Team:Pasteur Paris/Results
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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TPA toxicity
- Assessment of the toxicity
- Assessment of the toxicity
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.
^
Page up
- Succesful assembly of 2 of our 4 gene clusters (BBa_K1622000 and BBa_K1622001).
- Optimization of DNA sequences for E.coli.
- 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
Interlab Study
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.
^
Page up