Team:Aix-Marseille/Notebook 09

From 2014.igem.org

(Difference between revisions)
Line 295: Line 295:
                 </ul>
                 </ul>
               </div>
               </div>
-
             
 
             </div>
             </div>
 +
          </div>
 +
        </div>
 +
 +
                  <!-- ======= WEEK 13 ======= -->
 +
        <div class="panel panel-info notes-panel">
 +
          <span class="note-tag" id="week13"></span>
 +
          <div class="panel-heading">
 +
            <h3 class="panel-title">Week 13 : 09/22/2014 &#10145; 09/28/2014 <div class="pull-right"></div></h3>
 +
          </div>
 +
          <div class="panel-body">
 +
            <b>Attribution:</b> Emmanuelle Bouveret<br>
 +
            <b>Objective:</b> Test the functionality of our RelA part.<br>
 +
            <p>The pSB1K3-relA plasmid constructed by the iGEM-AMU team is expected to be able to rescue the phenotype of a MG1655∆relA mutant grown on SMG plates, a medium known to induce a strong amino acid depletion in the cells (Rudd et al.,1985) that is toxic to a ∆relA mutant.</p>
 +
            <br><br>
 +
            <ul>
 +
              <li>
 +
                <u>Day 1:</u><br>
 +
                <p>Isolated clones were restreaked on SMG plates supplemented with 50 uM Kanamycin and 0.5mM IPTG. The plates were incubated at 37°C during 48 hrs.</p>
 +
              </li>
 +
              <li>
 +
                <u>Day 2:</u><br>
 +
                <p>Electrocompetent MG1655∆<i>relA</i> (EB421 strain, Wahl <i>et al</i>. 2011) cells were electroporated with either plasmid pSB1K3-RFP as a control or pSB1K3-<i>relA</i>.</p>
 +
              </li>
 +
              <li>
 +
                <u>Day 4:</u><br>
 +
                <p>As expected, we observed that the relA construct was able to complement the MG1655∆relA strain, as seen on the following figure.</p>
 +
                <div class="media notes-media">
 +
                  <img class="media-object img-rounded pull-right" src="https://static.igem.org/mediawiki/2014/6/63/AMU_Team-RelA_part.png" style="width:350px">
 +
                </div>
 +
              </li>
 +
              <li>
 +
                <p><u>SMG plate composition:</u></p>
 +
                <table class="table table-hover table-bordered notes-table">
 +
                  <tbody>
 +
                    <tr>
 +
                      <td>M9 salts</td> <td>1X</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>MgSO<sub>4</sub></td> <td>1mM</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>CaCl<sub>2</sub></td> <td>0.1mM</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>VitB1</td> <td>0.5µg/ml</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>Glucose</td> <td>0.2%</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>Serine</td> <td>1mM</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>Methionine</td> <td>1mM</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>Glycine</td> <td>1mM</td>
 +
                    </tr>
 +
                    <tr>
 +
                      <td>Bactoagar</td> <td>15g/L</td>
 +
                    </tr>
 +
                  </tbody>
 +
                </table>
 +
                <p><u>References:</u></p>
 +
                <ul>
 +
                  <li>Rudd KE, Bochner BR, Cashel M, Roth JR. Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression. <i>J Bacteriol</i>. ;163(2):534-42, 1985.</li>
 +
                  <li>Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in <i>Escherichia coli. Mol Microbiol</i>. 80(5):1260-75, 2011.</li>
 +
                </ul>
 +
              </li>
 +
            </ul>
           </div>
           </div>
         </div>
         </div>
Line 313: Line 382:
             <h4 class="list-group-item-heading">Week 11
             <h4 class="list-group-item-heading">Week 11
               <small style="padding-left:20px">09/08/14 &#8594; 09/14/14</small>
               <small style="padding-left:20px">09/08/14 &#8594; 09/14/14</small>
 +
            </h4>
 +
          </a>
 +
          <a data-scroll href="#week13" class="list-group-item">
 +
            <h4 class="list-group-item-heading">Week 13
 +
              <small style="padding-left:20px">09/22/14 &#8594; 09/28/14</small>
             </h4>
             </h4>
           </a>
           </a>

Revision as of 22:03, 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.

Week 13 : 09/22/2014 ➡ 09/28/2014

Attribution: Emmanuelle Bouveret
Objective: Test the functionality of our RelA part.

The pSB1K3-relA plasmid constructed by the iGEM-AMU team is expected to be able to rescue the phenotype of a MG1655∆relA mutant grown on SMG plates, a medium known to induce a strong amino acid depletion in the cells (Rudd et al.,1985) that is toxic to a ∆relA mutant.



  • Day 1:

    Isolated clones were restreaked on SMG plates supplemented with 50 uM Kanamycin and 0.5mM IPTG. The plates were incubated at 37°C during 48 hrs.

  • Day 2:

    Electrocompetent MG1655∆relA (EB421 strain, Wahl et al. 2011) cells were electroporated with either plasmid pSB1K3-RFP as a control or pSB1K3-relA.

  • Day 4:

    As expected, we observed that the relA construct was able to complement the MG1655∆relA strain, as seen on the following figure.

  • SMG plate composition:

    M9 salts 1X
    MgSO4 1mM
    CaCl2 0.1mM
    VitB1 0.5µg/ml
    Glucose 0.2%
    Serine 1mM
    Methionine 1mM
    Glycine 1mM
    Bactoagar 15g/L

    References:

    • Rudd KE, Bochner BR, Cashel M, Roth JR. Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression. J Bacteriol. ;163(2):534-42, 1985.
    • Wahl A, My L, Dumoulin R, Sturgis JN, Bouveret E.Antagonistic regulation of dgkA and plsB genes of phospholipid synthesis by multiple stress responses in Escherichia coli. Mol Microbiol. 80(5):1260-75, 2011.

Contact us

Email : equipe.igem.amu@gmail.com

Follow us

iGEM_AMU   iGEM.AMU

Our sponsors

   

Retrieved from "http://2014.igem.org/Team:Aix-Marseille/Notebook_09"