Difference between revisions of "Team:Bordeaux/Results"

crdS, crdA and crdC genes

✵ crdS gene codes the Curdlan synthase.
✵ crdA gene codes a protein which assists translocation of nascent polymer across cytoplasmic membrane.
✵ crdC gene codes a protein which assists translocation of nascent polymer across the periplasm.
crdA, crdC, crdS genes occupy a contiguous 4,948-bp region in Agrobacterium sp. ATCC31749.

N.B : We tried to work on these three genes. However, amplification attempts by PCR were unsuccessful for crdA and crdC genes. So, in a first time, we focused on cloning crdS gene only.

OsmY promoter

In Agrobacterium sp.ATCC31749, Curdlan production is started after a nitrogen starvation in stationary phase. So we decided to use OsmY promoter (BBa_J45992) characterized by MIT 2006 iGEM team which is active in stationary phase and under high osmotic pressure condition. This promoter imitates Curdlan biosynthesis in E.coli without the nitrogen stress.

Figure 1: Growth dependent regulation with three promoters
(Property of MIT 2006 iGEM team)

M63 and LB media

Curdlan production was carried out in two different media: LB medium and M63 medium.
✵ We worked on M63 medium because this one is cited in literature. M63 is a minimal, low osmolarity medium for E.coli, resulting in slower growth rate of these cells. (XXX Mettre la reference de la publication XXX)
→With this medium of known composition we were able to control parameters for the production of our molecule of interest.
✵We worked also on LB medium because this is the most common medium used in the laboratory.

Figure 2: Bacteria growth in two media

Optical Density cultures were analysed each hour after inoculation. As we can see, the growth is much lower in M63 than in LB medium.
✵ For LB medium, we obtained 0.8 OD after 5h.
✵ For M63 medium, we obtained 0.8 OD after 20h.
So, entire process of production goes on 2 days in LB medium and 3 days in M63 medium.