Difference between revisions of "Team:Aix-Marseille/Results"

 
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=== Proof of fonctionnality===
 
=== Proof of fonctionnality===
 
We investigated if DesA (Lysine decarboxylase) was fonctionnal by measurement of cadaverine using HPLC with C18 column and we proved that our biobrick allows the production of DesA which enables to produce the cadaverine and makes it fonctionnal.   
 
We investigated if DesA (Lysine decarboxylase) was fonctionnal by measurement of cadaverine using HPLC with C18 column and we proved that our biobrick allows the production of DesA which enables to produce the cadaverine and makes it fonctionnal.   
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[[File:T--Aix-Marseille--result5.jpeg|500px|tight|thumb| Investigation of the cadaverine production by the lysine decarboxylase DesA. The cadaverin production has been detected by HPLC using C18 column after induction of the <i>des</i> genes. Different backgrounds were analysed : wild type <i>Escherichia coli</i> TG1 strain (yellow column), <i>cadA</i> mutant from Keio bank (blue column), complemented <i> cadA </i> mutant from Keio bank with p<i>desA</i> ([http://parts.igem.org/Part:BBa_K1951004 Bba_K1951004]) (orange column), complemented <i>cadA</i> mutant from Keio bank with <i>des</i> operon ([http://parts.igem.org/Part:BBa_K1951011 Bba_K1951011]) (grey column).]]
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[[File:T--Aix-Marseille--result5.jpeg|500px|tight|thumb| Investigation of the cadaverine production by the lysine decarboxylase DesA. The cadaverin production has been detected by HPLC using C18 column after induction of the <i>des</i> genes. Different backgrounds were analysed : wild type <i>Escherichia coli</i> TG1 strain (yellow column), <i>cadA</i> mutant from Keio bank (blue column), complemented <i> cadA </i> mutant from Keio bank with p<i>desA</i> ([http://parts.igem.org/Part:BBa_K1951004 BBa_K1951004]) (orange column), complemented <i>cadA</i> mutant from Keio bank with <i>des</i> operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011]) (grey column).]]
  
 
Results showed cadaverine detection in the wild type meaning the original strain well produces the cadaverine.
 
Results showed cadaverine detection in the wild type meaning the original strain well produces the cadaverine.
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In <i>cadA</i> mutant from Keio bank, cadaverine was also produced in a least quantity showing that an other pathway is responsible for the production of cadaverine (existence of an other constitutive lysine decarboxylase in the <i> E. coli </i> genome.  
 
In <i>cadA</i> mutant from Keio bank, cadaverine was also produced in a least quantity showing that an other pathway is responsible for the production of cadaverine (existence of an other constitutive lysine decarboxylase in the <i> E. coli </i> genome.  
  
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In the <i>cadA</i> mutant complemented by p<i>desA</i> ([http://parts.igem.org/Part:BBa_K1951004 Bba_K1951004]), the amount of cadaverine was recovered and even beyond the wild type production.
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In the <i>cadA</i> mutant complemented by p<i>desA</i> ([http://parts.igem.org/Part:BBa_K1951004 BBa_K1951004]), the amount of cadaverine was recovered and even beyond the wild type production.
  
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Moreover, in the <i>cadA</i> mutant complemented by the des operon ([http://parts.igem.org/Part:BBa_K1951011 Bba_K1951011]), the cadaverine level produced was even over the wild type and complemented Bba_K1951004 production. We explain this result because of the higher stability of this big composite part.
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Moreover, in the <i>cadA</i> mutant complemented by the des operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011]), the cadaverine level produced was even over the wild type and complemented Bba_K1951004 production. We explain this result because of the higher stability of this big composite part.
  
 
To conclude, we made a big composite part able to produce every proteins involved in the biosynthetic pathway of the desferrioxamine B.
 
To conclude, we made a big composite part able to produce every proteins involved in the biosynthetic pathway of the desferrioxamine B.
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[[File:T--Aix-Marseille--result3.jpeg|770px|center|thumb|We investigated if swimming motility was recovered by a knockout <i>fliC</i> strain.  
 
[[File:T--Aix-Marseille--result3.jpeg|770px|center|thumb|We investigated if swimming motility was recovered by a knockout <i>fliC</i> strain.  
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To test complementation with our biobrick([http://parts.igem.org/Part:BBa_K1951008 BBa_K1951008]), strains were stabbed into soft (0.3%) LB agar plates,
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To test complementation with our biobrick ([http://parts.igem.org/Part:BBa_K1951008 BBa_K1951008]), strains were stabbed into soft (0.3%) LB agar plates,
 
and incubated at 37°C for 4 hours.
 
and incubated at 37°C for 4 hours.
 
Three strains are shown:  
 
Three strains are shown:  
 
<i> Escherichia coli</i> W3110 wild-type strain, which has a good swimming capacity (lower right);
 
<i> Escherichia coli</i> W3110 wild-type strain, which has a good swimming capacity (lower right);
 
a fliC deletion mutant of W3110 (lower left);
 
a fliC deletion mutant of W3110 (lower left);
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and the fliC mutant complemented with [http://parts.igem.org/Part:BBa_K1951008 BBa_K1951008](top).  
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and the fliC mutant complemented with [http://parts.igem.org/Part:BBa_K1951008 BBa_K1951008] (top).  
 
]]
 
]]
  
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This check the flagella assembly and the integration of the flagellin protein expressed from our biobrick BBa_K1951008 we have observed bacteria with an electron microscope.  
 
This check the flagella assembly and the integration of the flagellin protein expressed from our biobrick BBa_K1951008 we have observed bacteria with an electron microscope.  
  
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The image shows mutiple polar flagella in an <i>E. coli fliC</i> deletion mutant containing our biobrick. We saw that these cells had more flagella than wild-type (W3110) cells.
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The image shows mutiple polar flagella in an <i>E. coli fliC</i> deletion mutant containing our biobrick. We saw that these cells had more flagella than wild-type (W3110) cells. The experiments suggest the functionality of our biobrick even if this experiment need a proper negative control i.e. a deletion mutant with no flagella in order to prove effect of our biobrick.
  
 
===In the futur===
 
===In the futur===
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This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team.  
 
This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team.  
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[http://parts.igem.org/Part:BBa_K1463604 K1463604]
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[http://parts.igem.org/Part:BBa_K1463604 BBa_K1463604]
  
 
The improvement of this part is multiple.
 
The improvement of this part is multiple.

Latest revision as of 01:16, 20 October 2016