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Optimisation of the Schizosaccharomyces pombe urg1 expression system

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posted on 2023-06-08, 16:40 authored by Adam WatsonAdam Watson, Yasukazu Daigaku, Saed Mohebi, Thomas Etheridge, Charley Chahwan, Jo Murray, Antony CarrAntony Carr
The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down P urg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining P urg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair.

Funding

G1100074; MRC

268788-SMI-DDR; ERC

History

Publication status

  • Published

File Version

  • Published version

Journal

PLoS ONE

ISSN

1932-6203

Publisher

Public Library of Science

Issue

12

Volume

8

Article number

e83800

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2014-03-12

First Open Access (FOA) Date

2014-03-12

First Compliant Deposit (FCD) Date

2014-03-02

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