'''Goal''':
to transfer the system of biosynthesis of the holdfast (see article “Characterization off the Caulobacter crescentus holdfast polysaccharide biosynthesis pathway reveals significant fedundancy in the initiating glycosyltransferase and polymerase steps” page 7229” http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2580695” ) in E.Coli.

The E.Coli bacterium is of γ type whereas Caulobacter crescentus is of α type. The problem when we expresse a gene of Caulobacter in E.Coli is that the signal of recognition is not the same one. It is necessary to Cloner gene of Caulobacter without promoter and to place the cloned part with a promoter of E.coli.

The genes which interest us are probably organized in operon. It is necessary to insert a promoter by vector.

We distinguishes 3 clusters different:

It is necessary to express the clusters responsible for the synthesis and export in E.Coli. we needs two plasmids (for each). Both plasmids must be able to be retorted together in the same bacterium. One of genes is expressed in the other direction (see article “Identidication off constrained required for synthesis off the adhesive holdfast in Caulobacter crescentus” page 1437, 1st part of the last diagram). This gene “will be turned over” by PCR and will be inserted in one of both plasmids previously quoted.

It is thus necessary: - different systems of replication for each plasmid - different promoters to express differently the systems (either one or the other or both at the same time) - the plasmids must have a resistance to a different antibiotic.

On the site of UNCBI, if we makes “blast” between two proteinics sequences, we obtains percentage of similarity, percentage of identity and “the univalue”. The weaker “the univalue” is, the more the sequences are similar.

When we add genes on the vector, it is necessary to pay attention to proportioning. If we place a gene naturally absent at E.Coli with a promoter of E.Coli, this protein will be expressed more strongly than the remainder. It is thus more interesting to insert a block of genes.

Practically, it will be necessary: - a promoter (already in the vector of expression of the biobricks) - a sequence RBS (ribosome binding site); this sequence is rich in has and in G, it is thus necessary to find a sequence consensus at E.Coli - starts complementary to the bit of the lower part and starts complementary to the bit of the top - if we does not have a vector containing a sequence RBS, it is necessary to add a RBS on the starter - once the amplified gene, one it insert in the vector via sites of restriction (to be also put in the starters) - GFP (to make sure that the genes are actually expressed in E.Coli) - terminating (there is of two types at E.Coli)

To use a possible maximum of biobricks. To analyze the vectors:

Stages of checking for the cloning: once the vector selected and ready to incorporate the fragment of interest, we operate a ligation. This all the vectors do not containreaction does not function at 100% . Then the cells are transferred.