From 2009.igem.org

Revision as of 08:10, 19 August 2009

July 7, 2009

As mention in Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps 1, a cluster of polysaccharide biosynthesis genes (hfsEFGH) involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide has been identified. hfsC and hfsI genes serve to polymerization.

Genes involved in the export and anchoring of the holdfast polysaccharide have been neglected for our project.

All protein names in parentheses are the E. coli homologs

First idea 

We would like to transfer all the genes needed for a good holdfast synthesis As hfsEFGH and hfsCBAD gene are clusters, 2 plasmids are required :

• one containing hfsA, B, C , D
• the other hfsE F G H

Both plasmids would have a different antibiotic resistance and each gene would have its own ORF.

Primers are designed.

The assembly standart 21 plasmid has been chosen.

We’ll pay attention that genes do not contain any restriction enzyme cutting site corresponding to the assembly standart 21.

References

• Evelyn Toh, Harry D. Kurtz, Jr., and Yves V. Brun. November 2008. "Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps" Journal of Bacteriology 190(21) : 7219–7231

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