journal.pone.0083800.pdf (4.39 MB)
Optimisation of the Schizosaccharomyces pombe urg1 expression system
journal contribution
posted on 2023-06-08, 16:40 authored by Adam WatsonAdam Watson, Yasukazu Daigaku, Saed Mohebi, Thomas Etheridge, Charley Chahwan, Jo Murray, Antony CarrAntony CarrThe 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 ONEISSN
1932-6203Publisher
Public Library of ScienceExternal DOI
Issue
12Volume
8Article number
e83800Department affiliated with
- Sussex Centre for Genome Damage Stability Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2014-03-12First Open Access (FOA) Date
2014-03-12First Compliant Deposit (FCD) Date
2014-03-02Usage metrics
Categories
No categories selectedKeywords
Licence
Exports
RefWorks
BibTeX
Ref. manager
Endnote
DataCite
NLM
DC