, we carried out the Standard Assembly but results are not significant.<br/>We are having to many problem with cloning, we have to check every steps (Minipreps concentration, digestion results by PCR , purification of the insert, proportion of vector (X1)/insert(X3) during the ligation...) </p>
 
-
 
-
<ul>
 
-
 
-
  <li>New cloning trial CinI-RBS (Standard Assembly)</li><p>-> 2 different assemblies were achieved<br/>Digestion of the 2 biobricks :<br/>
 
-
  -> RBS (S-P), the plasmid of RBS remains.<br/>
 
-
  -> CinI (X-P) is inserted into the plasmid of RBS.<br/><br/>
 
-
  Digestion of the 2 biobricks :<br/>
 
-
  -> CinI (S-P), the plasmid of CinI remains.<br/>
 
-
  -> RBS (X-P) is inserted into the plasmid of CinI.<br/><br/>
 
-
  Ligation : <br/>
 
-
  First digestion results were individually heat-inactivated at 80°C to eliminate enzymes remaining and then mixed all together.<br/><br/>
 
-
  Spreading over Petri dish<br/><br/>
 
-
  Colonies grown only on the Petri dish containing the construction of CinI inserted into RBS plasmid.<br/>
 
-
  PCRs were performed on 4 colonies from this dish. None of them gave a conclusive result. All inserts are much shorter than expected (400 bps instead of 1040 bps)
 
-
  </p>
 
-
 
-
  <li>pTet/TetR Miniprep</li><p>Petri dishes full of colonies.</p>
 
-
  <li>MerR receipt</li><p>MerR was delivered into a small flask containing bacteria already transformed with MerR.</p>
 
-
  <li>MerR culture and PCR checking</li><p>The insert amplified by PCR had the expected length.</p>
 
-
  <li>Cloning of every first steps of our construction : (Standard Assembly Method)</li><p>-> 8 clonings<br/>We used the same process as above inserting the biggest Biobrick into the plasmid of the shortest. Thus, the risk to loose the shortest piece is avoided.</p>
 
-
  <li>Analysis of Sequencing Results :</li><p>-> pMerT-GFP<br/>-> RBS-CinR<br/>Sequences from GATC and Computer Sequencing were compared.<br/>No match between both sequences from GATC and Computer Sequencing for pMerT-GFP construction.
 
-
Whereas sequences comparison of RBS-CinR demonstrates a strong similitude in the CinR region. But, RBS region seems to be absent or to have received mutations.</p>
 
-
 
-
</ul>
 
-
  </div>
 
-
 
-
  <h1>June 22<SUP>nd</SUP> to 28<SUP>th</SUP></h1>
 
-
 
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/3/38/Geof.jpg" class="icon"/>
 
-
      <h2 >Team Godlike<span>Geoffrey</span></h2>
 
-
      <p>Working on Quorum Sensing</p>
 
-
     
 
-
<ul>
 
-
  <li>Parameters</li><p>We now have a complete set of parameters for our whole network. However physical parameters such as viscosity of AHL are still missing. Accurate predictions are therefore still not possible for the whole system.</p>
 
-
  <li>Models and results</li><p>We have worked on the whole set of equations for the Quorum Sensing part of our network. We also demonstrated that our system can switch from one way to another with sufficient amount of IPTG/pollutant.</p>
 
-
</ul>
 
-
  </div>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/c/c6/Feriel.png" class="icon"/>
 
-
      <h2 >Team Marmottes<span>Feriel</span></h2>
 
-
      <p>3A-Assembly were carried out for all first steps of our constructions but results were inconclusive. Alternative methods should be thought off like inactivate enzymes before mixing them together.</p>
 
-
 
-
<ul>
 
-
 
-
  <li>Stock of electro competent cells </li><p>Great Colony Rate on Petri dishes</p>
 
-
  <li>Cloning training with RBS-CinI: (3A Assembly Method)</li><p>Digestion of the 3 biobricks<br/>
 
-
  -> RBS (E-S)<br/>
 
-
  -> CinI (X-P) <br/>
 
-
  -> pSB1AC3 (E-P)<br/><br/>
 
-
  Ligation: <br/>
 
-
  digestion results were mixed all together and then enzymes were heat-inactived at 80°C<br/><br/>
 
-
  Growth of red and white colonies.PCR checkings were performed on 3 white colonies. The results demonstrate that the insert is shorter than expected.</p>
 
-
  <li>Cloning of every first steps of our construction:(3A Assembly Method)</li><p>-> 10 clonings<br/>Same process as above<br/>Growth of red and white colonies for 6 out of 10 ligation results. PCR checks were performed as above for the 6 valid ligations. Only 2 appeared to have the right size. The sequencing of the 2 valid constructions should corroborate our results.</p>
 
-
  <li>Sequencing order of the two valid constructions :</li><p>-> pMerT-GFP<br/>-> RBS-CinR<br/>We ordered on GATC company.</p>
 
-
 
-
</ul>
 
-
  </div>
 
-
 
-
  <h1>June 15<SUP>th</SUP> to 21<SUP>st</SUP></h1>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/3/38/Jb.png" class="icon"/>
 
-
      <h2 >Team Godlike<span>JB</span></h2>
 
-
      <p>Working on parameters</p>
 
-
     
 
-
<ul>
 
-
  <li>Parameters</li><p>We now have enough parameters for simulating our toggle switch system. But still many parameters missing for a complete model.<br/>
 
-
  However, with such parameters we can at least start working on the final device main features. </p>
 
-
  <li>Simulations</li><p>Obtained our first (meaningful) curves ! The simulated genetical network does switch indeed : <br/>
 
-
  <img src="https://static.igem.org/mediawiki/2011/d/df/Toggleswitch4wiki_grenoble.png"/></p>
 
-
  </div>
 
-
<div class="clearboth">  </div>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/igem.org/7/73/Clement.png" class="icon"/>
 
-
      <h2 >Team Marmottes<span>Clement</span></h2>
 
-
      <p>Still working on Biobrick Transformations. MerR transformations is still giving nothing. pTet/TetR has been considered as a substitut.</p>
 
-
 
-
<ul>
 
-
 
-
  <li>Plasmids and MerR Transformation</li><p>In case our colony issues come from a manipulation mistake, we tried again to Transform our Biobricks with the Standard protocole.<br/>MerR transformation gave nothing. From plasmid backbones resulted Red colonies. But after looking on iGEM website plasmid backbones appears to have a RFP-gene as default Biobrick.</p>
 
-
  <li>Miniprep</li><p>The kit Macherey-Nagel NucleoSpin Extract II was used.</p>
 
-
  <li>MerR Electroporation Transformation</li><p>Because the efficiency of the Standard Transformation is much lower than the Electroporation Transformation, we performed the latter.<br/>As previously, we  nothing on our Petri dish.</p>
 
-
  <li>MerR PCR</li><p>To verify if there is something in the well.<br/>Nothing on the gel.</p>
 
-
  <li>pTet/TetR Transformation</li><p>Because we still have nothing with merR, we are looking for alternative to the couple pMerT/MerR. As usual, the Standard protocole was used.<br/>Petri dishes full of colonies.</p>
 
-
  <li>MerR order</li><p>After many trial, merR seems to be absent from the well 7C of the plate 4.</p>
 
-
  <li></li><p></p>
 
-
 
-
</ul>
 
-
  </div>
 
-
 
-
  <h1>June 8<SUP>th</SUP> to 14<SUP>th</SUP></h1>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/3/38/Jb.png" class="icon"/>
 
-
      <h2 >Team Godlike<span>JB</span></h2>
 
-
      <p>Still worked on model equations</p>     
 
-
<ul>
 
-
  <li>Model equations</li><p>We deducted our equations from chemical and physical mechanisms and worked on simplifications.</p>
 
-
  <li>Parameters</li><p>One of the most importants part of our work is finding parameters for our model in order to match
 
-
  real behaviour of our cells as precisely as possible. Half of the needed parameters obtained up to now.</p>
 
-
  <li>Programs</li><p>Minor changes on the algorithm, minor bugs fixed and some plotting features added.</p>
 
-
</ul>
 
-
  </div>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/5/56/Marion.png" class="icon"/>
 
-
      <h2 >Team Marmottes<span>Marion</span></h2>
 
-
      <p>Computer sequencing.<br/>First manipulations: transformation of the Biobrick just arrived.</p>
 
-
 
-
<ul>
 
-
 
-
  <li>Computer Sequencing</li><p>Every intermediate, final and test constructions were listed on DNA Workbench in order to compare PCR and Sequencing Results with this data bank.</p>
 
-
  <li>Biobricks Transformation</li><p>To achieve it, the Standard Transformation protocole was performed.<br/>3 out 21 transformation gave colonies on Petri dishes:<br/>
 
-
  - MerR transformation<br/>
 
-
  - 2 plasmid backbone<br/>
 
-
  Red colonies resulted from all plasmid backbones, looks odd!!</p>
 
-
  <li>Culture on liquid media</li><p>All transformation that gave colonnies were resuspended except plasmid backbones.<br/>A stock of " to use biobricks" is waiting at 4°C and an other one is remaining at -80°C.</p>
 
-
 
-
</ul>
 
-
  </div>
 
-
 
-
  <h1>June 1<SUP>st</SUP> to 7<SUP>th</SUP></h1>
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/5/5c/Morgane.png" class="icon"/>
 
-
      <h2 >Team Marmottes<span>Morgane</span></h2>
 
-
      <p>No manipulation, we just planned the best way to work. </p>
 
-
 
-
<ul>
 
-
 
-
  <li>Plasmid Mapping</li><p>Toggle Switch: (4 final plasmids)
 
-
  1 plasmid was considered for each toggle switch way (MerR or LacI), so 2 final plasmids.
 
-
  But those 2 plasmids will include the 2 different couples cinI/cinR and luxI/luxR. The most efficient construction will be used.</br><br/>
 
-
 
-
  Coloration Generator: (4 plasmids)
 
-
  Coloration is induced by the activation of the promoter pLux or pCin depending on which couple cinI/cinR or luxI/luxR picked. As previously, the best coloring agent hasn't been decided yet. So, 4 plasmids will be produced: 1 for pCin, 1 for pLux and both followed either by GFP or Lycopène.</br><br/>
 
-
 
-
  Tests: (6 plasmids)
 
-
  The test of the two ways (MerR and LacI) of our toggle will be achieved by replacing pLac or pMerT by a constitutive promoter. So, 4 plasmids: 2 ways of the toggle switch, 2 different couples (luxI/luxR, cinI/cinR).
 
-
  Efficiency of both promoters will be tested separately by associating them to GFP.
 
-
  </p>
 
-
 
-
  <li>Biobrick Listing</li><p>21 Biobricks will be necessary to achieve only the toggle switch and the coloration generator:<br/>
 
-
  - 14 Biobricks<br/>
 
-
  - 7 plasmid backbones<br/>
 
-
  This includes biobricks which will be used for the tests (for example for the promoters) and as intermediate constructions.
 
-
  </p>
 
-
 
-
  <li>Manipulation schedule</li><p>Steps were defined for each construction depending on the level of assembly required. For example the assembly of two existing biobricks corresponds to a first level. Whereas the assembly of two newly obtained corresponds to a 2nd our 3rd level.
 
-
  Each level should be achieve at the time.</p>
 
-
 
-
      </ul>
 
-
  </div>
 
-
 
-
  <div class="blocbackground">
 
-
    <img src="https://static.igem.org/mediawiki/2011/3/38/Geof.jpg" class="icon"/>
 
-
      <h2 >Team Godlike<span>Geoffrey</span></h2>
 
-
      <p>Essentially worked on the models to use and how to simulate them</p>
 
-
 
-
<ul>
 
-
 
-
  <li>Models</li><p>Toggle Switch : Worked on the equations that would modelize our system the best.
 
-
  We now have the equations for the toggle switch and a first Matlab script that we can base our work on.
 
-
  <i>reference : Gardner, T.R., Cantor, C.R. & Collins, J.J., Construction of a genetic toggle switch in Escherichia coli,  403, 339 - 342 (2000)</i>
 
-
  </p>
 
-
  <li>Programs</li><p>Simulations : We are mainly working on a deterministic model for our network, we use basic Matlab
 
-
  ODE solvers for now. Our plan for the final device is a plate with our bacteria. We will have to adapt our code for
 
-
  this particular device</p>
 
-
</ul>
 
-
  </div>
 
</div>
</div>
</div>
</div>
 +
<script>
 +
document.getElementById('submenu').innerHTML = '<h3><span class="vert">Notebook:</span>June</h3><ul ><li><a href="/Team:Grenoble/Notebook_June#week1">July 7<SUP>th</SUP> to 13<SUP>th</SUP></a></li><li><a href="/Team:Grenoble/Notebook_June#week2">July 14<SUP>th</SUP> to 21<SUP>st</SUP></a></li><li><a href="/Team:Grenoble/Notebook_June#week3">July 22<SUP>nd</SUP> to 27<SUP>th</SUP></a></li><li><a href="/Team:Grenoble/Notebook_June#week4">July 22<SUP>nd</SUP> to 27<SUP>th</SUP></a></li></ul>'
 +
</script>
</html>
</html>

Latest revision as of 12:43, 8 August 2011


Grenoble 2011, Mercuro-Coli iGEM

July 28th to 3st

Team MarmottesMarion

According to the results from the sequencing order will have to start over constructions since the 1st-steps. A Purification step will be add for all 1st-step construction in order to get round our resistance issues.

  • Cloning session :
    -> 1st-step construction: RBS-CinI
    -> 2nd-step constructions:
    TetR-CinR
    MerR-CinR
    MerR-LuxR
    LacI-LuxI
    -> 3rd-step construction
    pLac + TetR-LuxR

    • Digestions: Because we are having issues to get 2nd-step constructions. We tried both 3A Assembly and Standard protocols.

    Cheking and purification gels: We add a cheking step, before performing ligations on Standard Protocol. To maximise our chance to get 2nd-step constructions, we tried to purified the inserts.

    On purification gel, plasmids seem to be digested but inserts is missing. On checking gel, cinI wasn't digested correctly.

  • Sequencing Order
    ->RESULTS

    • RBS-TetR RBS-LuxI RBS-CinR RBS-LacI pLac-GFP pCin-GFP pMerT-GFP pLux-Lyco

    6 out of 8 sequence alignments failed.
    To perform this constructions, we used standard protocol: we kept the plasmid of the shortest biobrick which we inserted the longest biobrick.
    The problem was identified as coming from a wrong identification of antibiotic resistants. In most constructions below, vector and inserts had the same antibiotic resistance. So, even wrong constructions were selected on Petri Dishes.

  • Enzyme checking

    • Because we had no results with the latest cloning session, we tested our enzymes.

    We tried to digest each site individually. We obtained linearized plasmid. So, enzymes are well working.

  • Cloning session:
    -> Biobricks which require an RBS and have the same antibiotic resistance as RBS plasmid, were transfered on pSB1AC3 plasmid with chloramphenicol resistance:
    TetR
    LuxR
    LuxI
    LacI
    GFP
    Lycopen
    -> Fha1 was also transfered on pSB1AT3 plasmid to simplify selection of the right constructions.

    • Digestions: a gel checking of the restriction results were performed.

    Spreading over Petri dish

    On the Restriction Gel, We obtain the right size for all inserts.
    Because pSB1AC3 is a double resistance plasmid (ampicillin and chloramphenicol) and RBS plasmid is on ampicillin, as previously the right constructions won't be selected.

  • Cloning session:
    -> 3rd-step construction:
    pLac + MerR-CinR
    pConst + MerR-CinR
    -> 2nd-step construction:
    RBS-CinI

    • Preculture: To perform this new cloning session, we first selected 5 colonies from the Petri dish of MerR-CinR construction.

    Digestions: a gel checking of the restriction results were performed.

    Purification: In order to check and extract the wright insert.

    Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids.

    Spreading over Petri dish

  • Cloning Session:
    -> 1st-step construction START OVER:
    RBS-LacI RBS-TetR RBS-LuxI Fha-LacI Fha-LuxI Fha-LuxR Fha-CinI Fha-CinR Fha-TetR pLux-Lycopene pCin-Lycopene
    -> Construction Test
    pCin-GFP pLux-GFP pTet-GFP pConst-GFP pLac-GFP pConst-RBS-CinR pConst-RBS-LuxR

    • Digestions: a gel checking of the restriction results were performed.

    Purification: In order to check and extract the wright insert.

    Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids.

    Spreading over Petri dish

    On Puritication gel, 4 biobricks were not correctly digested: CinI, LuxI, LuxR, RBS-LuxR. All the other were apparently well purified.
    Ligations were performed as usual.
    Results on Petri Dishes were unsatifying: we obtained an agglomerate of colonies. And this in all Petri Dishes. Does the issue come from too old Petri Dishes? Or LB pre culture?
    So, we tried a 2nd-spreding on Petri Dishes and the result was the same.
    We also checked by PCR if we could work anyway with those colonies. But the results were not conclusive.

  • New stock on Petri Dishes

    • We spread again -80°C stock in order to start new miniprep of the troubling biobricks:
    Lycopene
    RBS
    Fha
    TetR
    LuxR
    LuxI
    CinI
    CinR
    pTet
    pMerT
    pCin
    pConst
    pLux

  • Cloning Session:
    We started over constructions which couldn't be performed during the previous cloning because of wrong digestion.
    Fha-LuxR
    Fha-LuxI
    Fha-CinR
    Fha-CinI
    pCin-Lycopene
    pLux-Lycopene
    pConst-RBS-LuxR
    RBS-LuxI
    RBS-LuxR

    • Digestions: a gel checking of the restriction results were performed.

    Purification: In order to check and extract the wright insert.

    Ligations : Standard Protocol was performed. MerR-CinR was inserted into pLac and pConst plasmids. We add a control for the ligations: plasmids without its inserts (pLux, pCin, Fha, RBS).

    Spreading over Petri dish

Team GodlikeGeoffrey

It's all about patience

  • Models

    • Stochastic program is implemented for the toggle switch and works fine. We adapted the program for the whole system so we can implement the quorum sensing stochastic model very quickly when we have the parameters (about 1/2 day of work)

  • Parameters

    • We have coherent sets of parameters, but we still can't compare our models to the real system and are waiting for the results of characterization experiments.

July 22nd to 27th

Stock of electro competent cells

Team GodlikeJB

Stochastic model done

  • Models

    • Stochastic program is implemented for the toggle switch and works fine. We adapted the program for the whole system so we can implement the quorum sensing stochastic model very quickly when we have the parameters (about 1/2 day of work). The current model and parameters prove that we do have a bi-modal distribution when IPTG and pollutant concentrations are equal :

    On the X-axis we can see the concentration of lacI (neg) or merR (positive) in cells. On the Y-axis we can see the amount of cells in one way or another.

  • Parameters

    • We have coherent sets of parameters, but we still can't compare our models to the real system and are waiting for the results of characterization experiments.

Team MarmottesMarion

Moving in to the CIME, a biology teaching platform, free for the summer vacation. Preparation of new storage. PCR results from last week cloning.

  • Cloning session :
    -> every 1st-step constructions that failed last time, so 6 constructions.
    -> 2 2nd-step constructions:
    MerR-LuxR
    TetR-LuxR

    • Ligations:
    Because we still have a low cloning rate we tried different proportions between vectors and inserts:
    1 vector/1 insert
    1 vector/2 inserts
    1 vector/3 inserts

    Transformations with electrocompetent cells
    Spreading over Petri Dish

    RESULTS :
    Best results on Petri Dishes with the proportion 1/3. All constructions seem to have the right size except MerR-LuxR.

  • Sequencing Order

    • RBS-TetR RBS-LuxI RBS-CinR RBS-LacI pLac-GFP pCin-GFP pMerT-GFP pLux-Lyco

  • Products Order

    • For ligations, digestions, PCR, MiniPreps, MidiPreps

  • Cloning Session :
    -> 5 constructions with Fha1
    -> 3 2nd-step constructions
    LacI-LuxI
    MerR-CinR
    TetR-CinR
    -> 1 construction for the tests
    pLux-GFP
    -> 1st-step construction
    RBS-CinI

    • The final RBS (Fha1) which allows to keep the toggle off has just been delivered. So, all first steps constructions including RBS were made once again.
    Because the efficiency of Fha1 hasn't been completly demonstrated, we keep on cloning with the standard RBS. So, RBS and Fha1 constructions are made in parallel.

    RESULTS :
    Every constructions gave colonies on Petri Dishes.
    But PCR cheking showed that nothing were amplified on every Fha1 constructions with VF2 and VR primers. Thus, Fha1 was provided in a PCR blent plasmid instead of an iGEM's plasmid.
    The insert of the test construction is having the right size.
    2 out of 3 2nd step constructions had the right size.
    We still have issues to clone the 1st step construction: RBS-CinI.

July 14th to 21st

Team MarmottesMorgane

Better clonings rate: Clonings performed with recommended enzymes by iGEM.

  • Stock of electro competent cells

    • RESULTS : Great Transformation Rate

  • New stock of Biobrick Miniprep

    • DO checking PCR cheking

  • Cloning of every first steps of our construction: (Standard Assembly Method)
    -> 11 clonings

    • We changed our strategy: clonings were performed with enzymes recommended by iGEM (New England Biolabs).

    One Petri Dish was free of colony. We performed PCRs on 5 colonies from each Petri Dish. According to PCR results, 5 constructions out of 11 seem to have the right size.

  • Cloning session :
    -> every 1st-step constructions that failed last time, so 6 constructions.
    -> 2 2nd-step constructions:
    MerR-LuxR
    TetR-LuxR

    • Only digestions were performed because of a lack of equipments.
    We decided to use the Standard Protocol for the first step constructions because the assembly combinates a short and a long gene. The long gene is inserted in the plasmid of the small gene.
    But the Second step constructions were performed with the 3A Assembly Protocol.

Team GodlikeMaxime

Stochastic model done

  • Models

    • First results with the stochastic model. Our whole model does work like we expected it. But parameters are needed more than ever now.

  • Parameters

    • We got incomplete sets of parameters from either litterature or iGem teams. Some very useful sets can be found in Aberdeen 09, Brown 10, ETHZ 07, BCCS Bristol 08

July 7th to 13th

Team MarmottesMarion

Moving in to the CIME, a biology teaching platform, free for the summer vacation. Preparation of new storage. PCR results from last week cloning.

  • Moving in of the whole team to a new lab

    • We are taking advantage that the school year is over to move in the CIME, a teaching platform of biology.

  • Cheking of DNA concentration into our Biobrick Miniprep

    • OD cheking was performed on all our Biobrick stock to have an idea of the DNA rate into our preparation. We also made a PCR cheking to make sure that the DNA rate really corresponds to our Biobrick.

    We got an average rate of 100 ng/µl. Which is a much lower than expect, so 10 times more DNA products would had been necessary to get great constructions.

  • Order of new enzymes -> New England Biolabs Company

    • We hope to increase the digestion efficiency.

  • PCR Checking of cloning from the last week

    • As previously, inserts were shorter than expected, except the construction RBS-TetR.

  • Making new storage

    • Petri Dishes with Antibiotic (Amp, Kan, Cm, Tet). LB Culture medium

  • RBS-TetR Miniprep

    • In order to send it to the sequencing.

Team GodlikeGeoffrey

Learning Gillespie and looking for parameters.

  • Models

    • Stochastic model is on its way. We base it on Gillespie's algorithm. It's mainly about getting familiar with the math for now. Also working on Hysteresis and isoclines subparts of the deterministic models.

  • Parameters

    • Still a lot of work going on there. The problem is we can not compare our sets to experimental results as the whole system is not finished yet. We will then try to characterize some of the promoters we use and figure out how to obtain some degradation rates as well

Retrieved from "http://2011.igem.org/Team:Grenoble/Notebook_Rawhide"