✵ 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.
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.
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.
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.