Difference between revisions of "Team:Bordeaux/Yeast results"

CHOICE OF MATERIALS

FKS1 gene

FKS1 gene codes a D-glucan synthase.

pYES2 plasmid

The plasmid used is the pYES2 plasmid provided by M. Didier Thoraval (membrane biogenesis laboratory). It contains an inducible promoter to galactose (GAL1) and a selection gene URA3.

Saccharomyces cerevisiae strain

All results were obtained with haploid Lnvsc1 strain of Saccharomyces cerevisiae. Its genotype is Leu / Trp / Ura- / Hist- mat α. This genotype allows us an easy selection of transformed cells.

LABORATORY WORK

1.Overexpression of the gene by plasmidic transformation

One of the first strategies to overexpress the FKS1 gene encoding a D-glucan synthase was to insert it into a plasmid compatible with Saccharomyces cerevisiae.

To insert FKS1 gene into the chosen plasmid, it was necessary to amplify this gene by PCR on yeast genomic DNA. For that, we designed PCR primers that flank the gene of interest taking into account the issues of specific primers to the target sequences, self-aliasing or primer annealing there between.

We have designed two types of primers that differ in their floating tail. Each of the primer present floating tails with restriction sequences.
→ A first pair of primer containing sequences recognized by Not1 restriction enzyme on the forward primer and BamH1 restriction enzyme on the reverse primer, which will allow us to integrate the gene in the pYES2 plasmid
→ A second pair of primer containing sequences recognized by EcoR1 and Xba1 restriction enzyme on the forward primer then, Spe1 and Pst1 on the reverse primer, which will allow us to integrate the gene in pSB1C3 plasmid to constitute a biobrick.

N.B. Unfortunately this strategy could not be completed because PCR amplifications never worked. We tried different process by modeling stringency and with primers hybridizing in the target gene but nothing worked.

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