title: Engineering a portable riboswitch-LacP hybrid device for two-way gene regulation.
abstract: Riboswitches are RNA-based regulatory devices that mediate ligand-dependent control of gene expression. However, there has been limited success in rationally designing riboswitches. Moreover, most previous riboswitches are confined to a particular gene and only perform one-way regulation. Here, we used a library screening strategy for efficient creation of ON and OFF riboswitches of lacI on the chromosome of Escherichia coli. We then engineered a riboswitch-LacP hybrid device to achieve portable gene control in response to theophylline and IPTG. Moreover, this device regulated target expression in a 'two-way' manner: the default state of target expression was ON; the expression was switched off by adding theophylline and restored to the ON state by adding IPTG without changing growth medium. We showcased the portability and two-way regulation of this device by applying it to the small RNA CsrB and the RpoS protein. Finally, the use of the hybrid device uncovered an inhibitory role of RpoS in acetate assimilation, a function which is otherwise neglected using conventional genetic approaches. Overall, this work establishes a portable riboswitch-LacP device that achieves sequential OFF-and-ON gene regulation. The two-way control of gene expression has various potential scientific and biotechnological applications and helps reveal novel gene functions.
authors.affiliation: Department of Biochemistry, Li Ka Shing Faculty of Medicine, University of Hong Kong, Pok Fu Lam, Hong Kong SAR, People's Republic of China.
authors.forename: Ye
authors.initials: Y
authors.lastname: Jin
authors.forename: Jian-Dong
authors.initials: JD
authors.lastname: Huang
mesh_terms: D000085:Acetates / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D018441:Biofilms / Q000254:growth & development; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D056968:Lac Repressors / Q000235:genetics; D008969:Molecular Sequence Data; D012313:RNA / Q000737:chemistry; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D058928:Riboswitch; D012808:Sigma Factor / Q000378:metabolism
chemical_list: D000085:Acetates; D001426:Bacterial Proteins; C107123:CsrB RNA, E coli; D056968:Lac Repressors; D062085:RNA, Long Noncoding; D022661:RNA, Untranslated; D058928:Riboswitch; D012808:Sigma Factor; C069567:sigma factor KatF protein, Bacteria; D012313:RNA