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

 
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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 with pSB1C3 containing the RFP coding sequence (negative control)
  
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- <i>E. coli</i>  TG1 strain with des operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011) before and after induction. (You can observe the production of the 4 proteins on the figure on the right; left : before induction, right : after induction)
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- <i>E. coli</i>  TG1 strain with <i>des</i> operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011]) before and after induction. (You can observe the production of the 4 proteins on the figure on the right; left : before induction, right : after induction)
  
 
Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway.
 
Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway.
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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 strain. Different backgrounds were analysed : wild type <i>Escherichia coli</i> TG1 strain (yellow column), cadA mutant from Keio bank (blue column), complemented <i> cadA </i> mutant from Keio bank with [http://parts.igem.org/Part:BBa_K1951004 Bba_K1951004](orange column), complemented <i>cadA</i> mutant from Keio bank with [http://parts.igem.org/Part:BBa_K1951004Bba_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 cadA 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 <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 cadA mutant complemented by 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 cadA mutant complemented by 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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===In the futur===  
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===In the future===  
  
 
* Test the whole pathway
 
* Test the whole pathway
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- siderophore production (HPLC and absorption spectroscopy)
 
- siderophore production (HPLC and absorption spectroscopy)
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- Improve the siderophore production if needed (cofactors, protein stability, metabolic engineering)
 
- Improve the siderophore production if needed (cofactors, protein stability, metabolic engineering)
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* Binding affinity for platinum by our siderophore
 
* Binding affinity for platinum by our siderophore
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- ICPAES (ion coupled plasma atomic emission spectroscopy)
 
- ICPAES (ion coupled plasma atomic emission spectroscopy)
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* Importation of the siderophore in ''Streptomyces coelicolor''
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* Importation of the siderophore in <i>Streptomyces coelicolor</i>
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==Biosorption result==
 
==Biosorption result==
  
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=== Proof of protein production===
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=== Protein production===
  
  
 
[[File:T--Aix-Marseille--comassieflic.jpeg|left|400px|thumb|Test of proteins production with biobrick [http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008] using a SDS PAGE and coomassie blue stain]]
 
[[File:T--Aix-Marseille--comassieflic.jpeg|left|400px|thumb|Test of proteins production with biobrick [http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008] using a SDS PAGE and coomassie blue stain]]
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We investigated if the FliC protein was well produced by our biobrick using SDS PAGE.  
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We investigated if the FliC protein was well produced by our biobrick ([http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008]).  
  
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To to do this we performed SDS PAGE and stained with coomassie blue using cells containing this biobrick in plasmid backbone SDS page and coomassie blue.
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To do this we performed SDS PAGE and stained with coomassie blue using cells containing this biobrick.
  
 
From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=0.6.
 
From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=0.6.
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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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SDS page and coomassie blue The mixture was loaded onto a polyacrylamide gel and migrated during 50min at 180V.  
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The sample was loaded onto a polyacrylamide gel and migrated during 50min at 180V.  
  
 
Staining was done using coomassie blue.  
 
Staining was done using coomassie blue.  
  
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The FliC is at mass 51,3kDa, and can be clearly seen in the gel photograph.
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The FliC is at mass 51,3kDa, and can be seen in the gel photograph.
  
 
===Proof of swimming recovery===
 
===Proof of swimming recovery===
  
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We have made a biobrick BbaK1951008 ables to produce Flagellin (FliC protein of the flagellum). In the aim to test the flagellin integrity, we made a <i>fliC</i> mutant in a <i>E. coli</i> W3110 strain by transduction using phage P1 (protocol available on our website).  
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We have made a biobrick [http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008] ables to produce Flagellin (FliC protein of the flagellum). In the aim to test the flagellin integrity, we made a <i>fliC</i> mutant in a <i>E. coli</i> W3110 strain by transduction using phage P1 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Step2_:_Transduction_P1 protocol]).  
  
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In the figure the deletion mutant (lower left sector) shows no swimming motility as expected and a small white colony. In contrast the wild-type colony (lower right) has a diffuse halo due to swimming cells around the central white colony.  
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In the figure the deletion mutant (lower left sector) shows no swimming motility as expected and a small white colony.  
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Finally the complemented strain, the deletion mutant complemented with our biobrick, (top panel) shows two colonies with intense halos surrounding them. This illustrated clearly that our biobrick can restore motility and is functional.
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In contrast the wild-type colony (lower right) has a diffuse halo due to swimming cells around the central white colony.
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Finally the complemented strain, the deletion mutant complemented with our biobrick, (top panel) shows two colonies with intense
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halos surrounding them.  
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This illustrated clearly that our biobrick can restore motility and is functional.
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The intensity of the halo suggests that a greater proportion of the cells are mobile or swimming is in someway better than the wild-type.
 
The intensity of the halo suggests that a greater proportion of the cells are mobile or swimming is in someway better than the wild-type.
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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 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 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 and that the fliC deletion mutant did not have flagella.
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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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* Adsorption of platinum by our flagella
 
* Adsorption of platinum by our flagella
 
- election microscopy observation  
 
- election microscopy observation  
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* Finalise the insertion of platinum specific peptide
 
* Finalise the insertion of platinum specific peptide
  
 
- directed mutagenesis introducing a Bbs1 restriction site
 
- directed mutagenesis introducing a Bbs1 restriction site
  
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- insertion by annealing
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- insertion of coding sequence of peptide by annealing
  
 
* Metal recovery by calcination or proteolysis
 
* Metal recovery by calcination or proteolysis
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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.
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==Futur plan for our project==
 
==Futur plan for our project==
  
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Now that every biobricks have been created and are available, make proofs of concept could allow to envisage investigation about possibilities of an industrial application.  
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Now that every biobrick have been created and are available, make proof of concept could allow to envisage investigation about possibilities of an industrial application.  
  
 
[[File:T--Aix-Marseille--maquette2.jpeg|770px|center|thumb|Maquette of our process in the futur]]
 
[[File:T--Aix-Marseille--maquette2.jpeg|770px|center|thumb|Maquette of our process in the futur]]
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[[File:T--Aix-Marseille--igem.jpeg|500px|center|thumb|Igem Aix Marseille 2016 in a sustainable circle]]
 
[[File:T--Aix-Marseille--igem.jpeg|500px|center|thumb|Igem Aix Marseille 2016 in a sustainable circle]]
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<h5> Project Achievements </h5>
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Latest revision as of 01:16, 20 October 2016