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

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<h1><center> Results</center></h1>
 
<h1><center> Results</center></h1>
  
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<h3>pNP-Assay<h3/>
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<h3>pNP-Assay:<h3/>
 
<ul>
 
<ul>
 
   <li>Estimation of the activity of NB-Esterase, slowest enzyme of our degradation chain</li>
 
   <li>Estimation of the activity of NB-Esterase, slowest enzyme of our degradation chain</li>
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<br>
 
<br>
  
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<h3>TPA toxicity<h3>
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<h3>TPA toxicity:<h3>
 
<ul>
 
<ul>
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   <li>Assessment of the toxicity</il>
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   <li>Assessment of the toxicity</li>
 
   <li>Determination that TPA is not degraded </li>
 
   <li>Determination that TPA is not degraded </li>
 
</ul>
 
</ul>
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<br>
 
<br>
  
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<h3>Operon assembly<h3>
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<h3>Operon assembly:<h3>
 
<ul>
 
<ul>
 
   <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>
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<br>
 
<br>
  
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<h3>Interlab Study</h3>
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<h3>Interlab Study:</h3>
 
   <li>Successful building of the 3 devices</li>
 
   <li>Successful building of the 3 devices</li>
 
   <li>Characterization of the 3 devices using a Tecan micro-plate reader.</li>
 
   <li>Characterization of the 3 devices using a Tecan micro-plate reader.</li>
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<br><br>
 
<br><br>
  
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<p><strong>Problems: </strong></p>
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<h1>Problems: </h1>
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<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>
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<ul>
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<p><em>Interlab Study: </em></p>
+
<li><em>Gibson assembly:</em> the exonuclease contained in the Master Mix uncovered the Lox Recombination sites because they were too close to the overlapping ends.    </li>
 +
<li><em>Interlab Study: </em>
 
<p>Different strain for qPCR and Fluorescence test/    Modeling:</p>
 
<p>Different strain for qPCR and Fluorescence test/    Modeling:</p>
 
<p> Because of the absence of experimental results, we can't model our enzymatic system.    </p>
 
<p> Because of the absence of experimental results, we can't model our enzymatic system.    </p>
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<p><em>CRE:</em> - 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.</p>
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</li>
−
 
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<li><em>CRE:</em> the yeast assembly did not work.    </li>
 +
<li><em>pNP assay:</em>
 +
<p>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.</p>
 +
<p>Transformation of the construct very hard.</p>
 +
</li>
 +
</ul>
 
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Revision as of 10:25, 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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