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

Asli Azizoğlu, Roger Brent, Fabian Rudolf
doi: https://doi.org/10.1101/2019.12.12.874461
Asli Azizoğlu
aComputational Systems Biology and Swiss Institute of Bioinformatics, ETH Zurich D-BSSE, Mattenstr. 26, 4058 Basel, CH
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Roger Brent
bDivision of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
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  • For correspondence: rbrent@fredhutch.org fabian.rudolf@bsse.ethz.ch
Fabian Rudolf
aComputational Systems Biology and Swiss Institute of Bioinformatics, ETH Zurich D-BSSE, Mattenstr. 26, 4058 Basel, CH
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  • For correspondence: rbrent@fredhutch.org fabian.rudolf@bsse.ethz.ch
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Abstract

Methods to express genes conditionally into phenotype remain central to biological experimentation and biotechnology. Current methods enable either on/off or imprecisely controlled graded gene expression. We developed a “well-tempered” controller, WTC846, for precisely adjustable, graded and growth condition independent conditional expression of genes in Saccharomyces cerevisiae. In WTC846 strains, the controlled genes are expressed from a strong, native promoter engineered to be repressed by the prokaryotic TetR protein and induced by tetracycline and analogues. A second instance of this promoter drives TetR itself. This autorepression loop exhibits low cell-to-cell variation in gene expression and allows precise adjustment of the steady state abundance of any protein with inducer. A second, constitutively expressed zeroing repressor abolishes basal expression in the absence of inducer. WTC846-controlled, stable (Cdc42, Tpi1) and unstable (Ipl1) proteins recapitulated known knockout and overexpression phenotypes. WTC846::CDC20 strains enabled inducer regulated cell cycle synchronization. WTC846 alleles of CDC28, TOR1, PBR1 and PMA1 exhibited expected gene dosage-dependent growth rates and morphological phenotypes, and WTC846::WHI5 strains exhibited inducer controlled differences in cell volume. WTC846 controlled genes comprise a new kind of “expression clamped” allele, for which variation in expression is minimized and gene dosage can be set by the experimenter across the range of cellular protein abundances. In yeast, we expect WTC846 alleles to find use in assessment of phenotypes now incompletely penetrant due to variable dosage of the causative protein, and in genome-wide epistasis screens. Implementation in higher cells should enable experiments now impossible due to cell-to-cell variation and imprecise control.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Rewritten and shortened manuscript

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted September 08, 2020.
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A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery
Asli Azizoğlu, Roger Brent, Fabian Rudolf
bioRxiv 2019.12.12.874461; doi: https://doi.org/10.1101/2019.12.12.874461
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A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery
Asli Azizoğlu, Roger Brent, Fabian Rudolf
bioRxiv 2019.12.12.874461; doi: https://doi.org/10.1101/2019.12.12.874461

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