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

For more than 65 years, means to express genes conditionally into phenotype have remained central to biological experimentation and discovery. Current experimental methods typically enable either on/off gene expression or growth-condition-specific, imprecisely controlled graded expression in response to exogenous inducers. Here we describe a “well-tempered” controller, one that allows precise and graded conditional expression of genes in . This system, WTC, appropriates genetic and design elements from bacterial, eukaryotic, and engineered systems. Its main autorepressing circuitry relies on two identical instances of a strong, growth rate regulated promoter P engineered to be repressible by the TetR protein. This allows for titration of inducer concentration to drive variation-reduced expression of regulated genes across the entire range of the proteome. To reduce basal expression to zero, a TetR-Tup1 fusion protein is expressed at a low, constitutive level. We showed that strains carrying conditional, WTC allelic forms of genes encoding stable low abundance proteins (eg. Cdc42), unstable low abundance proteins (eg. Ipl1), and highly expressed proteins including the glycolytic enzyme Tpi1 recapitulated known knockout and overexpression phenotypes. Strains bearing WTC alleles allowed inducer controlled cell cycle synchronization of batch cultures and release. Chemical titration of WTC allelic strains of CDC28, TOR1, PBR1 and PMA1 brought about distinct, gene dosage dependent controlled growth rates, and highly penetrant morphological phenotypes expected for the different gene doses. The ability to generate WTC controlled, “expression-clamped” genes may operationally define a new kind of conditional allele whose cell-to-cell variation in expression is minimized and whose effective dosage is under the experimenter’s control. We expect Well-tempered Controller (WTC) strains to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative gene product, in targeted cell biological experimentation, and in genome-wide studies such as gene by gene epistasis screens. In higher cells, we hope that implementation of titratable, expression clamped control logic via mammalian specific transcription elements will enable experiments now impossible due to cell-to-cell variation and imprecise control.