Difference between revisions of "Team:Pasteur Paris/Results"
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| + | <img src="https://static.igem.org/mediawiki/2015/4/42/Plasticure.jpg" style="width: 100%;"/> | ||
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| − | <h1 | + | <h1 class="titrepage">Results</h1> |
| − | <h3>pNP-Assay:< | + | <h3><a href="https://2015.igem.org/Team:Pasteur_Paris/Measurement">pNP-Assay:</a></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> |
| + | <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> | </ul> | ||
<br> | <br> | ||
| − | <h3>TPA toxicity:<h3> | + | <h3><a href="https://2015.igem.org/Team:Pasteur_Paris/Measurement">TPA toxicity:</a></h3> |
<ul> | <ul> | ||
| − | <li>Assessment of the toxicity</li> | + | <li>Assessment of the toxicity: Test of the TPA toxicity thanks to a variation of the TPA concentration. </li> |
| − | <li>Determination that TPA is not degraded </li> | + | <li>Determination that TPA is not degraded.</li> |
| + | <li>Solubilization of TPA and amelioration of the method.</li> | ||
</ul> | </ul> | ||
<br> | <br> | ||
| − | <h3>Operon assembly:<h3> | + | <h3><a href="https://2015.igem.org/Team:Pasteur_Paris/Design">Operon assembly:</a></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> | ||
| − | <h3> | + | <h3><a href="https://2015.igem.org/Team:Pasteur_Paris/Design">Gibson assembly:</a></h3> |
| − | + | ||
| − | + | ||
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| − | </ | + | |
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<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><a href="https://2015.igem.org/Team:Pasteur_Paris/Interlab_study">Interlab Study:</a></h3> |
<ul> | <ul> | ||
| − | <li> | + | <li>Successful building of the 3 devices.</li> |
| − | <li> | + | <li>Characterization of the 3 devices using a Tecan micro-plate reader.</li> |
| − | <li> | + | <li>Quantification of the number of Plasmids in each bacteria.</li> |
| − | + | <li>Determination of the best Promoter.</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>Fluorescence test of the GFP expression and determination of the fluorescence per plasmid.</li> | |
| − | + | ||
| − | + | ||
| − | <li> | + | |
| − | <li>Transformation | + | |
| − | + | ||
| − | <li> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
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| − | <li> | + | |
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</ul> | </ul> | ||
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<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></li> |
| − | <p>Different strain for qPCR and Fluorescence test/ | + | <li><em>Modeling:</em><p> Because of the absence of experimental results, we can't model our enzymatic system.</p></li> |
| − | <p> Because of the absence of experimental results, we can't model our enzymatic system. | + | <li><em>CRE:</em><p>the yeast assembly did not work.</p> </li> |
| − | </li> | + | |
| − | <li><em>CRE:</em> the yeast assembly did not work. </li> | + | |
<li><em>pNP assay:</em> | <li><em>pNP assay:</em> | ||
| − | < | + | <ul><li>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.</li><li>Transformation of the construct very hard.</li></ul></li> |
| − | < | + | |
| − | </li> | + | |
</ul> | </ul> | ||
<!-- Renvoie haut de page --> | <!-- Renvoie haut de page --> | ||
Latest revision as of 12:21, 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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