Design
Our lab experience enabled us to create the final Biobrocks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.
MObilisation result
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 strain without any plasmide
- E. coli Tg1 strain without pSB1C3 containing the RFP coding sequence
- 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)
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.
In the futur
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.
Biosorption result
Proof of recovered swimming
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.
Microscopie of the flagellum
We analysed the fliC mutant complemented by BbaK1951008 using electronic microscopy. This tools allowed us to observe the flagellum integrity recovered and to obtain phenotypic image of our work.
In the futur
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.
Improvement of CsgA BBa_K342000
From the Biobrick of CsgA BBa_K342000, we made a new improved biobrick. We change p70 promotor into an inductible pBad/araC promotor allowing a controled production of CsgA.
Project achievement in a table
Futur plan for our project
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. The purely biologic production of high precious metal adsorber could reduce environnemental impact of the precious metal production and its cost while renew essential precious metals like platinum, for a sustainable production.
Project Achievements
You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.
- A list of linked bullet points of the successful results during your project
- A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.
Inspiration
See how other teams presented their results.
- <a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a>
- <a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a>
- <a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a>
