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)

Figure 2. Optical Densities were analysed each hour after culture 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, 0.8 OD is obtained much later.
→ So, entire process of production goes on 2 days in LB medium and 3 days in M63 medium.

Figure 2: Bacteria growth in two media

1.Cloning

To achieve our Curdlan production by Escherichia coli, it was necessary to integrate our interest gene crdS controlled by the promoter OsmY in two types of plasmids:
✵ in pSB1C3 plasmid for the characterization of our biobricks
• OsmY promoter only
• crdS gene only
• promoter and gene
✵ in pUC for production steps
• promoter and gene

3.Curdlan production

The production is done on two steps (in two Erlenmeyer flasks) in order to scale up bacterial biomass and then, to obtain a lot of Curdlan.

In stationary phase, we proceed to a temperature change in order to minimize the persistent bacterial growth, the Curdlan being a secondary metabolite.

4.Purification and Quantitative analysis

To obtain Curdlan, cells were chemically destroyed by NaOH and many centrifugations.
Curdlan purification was performed by neutralization after adding acetic acid.
Quantitative analysis was done before and after purification to compare and eliminate non significant measures (background signal).

N.B. Also, we use a polarimeter to characterize our Curdlan molecule produced.

2.Transformation

These plasmids are then transferred into competent bacteria E. coli DH5α by transformation. The selection of transformed bacteria is done by chloramphenicol resistance for pSB1C3 plasmid and by ampicillin resistance for pUC plasmid.

To check that cloning work, plasmids are digested with EcoR1 and Pst1 restriction enzymes.

Figure 3:Agarose electrophoresis gel

Figure 3. As we can see, the band corresponding to the piece of linearized plasmid containing OsmY promoter and crdS gene is a bit higher than the band corresponding to the piece of linearized plasmid containing crdS gene only.