Part:BBa_K1951011:Experience

Demonstration of DesA, DesB, DesC and DesD protein production

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 [http://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. After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer. We heated the mix at 95°C during 15min [http://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. Staining was done using coomassie blue.

We compared the production of proteins in different background :

- E. coli Tg1 strain without any plasmide (negative temoin)

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

- E. coli 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)

Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway.

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.

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 Escherichia coli 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.

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.

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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