A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery

Methods to express genes conditionally into phenotype remain central to biological experimentation and biotech- nology. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a ′well-tempered′ controller, WTC846, for precisely adjustable, graded and growth condition independent conditional expression of genes in Saccharomyces cerevisiae. In WTC846 strains, the controlled genes are expressed from a strong, native promoter engineered to be repressed by the prokaryotic TetR protein and induced by tetracycline and analogues. A second instance of this promoter drives TetR itself. This autorepression loop exhibits low cell-to- cell variation in gene expression and allows precise adjustment of the steady state abundance of any protein with inducer. A second, constitutively expressed zeroing repressor abolishes basal expression in the absence of inducer. WTC846-controlled, stable (Cdc42, Tpi1) and unstable (Ipl1) proteins recapitulated known knockout and overexpres- sion phenotypes. WTC846::CDC20 strains enabled inducer regulated cell cycle synchronization. WTC846 alleles of CDC28, TOR1, PBR1 and PMA1 exhibited expected gene dosage-dependent growth rates and morphological pheno- types, and WTC846::WHI5 strains exhibited inducer controlled differences in cell volume. WTC846 controlled genes comprise a new kind of ′expression clamped′ allele, for which variation in expression is minimized and gene dosage can be set by the experimenter across the range of cellular protein abundances. In yeast, we expect WTC846 alleles to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative protein, and in genome-wide epistasis screens. Implementation in higher cells should enable experiments now impossible due to cell-to-cell variation and imprecise control.