Heterologous Expression of Candida Antifungal Target Genes in the Model Organism Saccharomyces cerevisiae.

Understanding how a gene variant may influence antifungal resistance, or other phenotypic characteristics, is an important step in determining or dissecting resistance mechanisms. The influence of specific genes or gene alleles on a phenotype can initially be assessed within the model organism, Saccharomyces cerevisiae. S. cerevisiae exhibits efficient rates of homologous recombination making it amendable for heterologous expression and represents a susceptible organism that can be used to determine changes in antifungal susceptibilities. Many groups have developed different methodologies for the cloning, expression, and screening processes. In this chapter, we present straightforward methodology that utilizes gap-repair cloning to express a plasmid-borne copy of Candida auris ERG11 within S. cerevisiae. Multiple alleles can be compared in order to determine how specific alterations influence triazole susceptibility. Primer design, gap-repair co-transformation, and colony PCR screening are detailed.