title: Using riboswitches to regulate gene expression and define gene function in mycobacteria.
abstract: Mycobacteria include both environmental species and many pathogenic species such as Mycobacterium tuberculosis, an intracellular pathogen that is the causative agent of tuberculosis in humans. Inducible gene expression is a powerful tool for examining gene function and essentiality, both in in vitro culture and in host cell infections. The theophylline-inducible artificial riboswitch has recently emerged as an alternative to protein repressor-based systems. The riboswitch is translationally regulated and is combined with a mycobacterial promoter that provides transcriptional control. We here provide methods used by our laboratory to characterize the riboswitch response to theophylline in reporter strains, recombinant organisms containing riboswitch-regulated endogenous genes, and in host cell infections. These protocols should facilitate the application of both existing and novel artificial riboswitches to the exploration of gene function in mycobacteria.
authors.affiliation: Department of Chemistry, Stony Brook University, Stony Brook, New York, USA.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Van Vlack
authors.affiliation: Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York, USA. Electronic address: jessica.seeliger@stonybrook.edu.
authors.forename: Jessica C
authors.initials: JC
authors.lastname: Seeliger
mesh_terms: D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D058928:Riboswitch / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D058928:Riboswitch; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase
keywords: Flow cytometry; GFP; Homologous recombination; Inducible; Infection; Mycobacteria; Theophylline; β-Galactosidase