Difference between revisions of "Team:Pasteur Paris/Results"

Line 18: Line 18:
 
<h3>pNP-Assay<h3/>
 
<h3>pNP-Assay<h3/>
 
<ul>
 
<ul>
−
   <il>Estimation of the activity of NB-Esterase, slowest enzyme of our degradation chain</il>
+
   <li>Estimation of the activity of NB-Esterase, slowest enzyme of our degradation chain</li>
−
  <il>
+
</ul>
 +
 
 +
<br>
  
 
<h3>TPA toxicity<h3>
 
<h3>TPA toxicity<h3>
 
<ul>
 
<ul>
 
   <li>Assessment of the toxicity</il>
 
   <li>Assessment of the toxicity</il>
−
   <li>Determination that TPA is not degraded  
+
   <li>Determination that TPA is not degraded </li>
 +
</ul>
 +
 
 +
<br>
  
 
<h3>Operon assembly<h3>
 
<h3>Operon assembly<h3>
Line 30: Line 35:
 
   <li>Succesful assembly of 2 of our 4 gene clusters (BBa_K1622000 and BBa_K1622001).</li>
 
   <li>Succesful assembly of 2 of our 4 gene clusters (BBa_K1622000 and BBa_K1622001).</li>
 
   <li>Optimization of DNA sequences for <i>E.coli</i>.</li>
 
   <li>Optimization of DNA sequences for <i>E.coli</i>.</li>
 +
</ul>
 +
 +
<br>
  
 
<h3>Interlab Study</h3>
 
<h3>Interlab Study</h3>
Line 36: Line 44:
 
   <li>Quantification of the number of Plasmids in each bacteria.</li>
 
   <li>Quantification of the number of Plasmids in each bacteria.</li>
 
   <li>Determination of the best Promoter</li>
 
   <li>Determination of the best Promoter</li>
 +
</ul>
 +
 +
<br>
 +
 +
<h3>Gibson assembly:</h3>
 +
<ul>
 +
<li>BioBricks submitted to be BioBrick registry:  </li>
 +
<li>Bba_K1622000: assembly of the Glycoaldehyde Dehydrogenase (BBa_K936011) and the Glycoaldehyde Reductase (BBa_K936023) in the plasmid PSB1C3 :</li>
 +
<li>Bba_K1622001: assembly of the NB-Esterase (BBa_K808030) and the TPA transporteur (BBa_K808007)      </li>
 +
</ul>
 +
 +
<br>
 +
 +
<h3>Interlab Study: </h3>
 +
<ul>
 +
<li>Transformation in DH5-alpha of each promoter (BBa_J23101, BBa_J23106, BBa_J23117) and the controls GFP (BBa_I13405) and WT. </li>
 +
<li>qPCR of the transformed cells to determinate the plasmid copy number per strain.  </li>>
 +
<li>Fluorescence test of the GFP expression and determination of the fluorescence per plasmid.</li>
 +
</ul>
 +
 +
<br>
 +
 +
<h3>pNP assay: </h3>
 +
<ul>
 +
<li>Insertion of the NB-Esterase (BBa_K808030) in the plasmid pDG011.</li>
 +
<li>Transformation of BAP1 with the construct NB-Esterase (Bba_K808030) in the plasmid pDG011.  </li>
 +
<li>Isolation of three clones who have our construct NB-Esterase (Bba_K808030) in the plasmid pDG011.  </li>
 +
<li>Demonstration of the degradation of the 4-NitroPhenyl Butyrate by our modified BAP1 with NB-Esterase in the pDG011 plasmid.    </li>
 +
</ul>
 +
 +
<br>
 +
 +
<h3>TPA:</h3>
 +
<ul>
 +
<li>Test of the TPA toxicity thanks to a variation of the TPA concentration.</li>
 +
<li>Solubilization of TPA and amelioration of the method.      </li>
 +
</ul>
 +
 +
<br><br>
  
−
<p><em>Gibson assembly:  </em></p>
 
−
<p>- BioBricks submitted to be BioBrick registry:  </p>
 
−
<p>- Bba_K1622000: assembly of the Glycoaldehyde Dehydrogenase (BBa_K936011) and the Glycoaldehyde Reductase (BBa_K936023) in the plasmid PSB1C3 :</p>
 
−
<p> - Bba_K1622001: assembly of the NB-Esterase (BBa_K808030) and the TPA transporteur (BBa_K808007)      </p>
 
−
<p>&nbsp;</p>
 
−
<p><em>Interlab Study: </em></p>
 
−
<p>- Transformation in DH5-alpha of each promoter (BBa_J23101, BBa_J23106, BBa_J23117) and the controls GFP (BBa_I13405) and WT. </p>
 
−
<p>- qPCR of the transformed cells to determinate the plasmid copy number per strain.  </p>
 
−
<p>- fluorescence test of the GFP expression and determination of the fluorescence per plasmid.</p>
 
−
<p>&nbsp;</p>
 
−
<p> <em>pNP assay: </em></p>
 
−
<p>- Insertion of the NB-Esterase (BBa_K808030) in the plasmid pDG011</p>
 
−
<p> - Transformation of BAP1 with the construct NB-Esterase (Bba_K808030) in the plasmid pDG011  </p>
 
−
<p>- Isolation of three clones who have our construct NB-Esterase (Bba_K808030) in the plasmid pDG011.  </p>
 
−
<p>- Demonstration of the degradation of the 4-NitroPhenyl Butyrate by our modified BAP1 with NB-Esterase in the pDG011 plasmid.    </p>
 
−
<p>&nbsp;</p>
 
−
<p><em>TPA  :</em></p>
 
−
<p>- Test of the TPA toxicity thanks to a variation of the TPA concentration.</p>
 
−
<p> - Solubilization of TPA and amelioration of the method.      </p>
 
−
<p>&nbsp;</p>
 
−
<p>&nbsp;</p>
 
 
<p><strong>Problems: </strong></p>
 
<p><strong>Problems: </strong></p>
 
<p> Gibson assembly: the exonuclease contained in the Master Mix uncovered the Lox Recombination sites because they were too close to the overlapping ends.    </p>
 
<p> Gibson assembly: the exonuclease contained in the Master Mix uncovered the Lox Recombination sites because they were too close to the overlapping ends.    </p>

Revision as of 10:02, 22 October 2015

Results

pNP-Assay

  • Estimation of the activity of NB-Esterase, slowest enzyme of our degradation chain

TPA toxicity

  • Assessment of the toxicity
  • Determination that TPA is not degraded

Operon assembly

  • Succesful assembly of 2 of our 4 gene clusters (BBa_K1622000 and BBa_K1622001).
  • Optimization of DNA sequences for E.coli.

Interlab Study

  • Successful building of the 3 devices
  • Characterization of the 3 devices using a Tecan micro-plate reader.
  • Quantification of the number of Plasmids in each bacteria.
  • Determination of the best Promoter

    • Gibson assembly:

    • BioBricks submitted to be BioBrick registry:
    • Bba_K1622000: assembly of the Glycoaldehyde Dehydrogenase (BBa_K936011) and the Glycoaldehyde Reductase (BBa_K936023) in the plasmid PSB1C3 :
    • Bba_K1622001: assembly of the NB-Esterase (BBa_K808030) and the TPA transporteur (BBa_K808007)

    Interlab Study:

    • Transformation in DH5-alpha of each promoter (BBa_J23101, BBa_J23106, BBa_J23117) and the controls GFP (BBa_I13405) and WT.
    • qPCR of the transformed cells to determinate the plasmid copy number per strain.
      • >
    • Fluorescence test of the GFP expression and determination of the fluorescence per plasmid.

    pNP assay:

    • Insertion of the NB-Esterase (BBa_K808030) in the plasmid pDG011.
    • Transformation of BAP1 with the construct NB-Esterase (Bba_K808030) in the plasmid pDG011.
    • Isolation of three clones who have our construct NB-Esterase (Bba_K808030) in the plasmid pDG011.
    • Demonstration of the degradation of the 4-NitroPhenyl Butyrate by our modified BAP1 with NB-Esterase in the pDG011 plasmid.

    TPA:

    • Test of the TPA toxicity thanks to a variation of the TPA concentration.
    • Solubilization of TPA and amelioration of the method.


    Problems:

    Gibson assembly: the exonuclease contained in the Master Mix uncovered the Lox Recombination sites because they were too close to the overlapping ends.

    Interlab Study:

    Different strain for qPCR and Fluorescence test/ Modeling:

    Because of the absence of experimental results, we can't model our enzymatic system.

    CRE: - the yeast assembly did not work. pNP assay: - Two of our tests are less decisive because we didn't wait for a long time to see a real difference enter control and modified bacteria. - transformation of the construct very hard.

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