Autorepression-based conditional gene expression system in yeast for variation-suppressed control of protein dosage

Abstract

Conditional control of gene expression allows an experimenter to investigate many aspects of a gene’s function. In the model organismSaccharomyces cerevisiae, a number of methods to control gene expression are widely practiced, including induction by metabolites, small molecules, and even light. However, all current methods suffer from at least one of a set of drawbacks, including need for specialized growth conditions, leaky expression, or the requirement of specialized equipment. Here we describe protocols using two transformations to construct strains that carry a new controller, in which all these drawbacks are overcome. In these strains, the expression of a controlled gene (gene of interest, or GOI) is repressed by the bacterial repressor TetR, and induced by anhydrotetracycline. TetR also regulates its own expression, creating an autorepression loop. This autorepression allows tight control of gene expression/ protein dosage with low cell to cell variation in expression. A second repressor, TetR-Tup1, prevents any leaky expression. We also present a protocol showing a particular workhorse application of such strains, to generate synchronized cell populations. We turn off the expression of the cell cycle regulatorCDC20completely, arresting the cell population, and then back on so that the synchronized cells resume cell cycle progression. This control system can be applied to any endogenous or exogenous gene for precise expression.Basic Protocol 1Generating a parent WTC846strain.Basic Protocol 2Generating a WTC846strain with controlled expression of the targeted geneAlternate Protocol 1CRISPR-mediated promoter replacementBasic Protocol 3Cell cycle synchronization/Arrest and Release using the WTC846-K3::CDC20 strain