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

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<h3><a href="https://2015.igem.org/Team:Pasteur_Paris/Interlab_study">Interlab Study:</a></h3>
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  <li>Successful building of the 3 devices.</li>
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  <li>Characterization of the 3 devices using a Tecan micro-plate reader.</li>
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  <li>Quantification of the number of Plasmids in each bacteria.</li>
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  <li>Determination of the best Promoter.</li>
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<h3>Gibson assembly:</h3>
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<li>BioBricks submitted to be BioBrick registry.</li>
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<h3>Interlab Study:</h3>
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<h3><a href="https://2015.igem.org/Team:Pasteur_Paris/Interlab_study">Interlab Study:</a></h3>
 
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<li>Transformation in DH5-alpha of each promoter (BBa_J23101, BBa_J23106, BBa_J23117) and the controls GFP (BBa_I13405) and WT. </li>
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  <li>Successful building of the 3 devices.</li>
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<li>qPCR of the transformed cells to determinate the plasmid copy number per strain.  </li>
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  <li>Characterization of the 3 devices using a Tecan micro-plate reader.</li>
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<li>Fluorescence test of the GFP expression and determination of the fluorescence per plasmid.</li>
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  <li>Quantification of the number of Plasmids in each bacteria.</li>
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  <li>Determination of the best Promoter.</li>
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  <li>Transformation in DH5-alpha of each promoter (BBa_J23101, BBa_J23106, BBa_J23117) and the controls GFP (BBa_I13405) and WT. </li>
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  <li>qPCR of the transformed cells to determinate the plasmid copy number per strain.  </li>
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  <li>Fluorescence test of the GFP expression and determination of the fluorescence per plasmid.</li>
 
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Revision as of 12:13, 22 October 2015

Results

pNP-Assay:

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

  • Assessment of the toxicity: Test of the TPA toxicity thanks to a variation of the TPA concentration.
  • Determination that TPA is not degraded.
  • Solubilization of TPA and amelioration of the method.

Operon assembly:

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

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:

  • 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.
  • 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.


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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