Yeast Engineering
Many of the molecular biology techniques used for genome manipulation in bacteria are also applied in S. cerevisiae. For genomic manipulation in yeast, we start by cloning our genes of interest in plasmids in E. coli, since we use bacteria as the main DNA factory. Yeast transformation proceeds with a heat-shock protocol dependent on polyethylene glycol and lithium acetate as components to optimize efficiency.
There are some essential nutrients that laboratory yeast cannot produce due to specific mutations in structural genes belonging to biosynthetic pathways. In rich media, such yeast strains can thrive, but they cannot grow in minimal media selectively depleted of some nutrients.
Therefore, for the selection of the properly transformed colonies, we exploit the auxotrophies from yeast strains by plating the cells in minimal media without some specific nutrient (e.g.: without uracil) that only the transformants can produce.
The yeast strains maintained in laboratories have been selected throughout generations in order to express higher-than-usual homologous recombination. We can exploit this as a strategy to promote the integration of DNA sequences in the yeast genome, resulting in either a knock-in or a knockout-depending sequence.
For genomic manipulation in yeast, we start by cloning our genes in plasmids in E. coli, since we use bacteria as the main DNA factory (let’s represent DNA molecules as circles and lines for simplicity)
For a knockout, we use a bacterial vector to construct a disruption cassette, which is a selection marker-flanked by the gene to be knocked out.
After yeast transformation, the disruption cassette will substitute the gene of interest after the endogenous recombination machinery acts. In this iGEM project, disruption cassettes are used to knockout genes associated with the unwanted glycosylation steps for humanization of this pathway in yeast
For a knock-in, we start by cloning the gene in a special plasmid called shuttle vector because they can be used both, in bacteria and yeast.
We linearize the shuttle vector by single-cutting it in the selection marker, which has a non-functional version of the selection marker gene to be knocked in high sequence identity with the mutated structural gene needed for an essential nutrient. After yeast transformation, the linearized shuttle vector will be integrated into the selection marker locus by the action of the endogenous recombination machinery.
Using this strategy, we can knock in glucocerebrosidase for S. cerevisiae to express. We designed glucocerebrosidase with a cleavable targeting peptide derived from the yeast protein Pdi1 (protein disulfide isomerase) that directs the protein to the endoplasmic reticulum and then to the secretory pathway.
