Our lab experience enabled us to create the final Biobrocks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.
===Proof of protein production===
Production of every enzymes involved in the pathway has been shown by a SDS page and Comassie Blue. We compared the production of proteins in different background :
- E. coli Tg1 wuthout any plasmide - E. coli Tg1 without pSB1C3 containing the RFP coding sequence - E. coli 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.
We plan to test the whole pathway viability in the first hand. In a second hand, the potential adsorption of platium by our siderophore would be investigate in a rich metal medium.
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 E.coli W3110 strain by transduction using phage P1 (protocol available on our website). fliC mutant has been complemented by our biobrick Bba_K1951008. On the following figure, we investigated if the complemented fliC mutant recovered swimming after complementation. To do this, we did a swimming test using soft gelose. WT W3110 strain was well swimming after 3 hours incubation at 37°C. However, FliC mutant wasn't able to swim whereas fliC mutant complemented recover the swimming ability, making a proof that our biobrick is production a fonctionnal flagellin protein.
We analysed the fliC mutant complemented by BbaK1951008 using electronic microscopy. This tools allowed us to observe the flagellum integrity recovered and to obtain image of our work.
We plan to make a proof of concept of the platinum absorption to the surface of the flagellin. In a second time, we want to finish the insertion of the restriction site to allow the cassette insertion ( peptide absorbing specifically precious metal) and test their affinity.
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