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Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor.

Rudolph, Martin M. ; Vockenhuber, Michael-Paul ; Suess, Beatrix (2015):
Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor.
In: Methods in enzymology, 550, pp. 283-99. ISSN 1557-7988,
[Article]

Abstract

Here we provide a step-by-step protocol for the application of synthetic theophylline-dependent riboswitches for conditional gene expression in Streptomyces coelicolor. Application of the method requires a sequence of only ~85nt to be inserted between the transcriptional start site and the start codon of a gene of interest. No auxiliary factors are needed. All tested riboswitch variants worked well in concert with the promoters galP2, ermEp1, and SF14. Moreover, they allowed theophylline-dependent expression not only of the heterologous β-glucuronidase reporter gene but also of dagA, an endogenous agarase gene. The right combination of the tested promoters with the riboswitch variants allows for the adjustment of the desired dynamic range of regulation in a highly specific and dose-dependent manner and underlines the orthogonality of riboswitch regulation. We anticipate that any additional natural or synthetic promoter can be combined with the presented riboswitches. Moreover, this system should easily be transferable to other Streptomyces species, and most likely to any other genetically manipulable bacteria.

Item Type: Article
Erschienen: 2015
Creators: Rudolph, Martin M. ; Vockenhuber, Michael-Paul ; Suess, Beatrix
Title: Conditional Control of Gene Expression by Synthetic Riboswitches in Streptomyces coelicolor.
Language: English
Abstract:

Here we provide a step-by-step protocol for the application of synthetic theophylline-dependent riboswitches for conditional gene expression in Streptomyces coelicolor. Application of the method requires a sequence of only ~85nt to be inserted between the transcriptional start site and the start codon of a gene of interest. No auxiliary factors are needed. All tested riboswitch variants worked well in concert with the promoters galP2, ermEp1, and SF14. Moreover, they allowed theophylline-dependent expression not only of the heterologous β-glucuronidase reporter gene but also of dagA, an endogenous agarase gene. The right combination of the tested promoters with the riboswitch variants allows for the adjustment of the desired dynamic range of regulation in a highly specific and dose-dependent manner and underlines the orthogonality of riboswitch regulation. We anticipate that any additional natural or synthetic promoter can be combined with the presented riboswitches. Moreover, this system should easily be transferable to other Streptomyces species, and most likely to any other genetically manipulable bacteria.

Journal or Publication Title: Methods in enzymology
Journal volume: 550
Divisions: 10 Department of Biology
10 Department of Biology > Synthetic Genetic Circuits
Date Deposited: 27 Jan 2015 11:34
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