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 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. WTC846 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). 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.
Highlights
Since the spectacular demonstration of suppression of nonsense mutations and its application to T4 development (Epstein et al, 1963), means to express genes conditionally to permit observation of the phenotype have remained central to biological experimentation and discovery
Conditional expression of genes and observation of phenotype remain central to biological discovery
Controlled genes are expressed from a strong semisynthetic promoter repressed by the prokaryotic TetR, which represses its own synthesis; with basal expression abolished by a second, ’zeroing’ repressor
Summary
Since the spectacular demonstration of suppression of nonsense mutations and its application to T4 development (Epstein et al, 1963), means to express genes conditionally to permit observation of the phenotype have remained central to biological experimentation and discovery. In 2021, contemporaneous approaches to conditional expression in wide use include construction of transgenes activated by chimeric activators controlled by promoters whose expression is temporally and spatially restricted to different cell lineages (Brand and Perrimon, 1993), hundreds of approaches based on production of DNA rearrangements by phage-derived site specific recombination (Sauer, 1987), and triggered induction of engineered genes by chimeric transcription regulators with DNA-binding moieties based on derivatives of TetR from Tn10 (Gossen et al, 1995; Garıet al., 1997) Most of these approaches are all-or-none, in the sense that they are not intended to bring about expression of intermediate levels of protein; and the observations they enable are often qualitative.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
