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

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{{:Team:Aix-Marseille/Template-Top| Result}}
 
{{:Team:Aix-Marseille/Template-Top| Result}}
  
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Our lab experience enabled us to create the final Biobrocks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.
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Our lab experience enabled us to create the final Biobricks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.
  
 
==Mobilisation results==
 
==Mobilisation results==
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===Protein production===
 
===Protein production===
  
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[[File:T--Aix-Marseille--result4.jpeg|500px|right|thumb|Test of our biobrick proteins production using a SDS page and comassie. - : no induction ; + : induction with 0.02% arabinose at Abs(600nm)=0.4]]
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[[File:T--Aix-Marseille--result4.jpeg|500px|right|thumb|Test of our biobrick proteins production using a SDS PAGE and coomassie blue stain. - : no induction ; + : induction with 0.02% arabinose at Abs(600nm)=0.4]]
 
We investigated if the DesA, DesB, DesC and DesD proteins were well produced by our biobrick using SDS PAGE.  
 
We investigated if the DesA, DesB, DesC and DesD proteins were well produced by our biobrick using SDS PAGE.  
  
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To do this we performed SDS PAGE and stained with comassie blue using cells containing this biobrick in plasmid backbone [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols Find here the protocol] From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=1.  
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To do this we performed SDS PAGE and stained with coomassie blue using cells containing this biobrick in plasmid backbone [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols Find here the protocol] From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=1.  
 
Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min.  
 
Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min.  
 
After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer.  
 
After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer.  
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We compared the production of proteins in different background :  
 
We compared the production of proteins in different background :  
  
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- <i>E. coli</i>  Tg1 strain without any plasmide (negative temoin)
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- <i>E. coli</i>  Tg1 strain without any plasmide (negative control)
  
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- <i>E. coli</i>  Tg1 strain with pSB1C3 containing the RFP coding sequence (negative temoin)
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- <i>E. coli</i>  Tg1 strain with pSB1C3 containing the RFP coding sequence (negative control)
  
 
- <i>E. coli</i>  Tg1 strain complemented with our biobrick Bba_K1951011 before and after induction. (you can observe the production of the 4 proteins on the figure below on the left; right : before induction, left : after induction)
 
- <i>E. coli</i>  Tg1 strain complemented with our biobrick Bba_K1951011 before and after induction. (you can observe the production of the 4 proteins on the figure below on the left; right : before induction, left : after induction)
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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 proofed our biobrick is a producer of this protein and makes it fonctionnal.   
 
We investigated if DesA (Lysine decarboxylase) was fonctionnal, by measurement of cadaverine using HPLC with C18 column and proofed our biobrick is a producer of this protein 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. Production has been detected by HPLC using C18 column after induction of the strain. Different backgrounds were analysed : wild type <i>Escherichia coli</i> Tg1 strain (yellow column), cadA mutant from keio bank (blue column), complemented cadA mutant from keio bank with Bba_K1951004(orange column), complemented cadA mutant from keio bank with 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 strain. Different backgrounds were analysed : wild type <i>Escherichia coli</i> Tg1 strain (yellow column), cadA mutant from Keio bank (blue column), complemented cadA mutant from Keio bank with Bba_K1951004(orange column), complemented cadA mutant from Keio bank with 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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[[File:T--Aix-Marseille--comassieflic.jpeg|left|400px|thumb|Test of our biobrick proteins production using a SDS page and comassie]]
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[[File:T--Aix-Marseille--comassieflic.jpeg|left|400px|thumb|Test of our biobrick proteins production using a SDS PAGE and coomassie blue stain]]
 
We investigated if the FliC protein was well produced by our biobrick using SDS PAGE.  
 
We investigated if the FliC protein was well produced by our biobrick using SDS PAGE.  
  

Revision as of 17:45, 19 October 2016