Difference between revisions of "Team:Pasteur Paris/Week 10"
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− | <h3><em>Toxicity and solubility of TPA (Terephthalic acid)</em></h3><br> | + | <center><h3><em>Toxicity and solubility of TPA (Terephthalic acid)</em></h3></center></br> |
<ol> | <ol> | ||
− | <li style="text-align:left">Dissolution of TPA in DMSO ( | + | <li style="text-align:left">Dissolution of TPA in DMSO (dimethyl sulfoxide). The best concentration obtained is 62 mg/l </li></br> |
<li style="text-align:left">Dilution of TPA stock in LB Broth => TPA precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is 0.9 mg/l </li></br> | <li style="text-align:left">Dilution of TPA stock in LB Broth => TPA precipitates at a concentration of 3.9 g/l, maximum concentration in LB Broth is 0.9 mg/l </li></br> | ||
<li style="text-align:left">Solubilization of TPA by NaOH</li></br> | <li style="text-align:left">Solubilization of TPA by NaOH</li></br> | ||
− | <li style="text-align:left">Creation of a stock solution of soluble TPA in water at 0 | + | <li style="text-align:left">Creation of a stock solution of soluble TPA in water at 0.5 mol/l</li></br> |
<li style="text-align:left">Test of the TPA Solubilization in LB Broth</li></br> | <li style="text-align:left">Test of the TPA Solubilization in LB Broth</li></br> | ||
− | <li style="text-align:left"><u>Preparation of different media containing (TPA) | + | <li style="text-align:left"><u>Preparation of different media containing terephthalic acid (TPA) and ethylene glycol (EG):</u></br> |
<ol> | <ol> | ||
+ | Media 1 to 10 are made in solid form: | ||
<li style="text-align:left">M9</li> | <li style="text-align:left">M9</li> | ||
− | <li style="text-align:left">M9 + TPA (1. | + | <li style="text-align:left">M9 + TPA (1.2 mM)</li> |
− | <li style="text-align:left">M9 + TPA ( | + | <li style="text-align:left">M9 + TPA (6.0 mM)</li> |
− | <li style="text-align:left">M9 + TPA ( | + | <li style="text-align:left">M9 + TPA (12.0 mM)</li> |
− | <li style="text-align:left">M9 + sucrose (1. | + | <li style="text-align:left">M9 + sucrose (1.2 mM)</li> |
− | <li style="text-align:left">M9 + sucrose ( | + | <li style="text-align:left">M9 + sucrose (6.0 mM)</li> |
− | <li style="text-align:left">M9 + sucrose ( | + | <li style="text-align:left">M9 + sucrose (12.0 mM)</li> |
− | <li style="text-align:left">M9 + sucrose + TPA ( | + | <li style="text-align:left">M9 + sucrose + TPA (6.0 mM)</li> |
− | <li style="text-align:left">M9 + sucrose + TPA (1 | + | <li style="text-align:left">M9 + sucrose + TPA (1.2 mM)</li> |
− | + | Media 11 to 15 are made in both solid and liquid form: | |
− | <li style="text-align:left"> | + | <li style="text-align:left">LB Broth</li> |
− | <li style="text-align:left"> | + | <li style="text-align:left">LB + TPA (1.2 mM)</li> |
− | <li style="text-align:left"> | + | <li style="text-align:left">LB + TPA (6.0 mM)</li> |
− | <li style="text-align:left"> | + | <li style="text-align:left">LB + TPA (12.0 mM)</li> |
− | <li style="text-align:left"> | + | <li style="text-align:left">LB + TPA (20.0 mM)</li> |
− | + | </ol> | |
− | + | </li> | |
− | + | </br> | |
− | + | <li style="text-align:left">Growth curve of BAP 1 (E. coli, kindly provided by Blaine Pfeifer, U. Buffalo)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> | </ol> | ||
− | < | + | <center><h3><em>Enzymatic Assay of NB-Esterase</em></h3></center><br> |
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− | <h3><em>Enzymatic Assay of NB-Esterase</em></h3><br> | + | |
<ol> | <ol> | ||
− | <li style="text-align:left">XbaI ans PstI enzymatic | + | <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> | + | <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> | + | <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> | + | <li style="text-align:left">Gel migration on 0.8% agarose gel. </li></br> |
− | + | </ol> | |
− | <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 /> | + | <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 ligation. | + | <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. | + | There is no band for the ligation sample.</center> |
</p> | </p> | ||
</br> | </br> | ||
− | <h3><em>Gibson Assembly</em></h3> | + | |
+ | <center><h3><em>Gibson Assembly</em></h3></center> | ||
</br> | </br> | ||
− | <li style="text-align:left">Ligation of | + | <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> | <li style="text-align:left">DNA concentration measurement using a spectrophotometer. </li> | ||
</ol> | </ol> | ||
<table border="1" cellspacing="0" cellpadding="0" width="643" align="center"> | <table border="1" cellspacing="0" cellpadding="0" width="643" align="center"> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="center"><p><strong>Biobrick</strong></p></td> |
− | <td width="106" valign="top"><p>DNA | + | <td width="106" valign="top"><p><strong>DNA Concentration (ng/µl)</strong></p></td> |
− | <td width="88" valign="top"><p>OD (230 nm)</strong></p></td> | + | <td width="88" valign="top"><p><strong>OD (230 nm)</strong></p></td> |
− | <td width="88" valign="top"><p>OD (260 nm)</strong></p></td> | + | <td width="88" valign="top"><p><strong>OD (260 nm)</strong></p></td> |
− | <td width="71" valign="top"><p>OD (280 nm)</strong></p></td> | + | <td width="71" valign="top"><p><strong>OD (280 nm)</strong></p></td> |
− | <td width="106" valign="top"><p>OD (260 nm) / 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>OD (260 nm) / OD (230 nm)</strong></p></td> | + | <td width="109" valign="top"><p><strong>OD (260 nm) / OD (230 nm)</strong></p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="top"><p><strong>808010</strong></p></td> |
<td width="106" valign="top"><p>30.00</p></td> | <td width="106" valign="top"><p>30.00</p></td> | ||
− | <td width=" | + | <td width="90" valign="top"><p>9.33</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.75</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0,30</p></td> |
<td width="106" valign="top"><p>1.87</p></td> | <td width="106" valign="top"><p>1.87</p></td> | ||
<td width="109" valign="top"><p>0.06</p></td> | <td width="109" valign="top"><p>0.06</p></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="top"><p><strong>808011</strong></p></td> |
<td width="106" valign="top"><p>50.00</p></td> | <td width="106" valign="top"><p>50.00</p></td> | ||
− | <td width=" | + | <td width="90" valign="top"><p>11.78</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>1.00</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.63</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>1.61</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>0.09</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="top"><p><strong>808012</strong></p></td> |
<td width="106" valign="top"><p>17.50</p></td> | <td width="106" valign="top"><p>17.50</p></td> | ||
− | <td width=" | + | <td width="90" valign="top"><p>10.93</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.33</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.18</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>0.84</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>0.03</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="top"><p><strong>808013</strong></p></td> |
<td width="106" valign="top"><p>5.00</p></td> | <td width="106" valign="top"><p>5.00</p></td> | ||
− | <td width=" | + | <td width="90" valign="top"><p>10.55</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.10</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.06</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>1.56</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>0.01</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="top"><p><strong>808014</strong></p></td> |
<td width="106" valign="top"><p>22.5</p></td> | <td width="106" valign="top"><p>22.5</p></td> | ||
− | <td width=" | + | <td width="90" valign="top"><p>12.13</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.47</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.30</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>1.54</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>0.04</p></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td width=" | + | <td width="90" valign="top"><p><strong>1095000</strong></p></td> |
<td width="106" valign="top"><p>37.50</p></td> | <td width="106" valign="top"><p>37.50</p></td> | ||
− | <td width=" | + | <td width="90" valign="top"><p>18.48</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.78</p></td> |
− | <td width=" | + | <td width="90" valign="top"><p>0.43</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>1.83</p></td> |
− | <td width=" | + | <td width="115" valign="top"><p>0.04</p></td> |
</tr> | </tr> | ||
</table> | </table> | ||
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Latest revision as of 02:15, 19 September 2015
Week 1Week 2Week 3Week 4Week 5Week 6Week 7Week 8Week 9Week 10Week 11Week 12Week 13Week 14Week 15 |
Week 10 Toxicity and solubility of TPA (Terephthalic acid)
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
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