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> | ||
<p></p><br><br><br> | <p></p><br><br><br> | ||
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− | + | <center><h3><em> Yeast Assembly </em></h3></center></br> | |
− | + | <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> | |
− | + | <ol> | |
− | < | + | <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 1Week 2Week 3Week 4Week 5Week 6Week 7Week 8Week 9Week 10Week 11Week 12Week 13Week 14Week 15 |
Week 11 Yeast AssemblyThe Bacteria was first sent to the iGEM Toulouse and could have suffered from the travel so we ordered it again.
Enzymatic Assay of NB-Esterase
![]() Figure 8: Gel for verification of the ligation of BBa_K808030 and pDG011 plasmid. There is no band for the ligation sample. Gibson Assembly
|