title: Rewiring Riboswitches to Create New Genetic Circuits in Bacteria.
abstract: Riboswitches are RNA elements that control the expression of genes through a variety of mechanisms in response to the specific binding of small-molecule ligands. Since their discovery, riboswitches have shown promise for the artificial control of transcription or translation of target genes, be it for industrial biotechnology, protein expression, metabolic engineering, antimicrobial target validation, or gene function discovery. However, natural riboswitches are often unsuitable for these purposes due to their regulation by small molecules which are already present within the cell. For this reason, research has focused on creating riboswitches that respond to alternative biologically inert ligands or to molecules which are of interest for biosensing. Here we present methods for the development of artificial riboswitches in Gram-negative and Gram-positive bacteria. These methods are based on reengineering natural aptamers to change their ligand specificity toward molecules which do not bind the original aptamer (ie, that are orthogonal to the original). The first approach involves targeted mutagenesis of native riboswitches to change their specificity toward rationally designed synthetic ligand analogs. The second approach involves the fusion of previously validated orthogonal aptamers with native expression platforms to create novel chimeric riboswitches for the microbial target. We establish the applicability of these methods both for the control of exogenous genes as well as for the control of native genes.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom; Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.
authors.forename: C J
authors.initials: CJ
authors.lastname: Robinson
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.
authors.forename: D
authors.initials: D
authors.lastname: Medina-Stacey
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.
authors.forename: M-C
authors.initials: MC
authors.lastname: Wu
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.
authors.forename: H A
authors.initials: HA
authors.lastname: Vincent
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom; Centre for Synthetic Biology of Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom. Electronic address: jason.micklefield@manchester.ac.uk.
authors.forename: J
authors.initials: J
authors.lastname: Micklefield
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics*; D001419:Bacteria / Q000187:drug effects / Q000235:genetics*; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007218:Industrial Microbiology / Q000379:methods; D008024:Ligands; D016296:Mutagenesis; D058928:Riboswitch / Q000187:drug effects; D052156:SELEX Aptamer Technique / Q000379:methods
chemical_list: D052157:Aptamers, Nucleotide; D008024:Ligands; D058928:Riboswitch
keywords: Aptamer; Expression platform; Gene circuit; Gene regulation; Morphology; Motility; Riboswitch