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Conditional expression of genes and observation of phenotype remain central to biological discovery. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a 'well-tempered' controller, WTC 846 , for precisely adjustable, graded, growth condition independent expression of genes in  Saccharomyces cerevisiae . Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which also represses its own synthesis; with basal expression abolished by a second, 'zeroing' repressor. The autorepression loop lowers cell-to-cell variation while enabling precise adjustment of protein expression by a chemical inducer. WTC 846  allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes ( CDC42, TPI1 ), exhibited novel overexpression phenotypes ( IPL1 ), showed protein dosage-dependent growth rates and morphological phenotypes ( CDC28, TOR2, PMA1  and the hitherto uncharacterized  PBR1 ), and enabled cell cycle synchronization ( CDC20 ). WTC 846  defines an 'expression clamp' allowing protein dosage to be adjusted by the experimenter across the range of cellular protein abundances, with limited variation around the setpoint.

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