In the iGEM team, Saccharomyces cerevisiae is often used as a host for cell factories to produce large quantities of valuable metabolites such as drugs, industrial alcohol, etc. To help more iGEM teams working on engineering yeast cell factories, our group provides some valuable tools and solutions for heterologous overexpression of certain key metabolic pathway enzymes in yeast cells for future iGME teams.
We submit information on the S. cerevisiae and E. coli shuttle plasmid backbone. If a future team is committed to genetic engineering related to S. cerevisiae, the plasmid-backbone can be used. Moreover, we provide a set of practical solutions for overexpression genes. Please refer to the experimental methods page for details. If there is a future team dedicated to constructing yeast cell factories, they can refer to the overexpression scheme we used.
YEP352-backbone is a plasmid vector we constructed used for overexpressing some genes in yeast, which can shuttle in E. coli and S. cerevisiae. The vector is a high-copy-number plasmid with 2μ ori. When expressed in the prokaryotic system, the Apramycin+ resistance can be used to screen the right colony, while transformed into the S. cerevisiae lacking the URA3 gene, the strain should be cultured in uracil-deficient minimal medium. This plasmid backbone can be used to express different proteins in yeast in the future.
Figure 1. The map of plasmid YEP352-backbone.
Take the protein FAS1 overexpressing by our project as an example, FAS1 is the gene we want to overexpress, and it is cloned into YEP352’ XhoI site through recombination. Overexpression strains can be successfully obtained by screening in the uracil-deficient minimal medium. The design of this basic part and the experimental protocol are useful for future iGEMers committed to overexpress some protein in S. cerevisiae.
