title: Dual transcriptional-translational cascade permits cellular level tuneable expression control.
abstract: The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Rosa
authors.initials: R
authors.lastname: Morra
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, UK School of Chemistry, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Jayendra
authors.initials: J
authors.lastname: Shankar
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK School of Chemistry, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Robinson
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Samantha
authors.initials: S
authors.lastname: Halliwell
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Butler
authors.affiliation: School of Biomedical & Healthcare Sciences, Plymouth University Peninsula Schools of Medicine and Dentistry, Plymouth, PL4 8AA, UK.
authors.forename: Mathew
authors.initials: M
authors.lastname: Upton
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Sam
authors.initials: S
authors.lastname: Hay
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK School of Chemistry, University of Manchester, Manchester, M13 9PL, UK SYNBIOCHEM, University of Manchester, Manchester, M13 9PL, UK.
authors.forename: Jason
authors.initials: J
authors.lastname: Micklefield
authors.affiliation: Manchester Institute of Biotechnology, University of Manchester, Manchester, M13 9PL, UK Faculty of Life Sciences, University of Manchester, Manchester, M13 9PL, UK School of Chemistry, University of Manchester, Manchester, M13 9PL, UK SYNBIOCHEM, University of Manchester, Manchester, M13 9PL, UK neil.dixon@manchester.ac.uk.
authors.forename: Neil
authors.initials: N
authors.lastname: Dixon
mesh_terms: D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D058928:Riboswitch; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D058928:Riboswitch; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases