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

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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.

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All relevant sequences are included in the supporting files for reproducibility. All raw flow cytometry data is publicly available at doi.org/10.3929/ethz-b-000488967. All other source data is included in the manuscript and supporting files.

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Author details

Asli Azizoglu

BSSE, ETH Zurich, Basel, Switzerland

For correspondence
asli.azizoglu@bsse.ethz.ch

Competing interests
The authors declare that no competing interests exist.
Roger Brent

Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States

For correspondence
rbrent@fhcrc.org

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8398-3273
Fabian Rudolf

D-BSSE, ETH Zurich, Basel, Switzerland

For correspondence
fabian.rudolf@bsse.ethz.ch

Competing interests
The authors declare that no competing interests exist.

Funding

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (NCCR Molecular Systems Engineering)

Asli Azizoglu
Fabian Rudolf

National Cancer Institute (R21CA223901)

Roger Brent

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.