Fig. 2: Generation of resistant yeast strains using a stepwise method of compound exposure.

a To determine the degree of growth inhibition of small molecules, cultures derived from single colonies of the ABC₁₆-Green Monster strain (GM) were exposed to various drugs and the IC₅₀s determined. b For in vitro selections single colonies of GM were picked and grown to saturation in YPD media. 50 μl cells of a saturated culture (OD₆₀₀ = 1) were inoculated into 50 mL tubes containing 20 mL of YPD media with a small-molecule inhibitor and grown until saturation. The starting drug concentration was the pre-determined IC₅₀. c Upon reaching saturation cultures were diluted (1:400) into fresh media with increasing drug concentrations. d Development of resistance was evaluated through regular IC₅₀ determinations. e Once cultures showed at least a 2-fold shift in IC₅₀ single clones were generated by plating an aliquot of the resistant strain onto compound-containing YPD plates. f Two independent clones were picked and the IC₅₀ shift confirmed. IC₅₀ values were calculated by subtracting OD₆₀₀ nm values at time 0 h from time 18 h. Nonlinear regression on log([inhibitor]) vs. response with variable slope was performed using GraphPad Prism. Cycloheximide was used as a negative control. g DNA from clones deemed to be resistant (through a combination of fold shift of IC₅₀ and p-value) was isolated and their whole-genome analyzed. h The genomes of the drug-naïve parents and the drug-resistant clones were compared and allele differences between these two clones were determined. Data from all in vitro evolutions was analyzed in great detail. To further validate the potential resistance of the identified mutations allelic replacement of these SNVs into the parental line through CRISPR-Cas9 was performed. Graphics created with Biorender.com under BioRender’s Academic License Terms.