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

Abstract

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, WTC₈₄₆, for precisely adjustable, graded, growth condition independent expression of genes in <i>Saccharomyces cerevisiae</i>. 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. WTC₈₄₆ allelic strains in which the controller replaced the native promoters recapitulated known null phenotypes (<i>CDC42, TPI1</i>), exhibited novel overexpression phenotypes (<i>IPL1</i>), showed protein dosage-dependent growth rates and morphological phenotypes (<i>CDC28, TOR2, PMA1</i> and the hitherto uncharacterized <i>PBR1</i>), and enabled cell cycle synchronization (<i>CDC20</i>). WTC₈₄₆ 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.