Team:Aix-Marseille/Notebook 09

From 2014.igem.org

(Difference between revisions)
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               <li class="active"><a href="#0903" role="tab" data-toggle="tab" class="date-notebook">09/03/14</a></li>
               <li class="active"><a href="#0903" role="tab" data-toggle="tab" class="date-notebook">09/03/14</a></li>
               <li><a href="#0904" role="tab" data-toggle="tab" class="date-notebook">09/04/14</a></li>
               <li><a href="#0904" role="tab" data-toggle="tab" class="date-notebook">09/04/14</a></li>
 +
              <li><a href="#0905" role="tab" data-toggle="tab" class="date-notebook">09/05/14</a></li>
 +
              <li><a href="#0907" role="tab" data-toggle="tab" class="date-notebook">09/07/14</a></li>
             </ul>
             </ul>
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                   <li>
                   <li>
                     <p>Clean-up of these PCR and elution with 30µL H2O at 70°C.</p>
                     <p>Clean-up of these PCR and elution with 30µL H2O at 70°C.</p>
 +
                  </li>
 +
                  <li>
 +
                    <p>PCR cheA-kan-cheA using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C, the elongation time is 1’30”.</p>
 +
                    <p>
 +
                      Mix:
 +
                      <ul>
 +
                        <li>1µL pKD4 vector</li>
 +
                        <li>2,5µL primer 3</li>
 +
                        <li>2,5µL primer 4</li>
 +
                        <li>2,5µL dNTP</li>
 +
                        <li>10µL Q5 buffer</li>
 +
                        <li>qsp 50µL H2O</li>
 +
                      </ul>
 +
                    </p>
 +
                    <div class="media notes-media">
 +
                      <img class="media-object img-rounded pull-right" src="https://static.igem.org/mediawiki/2014/9/94/AMU_Team-w3110-0903.png" style="height:350px">
 +
                    </div>
                   </li>
                   </li>
                 </ul>
                 </ul>
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                   <li>
                   <li>
                     <p>Transformation of 90µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Cm 30µg/mL.</p>
                     <p>Transformation of 90µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Cm 30µg/mL.</p>
 +
                  </li>
 +
                  <li>
 +
                    <p>Clean-up of the previous PCR. Elution in 45µL of water.</p>
 +
                  </li>
 +
                  <li>
 +
                    <p>Electroporation of W3110 ∆sdaBC electrocompetent cells from Aimeric (clone 43)</p>
 +
                    <p>
 +
                      40µL cells + 10µL PCR<br>
 +
                      40µL cells + 0,5µL pKT25 (negative control)<br>
 +
                      ==> Spread bacteria on LB-Kan
 +
                    </p>
 +
                  </li>
 +
                </ul>
 +
              </div>
 +
             
 +
              <div class="tab-pane fade in active" id="0905">
 +
                <ul class="list-notebook">
 +
                  <li>
 +
                    <p>Many clones have grown on the negative control but no clone has grown for the cheA mutant. So cells are competent but they don’t express the recombinase.</p>
 +
                  </li>
 +
                </ul>
 +
              </div>
 +
             
 +
              <div class="tab-pane fade in active" id="0907">
 +
                <ul class="list-notebook">
 +
                  <li>
 +
                    <p>Streak out W3110 ∆sdaBC 43 and 63 from Aimeric on LB and LB-Kan</p>
                   </li>
                   </li>
                 </ul>
                 </ul>
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             <ul class="nav nav-tabs" role="tablist">
             <ul class="nav nav-tabs" role="tablist">
               <li class="active"><a href="#0908" role="tab" data-toggle="tab" class="date-notebook">09/08/14</a></li>
               <li class="active"><a href="#0908" role="tab" data-toggle="tab" class="date-notebook">09/08/14</a></li>
 +
              <li><a href="#0911" role="tab" data-toggle="tab" class="date-notebook">09/11/14</a></li>
             </ul>
             </ul>
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                         <p>All clones are good except the K10512-08-2.</p>
                         <p>All clones are good except the K10512-08-2.</p>
                         <p>The pSB1C3-K10512-06-1, pSB1C3-K10512-07-1 and pSB1C3-K10512-08-1 are sent to sequencing. They are OK.</p>
                         <p>The pSB1C3-K10512-06-1, pSB1C3-K10512-07-1 and pSB1C3-K10512-08-1 are sent to sequencing. They are OK.</p>
 +
                      </div>
 +
                    </div>
 +
                  </li>
 +
                  <li>
 +
                    <p>The bacteria are sensitive to kanamicyn.</p>
 +
                  </li>
 +
                </ul>
 +
              </div>
 +
             
 +
              <div class="tab-pane fade in active" id="0911">
 +
                <ul class="list-notebook">
 +
                  <li>
 +
                    <p>Repreparation of W3110 ∆sdaBC 43 electrocompetent cells (see Lambda Red mutant construction protocol) and restart from this step (day 1).</p>
 +
                  </li>
 +
                  <li>
 +
                    <p>The clones obtained were Kan<sup>R</sup> and Ap<sup>S</sup> ==> OK
 +
                  </li>
 +
                  <li>
 +
                    <p>The clones are pooled 3 by 3 to be tested by PCR with primers 52 and 53.</p>
 +
                    <div class="media notes-media">
 +
                      <img class="media-object img-rounded pull-right" src="https://static.igem.org/mediawiki/2014/3/3f/AMU_Team-w3110-0911.png" style="width:350px">
 +
                      <div class="media-body" style="padding-left: 10px;">
 +
                        <p>The pools 1 and 2 seemed to contain mutants (1786bp).</p>
 +
                      </div>
 +
                    </div>
 +
                  </li>
 +
                  <li>
 +
                    <p>Restart PCR with primers 52 and 53 on the isolated clones from pools 1 and 2.</p>
 +
                    <div class="media notes-media">
 +
                      <img class="media-object img-rounded pull-right" src="https://static.igem.org/mediawiki/2014/e/e1/AMU_Team-w3110-0911-2.png" style="width:350px">
 +
                      <div class="media-body" style="padding-left: 10px;">
 +
                        <p>The clones 1a, 2a and 2b seemed good, they are kept at -80°C. So the W3110 ∆sdaBC ∆cheA  is OK.</p>
                       </div>
                       </div>
                     </div>
                     </div>

Revision as of 18:55, 13 October 2014

Notebook: September



Week 10 : 09/01/2014 ➡ 09/07/2014

  • Construction of stability epitopes

    The main idea is to use the primers 57-58 and 59 as matrices in order to synthesize and amplify the stability epitope. The PCR uses the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 55°C, the elongation time is 5", the number of cycle is 25.

    Mix:

    • 5µL OiGEM-57 or 58 or 59
    • 2,5µL OiGEM-60
    • 2,5µL OiGEM-44
    • 25µL Q5 2x Mix
    • 15µL H2O

  • Clean-up of these PCR and elution with 30µL H2O at 70°C.

  • PCR cheA-kan-cheA using the iGEM PCR protocol with Q5 polymerase. The temperature of annealing is 60°C, the elongation time is 1’30”.

    Mix:

    • 1µL pKD4 vector
    • 2,5µL primer 3
    • 2,5µL primer 4
    • 2,5µL dNTP
    • 10µL Q5 buffer
    • qsp 50µL H2O

  • Construction of stability epitopes

    Digestion ES of PCR products of the stability epitopes and of piGEM-01.13 (pSB1C3-BBa_J04450).

    Mix:

    • 10µL K10512-06 or 07 or 08 or 5µL piGEM-01.13
    • 1µL EcoRI
    • 1µL SpeI
    • 5µL Buffer 2.1
    • qsp 50µL of water


    Incubation 60' at 37°C.

  • Then, for K10512-06 or 07 or 08, inactivation of enzymes 20' at 80°C.

  • Clean-up of piGEM-01.13 and elution with 30µL of water. Add 1µL of SAP (Promega) + 2µL of SAP buffer (dephosphorylation). Incubation 30' at 37°C, then Inactivation of enzyme 20' at 65°C.

  • Ligation K10512-06 or 07 or 08 with piGEM-01.13.

    Mix:

    • 2µL piGEM-01.13
    • 4µL K10512-06 or 07 or 08
    • 1µL T4 ligase 10U/µL
    • 2µL T4 ligase buffer
    • 11µL H2O

  • Transformation of 90µL of E.coli TG1 competent cells with 10µL of ligation. The selective medium is LB-Cm 30µg/mL.

  • Clean-up of the previous PCR. Elution in 45µL of water.

  • Electroporation of W3110 ∆sdaBC electrocompetent cells from Aimeric (clone 43)

    40µL cells + 10µL PCR
    40µL cells + 0,5µL pKT25 (negative control)
    ==> Spread bacteria on LB-Kan

  • Many clones have grown on the negative control but no clone has grown for the cheA mutant. So cells are competent but they don’t express the recombinase.

  • Streak out W3110 ∆sdaBC 43 and 63 from Aimeric on LB and LB-Kan

Week 11 : 09/08/2014 ➡ 09/14/2014

  • Construction of stability epitopes

    Test 5 clones of each transformation (with pSB1C3- K10512-06 or 07 or 08) by PCR. The temperature of annealing is 55°C, the elongation time is 1'

    Mix:

    • 2µL OiGEM-60
    • 2µL OiGEM-44
    • 4µL dNTP
    • 1µL DMSO
    • 2µL buffer
    • 0,5µL Taq polymérase
    • 8,5µL H2O

    All clones are good except the K10512-08-2.

    The pSB1C3-K10512-06-1, pSB1C3-K10512-07-1 and pSB1C3-K10512-08-1 are sent to sequencing. They are OK.

  • The bacteria are sensitive to kanamicyn.

  • Repreparation of W3110 ∆sdaBC 43 electrocompetent cells (see Lambda Red mutant construction protocol) and restart from this step (day 1).

  • The clones obtained were KanR and ApS ==> OK

  • The clones are pooled 3 by 3 to be tested by PCR with primers 52 and 53.

    The pools 1 and 2 seemed to contain mutants (1786bp).

  • Restart PCR with primers 52 and 53 on the isolated clones from pools 1 and 2.

    The clones 1a, 2a and 2b seemed good, they are kept at -80°C. So the W3110 ∆sdaBC ∆cheA is OK.

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