Difference between revisions of "Team:Pasteur Paris/Week 11"

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               <span class="soustitrepage">08/10 - 08/14</span></p>
 
               <span class="soustitrepage">08/10 - 08/14</span></p>
 
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<center><h3><em> Yeast Assembly </em></h3></center></br>
  
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<p style="text-align:left"> The Bacteria was first sent to the iGEM Toulouse and could have suffered from the travel so we ordered it again.</p>
  
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<ol>
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<a class="hautdepage" href="#"><p><span style="font-size: 60px; font-family: Courier;">^</span><br/><span style="font-size: 20px;">Page up</span></p></a></p>
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  <li style="text-align:left">Reception of a STAB culture of the Biobrick BBa_J61047.</li></br>
 +
  <li style="text-align:left"> Spreading of BBa_J61047 on Petri dishes containing LB medium and LB + Cm 34 µg/ml.
 No colonies were observed on Petri dishes containing LB + Cm 34 µg/ml but the bacteria grew on LB media.</li></br>
 +
  <li style="text-align:left"> Bacterial growth of the bacteria in liquid medium (5 ml of LB + Cm 34 µg/ml) and on a Petri dish (LB + Cm 34 µg/ml) and overnight incubation at 37°C.</li></br>
 +
  <li style="text-align:left">Storage at 4°C.</li></br>
 +
  <li style="text-align:left">Bacterial growth of the Bacteria in LB: incubation at 37°C for 1h30 at 140rpm.</li></br>
 +
  <ol>
 +
    <li style="text-align:left">M9</li>
 +
    <li style="text-align:left">M9 + TPA (1.2mM)</li>
 +
    <li style="text-align:left">M9 + TPA (6mM)</li>
 +
    <li style="text-align:left">M9 + TPA (12mM)</li>
 +
    <li style="text-align:left">M9 + sucrose (1.2mM)</li>
 +
    <li style="text-align:left">M9 + sucrose (6mM)</li>
 +
    <li style="text-align:left">M9 + sucrose (12mM)</li>
 +
    <li style="text-align:left">M9 + sucrose + TPA (6mM)</li>
 +
    <li style="text-align:left">M9 + sucrose + TPA (1,2mM)</li>
 +
Media 11 to 15 are made in both solid and liquid form:
 +
    <li style="text-align:left">LB Broth</li>
 +
    <li style="text-align:left">LB + TPA (1.2mM)</li>
 +
    <li style="text-align:left">LB + TPA (6mM)</li>
 +
    <li style="text-align:left">LB + TPA (12mM)</li>
 +
    <li style="text-align:left">LB + TPA (20mM)</li>
 +
  </ol>
 +
  </li>
 +
  </br>
 +
  <li style="text-align:left">Growth curve of BAP 1 in media 11 to 14 in order to assess TPA toxicity.</li></br>
 +
  <li style="text-align:left">Plating of BAP 1 bacteria on all solid media to assess TPA toxicity and the ability of TPA to be used as a carbon source.</br></br>
 +
  </ol>
 +
 
 +
<center><h3><em>Enzymatic Assay of NB-Esterase</em></h3></center><br>
 +
<ol>
 +
<li style="text-align:left">XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.</li></br>
 +
<li style="text-align:left">Gel purification  of the digested fragments on 0.8% agarose gel. </li></br>
 +
<li style="text-align:left">Ligation of  pDG011 plasmid and BBa_K808030 using T4 DNA ligase. </li></br>
 +
<li style="text-align:left">Gel migration on  0.8% agarose gel. </li></br>
 +
</ol>
 +
<center><strong><img src="https://static.igem.org/mediawiki/2015/9/91/IGEM_Pasteur-gel--week-10.png" alt="k" width="292" height="455" /></strong><u> </u><br />
 +
<strong><u>Figure 8:</u></strong> Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.<br />
 +
There is no band for the ligation sample.</center>
 +
</p>
 +
</br>
 +
 
 +
<center><h3><em>Gibson Assembly</em></h3></center>
 +
</br>
 +
<ol>
 +
  <li style="text-align:left">Ligation of TphA1/,  Transp_, BBa_K1392932_, using T4 DNA ligase. </li></br>
 +
  <li style="text-align:left">DNA concentration  measurement using a spectrophotometer. </li>
 +
</ol>
 +
<table border="1" cellspacing="0" cellpadding="0" width="643" align="center">
 +
  <tr>
 +
    <td width="90" valign="center"><p><strong>Biobrick</strong></p></td>
 +
    <td width="106" valign="top"><p><strong>DNA Concentration (ng/µl)</strong></p></td>
 +
    <td width="88" valign="top"><p><strong>OD (230 nm)</strong></p></td>
 +
    <td width="88" valign="top"><p><strong>OD (260 nm)</strong></p></td>
 +
    <td width="71" valign="top"><p><strong>OD (280 nm)</strong></p></td>
 +
    <td width="106" valign="top"><p><strong>OD (260 nm) / OD (280 nm)</strong></p></td>
 +
    <td width="109" valign="top"><p><strong>OD (260 nm) / OD (230 nm)</strong></p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="90" valign="top"><p><strong>808010</strong></p></td>
 +
    <td width="106" valign="top"><p>30.00</p></td>
 +
    <td width="90" valign="top"><p>9.33</p></td>
 +
    <td width="90" valign="top"><p>0.75</p></td>
 +
    <td width="90" valign="top"><p>0,30</p></td>
 +
    <td width="106" valign="top"><p>1.87</p></td>
 +
    <td width="109" valign="top"><p>0.06</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="90" valign="top"><p><strong>808011</strong></p></td>
 +
    <td width="106" valign="top"><p>50.00</p></td>
 +
    <td width="90" valign="top"><p>11.78</p></td>
 +
    <td width="90" valign="top"><p>1.00</p></td>
 +
    <td width="90" valign="top"><p>0.63</p></td>
 +
    <td width="115" valign="top"><p>1.61</p></td>
 +
    <td width="115" valign="top"><p>0.09</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="90" valign="top"><p><strong>808012</strong></p></td>
 +
    <td width="106" valign="top"><p>17.50</p></td>
 +
    <td width="90" valign="top"><p>10.93</p></td>
 +
    <td width="90" valign="top"><p>0.33</p></td>
 +
    <td width="90" valign="top"><p>0.18</p></td>
 +
    <td width="115" valign="top"><p>0.84</p></td>
 +
    <td width="115" valign="top"><p>0.03</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="90" valign="top"><p><strong>808013</strong></p></td>
 +
    <td width="106" valign="top"><p>5.00</p></td>
 +
    <td width="90" valign="top"><p>10.55</p></td>
 +
    <td width="90" valign="top"><p>0.10</p></td>
 +
    <td width="90" valign="top"><p>0.06</p></td>
 +
    <td width="115" valign="top"><p>1.56</p></td>
 +
    <td width="115" valign="top"><p>0.01</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="90" valign="top"><p><strong>808014</strong></p></td>
 +
    <td width="106" valign="top"><p>22.5</p></td>
 +
    <td width="90" valign="top"><p>12.13</p></td>
 +
    <td width="90" valign="top"><p>0.47</p></td>
 +
    <td width="90" valign="top"><p>0.30</p></td>
 +
    <td width="115" valign="top"><p>1.54</p></td>
 +
    <td width="115" valign="top"><p>0.04</p></td>
 +
  </tr>
 +
  <tr>
 +
    <td width="90" valign="top"><p><strong>1095000</strong></p></td>
 +
    <td width="106" valign="top"><p>37.50</p></td>
 +
    <td width="90" valign="top"><p>18.48</p></td>
 +
    <td width="90" valign="top"><p>0.78</p></td>
 +
    <td width="90" valign="top"><p>0.43</p></td>
 +
    <td width="115" valign="top"><p>1.83</p></td>
 +
    <td width="115" valign="top"><p>0.04</p></td>
 +
  </tr>
 +
</table>
 
             </td>
 
             </td>
 
         </tr>
 
         </tr>
 
       </table>
 
       </table>
 
   </center>
 
   </center>
 +
<br>
 
</div>
 
</div>
 +
 +
 
<style type="text/css">
 
<style type="text/css">
 
.hautdepage {
 
.hautdepage {

Revision as of 15:26, 18 September 2015

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 11

08/10 - 08/14




Yeast Assembly


The Bacteria was first sent to the iGEM Toulouse and could have suffered from the travel so we ordered it again.

  1. Reception of a STAB culture of the Biobrick BBa_J61047.

  2. Spreading of BBa_J61047 on Petri dishes containing LB medium and LB + Cm 34 µg/ml.
 No colonies were observed on Petri dishes containing LB + Cm 34 µg/ml but the bacteria grew on LB media.

  3. Bacterial growth of the bacteria in liquid medium (5 ml of LB + Cm 34 µg/ml) and on a Petri dish (LB + Cm 34 µg/ml) and overnight incubation at 37°C.

  4. Storage at 4°C.

  5. Bacterial growth of the Bacteria in LB: incubation at 37°C for 1h30 at 140rpm.

    1. M9
    2. M9 + TPA (1.2mM)
    3. M9 + TPA (6mM)
    4. M9 + TPA (12mM)
    5. M9 + sucrose (1.2mM)
    6. M9 + sucrose (6mM)
    7. M9 + sucrose (12mM)
    8. M9 + sucrose + TPA (6mM)
    9. M9 + sucrose + TPA (1,2mM)
      10. Media 11 to 15 are made in both solid and liquid form:
    10. LB Broth
    11. LB + TPA (1.2mM)
    12. LB + TPA (6mM)
    13. LB + TPA (12mM)
    14. LB + TPA (20mM)

  6. Growth curve of BAP 1 in media 11 to 14 in order to assess TPA toxicity.

  7. Plating of BAP 1 bacteria on all solid media to assess TPA toxicity and the ability of TPA to be used as a carbon source.

Enzymatic Assay of NB-Esterase


  1. XbaI ans PstI enzymatic digestion of the Biobrick BBa_K808030 and pDG011 plasmid.

  2. Gel purification of the digested fragments on 0.8% agarose gel.

  3. Ligation of pDG011 plasmid and BBa_K808030 using T4 DNA ligase.

  4. Gel migration on 0.8% agarose gel.

k
Figure 8: Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid.
There is no band for the ligation sample.


Gibson Assembly


  1. Ligation of TphA1/, Transp_, BBa_K1392932_, using T4 DNA ligase.

  2. DNA concentration measurement using a spectrophotometer.

Biobrick

DNA Concentration (ng/µl)

OD (230 nm)

OD (260 nm)

OD (280 nm)

OD (260 nm) / OD (280 nm)

OD (260 nm) / OD (230 nm)

808010

30.00

9.33

0.75

0,30

1.87

0.06

808011

50.00

11.78

1.00

0.63

1.61

0.09

808012

17.50

10.93

0.33

0.18

0.84

0.03

808013

5.00

10.55

0.10

0.06

1.56

0.01

808014

22.5

12.13

0.47

0.30

1.54

0.04

1095000

37.50

18.48

0.78

0.43

1.83

0.04