{{:Team:Aix-Marseille/Template-Top| Result}}

Our lab experience enabled us to create the final Biobrocks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.

[[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]]

We investigated if the DesA, DesB, DesC and DesD proteins were well produced by our biobrick using SDS PAGE.  

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.  

We heated the mix at 95°C during 15min

[https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols Find here the protocol]

The mixture was loaded onto a polyacrylamide gel and migrated during 50min at 180V.  

- <i>E. coli</i>  Tg1 strain without any plasmide (negative temoin)

- <i>E. coli</i>  Tg1 strain with pSB1C3 containing the RFP coding sequence (negative temoin)

 

- <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)

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.   

[[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).]]

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.

In the cadA mutant complemented by Bba_K1951004, the amount of cadaverine was recovered and even beyond the wild type production.

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.

===In the futur===  

 

* Importation of the siderophore in <i>Streptomyces coelicolor</i>

=== Proof of protein production===

[[File:T--Aix-Marseille--comassieflic.jpeg|left|400px|thumb|Test of our biobrick proteins production using a SDS page and comassie]]

We investigated if the FliC protein was well produced by our biobrick using SDS PAGE.  

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.

SDS page and coomassie blue The mixture was loaded onto a polyacrylamide gel and migrated during 50min at 180V.  

The FliC is at mass 51,3kDa, and can be clearly seen in the gel photograph.

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 fliC mutant in a <i>E.coli W3110</i> strain by transduction using phage P1 (protocol available on our website).  

 

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.

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.

[[File:T--Aix-Marseille--result3.jpeg|770px|center|thumb|We investigated if swimming motility was recovered by a knockout FliC strain.  

To test complementation with our biobrick strains were stabbed into soft (0.3%) LB agar plates,

and the fliC mutant complemented with BBa_K1951008(top).  

The image shows mutiple polar flagella in an E.coli fliC 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.

- insertion by annealing

[http://parts.igem.org/Part:BBa_K1463604 K1463604]

* Second the sequence that we have used it a synthetic gene with codon optimisation, designed specifically for high level expression in <i>E.coli</i>. Thus as an expression part is improved over the initial design.

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.  

<h5> Project Achievements </h5>