Difference between revisions of "Team:Pasteur Paris/Results"
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{{Pasteur_Paris}} | {{Pasteur_Paris}} | ||
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| − | + | <!-- <div class="navbar navbar-default"> | |
<ul class="nav navbar-nav"> | <ul class="nav navbar-nav"> | ||
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| − | <h1><center> Results</center></h1> | + | <h1><center>Results</center></h1> |
<h3>pNP-Assay:<h3/> | <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> |
</ul> | </ul> | ||
<br> | <br> | ||
| − | <h3>TPA toxicity:<h3> | + | <h3>TPA toxicity:</h3> |
<ul> | <ul> | ||
| − | <li>Assessment of the toxicity</li> | + | <li>Assessment of the toxicity.</li> |
| − | <li>Determination that TPA is not degraded </li> | + | <li>Determination that TPA is not degraded.</li> |
</ul> | </ul> | ||
<br> | <br> | ||
| − | <h3>Operon assembly:<h3> | + | <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> | ||
| Line 41: | Line 41: | ||
<h3>Interlab Study:</h3> | <h3>Interlab Study:</h3> | ||
<ul> | <ul> | ||
| − | <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> | ||
<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> | </ul> | ||
| Line 51: | Line 51: | ||
<h3>Gibson assembly:</h3> | <h3>Gibson assembly:</h3> | ||
<ul> | <ul> | ||
| − | <li>BioBricks submitted to be BioBrick registry: | + | <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>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>Bba_K1622001: assembly of the NB-Esterase (BBa_K808030) and the TPA transporteur (BBa_K808007).</li> |
</ul> | </ul> | ||
<br> | <br> | ||
| − | <h3>Interlab Study: </h3> | + | <h3>Interlab Study:</h3> |
<ul> | <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>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>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> | <li>Fluorescence test of the GFP expression and determination of the fluorescence per plasmid.</li> | ||
</ul> | </ul> | ||
| Line 87: | Line 87: | ||
<h1>Problems: </h1> | <h1>Problems: </h1> | ||
<ul> | <ul> | ||
| − | <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><em>Gibson assembly:</em> |
| + | <p>the exonuclease contained in the Master Mix uncovered the Lox Recombination sites because they were too close to the overlapping ends.</p> | ||
| + | </li> | ||
<li><em>Interlab Study: </em> | <li><em>Interlab Study: </em> | ||
| − | <p>Different strain for qPCR and Fluorescence test</p> | + | <p>Different strain for qPCR and Fluorescence test.</p> |
</li> | </li> | ||
<li><em>Modeling:</em> | <li><em>Modeling:</em> | ||
Revision as of 11:56, 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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the exonuclease contained in the Master Mix uncovered the Lox Recombination sites because they were too close to the overlapping ends.
Different strain for qPCR and Fluorescence test.
Because of the absence of experimental results, we can't model our enzymatic system.
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.
^
Page up