A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery
© 2021, Azizoglu et al..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:10 |
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| Enthalten in: |
eLife - 10(2021) vom: 03. Aug. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Azizoglu, Asli [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 19.10.2021 Date Revised 19.10.2021 published: Electronic Citation Status MEDLINE |
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| doi: |
10.7554/eLife.69549 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 245 | 1 | 2 | |a A precisely adjustable, variation-suppressed eukaryotic transcriptional controller to enable genetic discovery |
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| 500 | |a Date Revised 19.10.2021 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a © 2021, Azizoglu et al. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a S. cerevisiae | |
| 650 | 4 | |a cell-to-cell variation | |
| 650 | 4 | |a chromosomes | |
| 650 | 4 | |a essential gene alleles | |
| 650 | 4 | |a gene expression | |
| 650 | 4 | |a genetic discovery | |
| 650 | 4 | |a genetics | |
| 650 | 4 | |a genomics | |
| 650 | 4 | |a inducible gene expression | |
| 650 | 4 | |a protein dosage | |
| 650 | 4 | |a transcriptional control | |
| 650 | 7 | |a CDC20 protein, S cerevisiae |2 NLM | |
| 650 | 7 | |a Cdc20 Proteins |2 NLM | |
| 650 | 7 | |a Cell Cycle Proteins |2 NLM | |
| 650 | 7 | |a Fungal Proteins |2 NLM | |
| 650 | 7 | |a Saccharomyces cerevisiae Proteins |2 NLM | |
| 650 | 7 | |a CDC28 Protein Kinase, S cerevisiae |2 NLM | |
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| 650 | 7 | |a cdc42 GTP-Binding Protein, Saccharomyces cerevisiae |2 NLM | |
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| 700 | 1 | |a Brent, Roger |e verfasserin |4 aut | |
| 700 | 1 | |a Rudolf, Fabian |e verfasserin |4 aut | |
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