A plasmid used was pYES2 plasmid provided by M. Didier Thoraval (membrane biogenesis laboratory). It contains an inducible promoter to galactose (GAL1) and a selection gene URA3.
A plasmid used was pFA 6a-HIS3MX6-pGAL-3HA plasmid because there is a gene cassette located into containing GAL1 promoter and HIS3 selection gene.
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.
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.
Thanks to Derek Mc Cluster we tried to overexpress FKS1 gene by integrating GAL1 inducible promoter upstream of it by the homologous recombination method.
To do that, we used a gene cassette located into pFA 6a-HIS3MX6-pGAL-3HA plasmid. This cassette contains GAL1 promoter and HIS3 selection gene. We amplified the cassette by PCR.
The primers used for PCR have floating tails allowing the integration of the cassette upstream FKS1 gene.
N.B. The amplification product containing the selection gene and the promoter flanked by the homologous recombination of sequences is purified.
After transformation and overnight growth, are picked in YNB liquid medium (Gal + His-).
We must then determine if the homologous recombination was successful. This verification will be carried out by PCR amplification. We designed a pair of primers specifically hybridizing upstream of the gene of interest and framing the location where construction must integrate.
PCR products: → 2kb if homologous recombination worked, → 1kb if homologous recombination does not take place at the desired location.
Figure 1: Gibson Assembly