title: Development of a 2,4-dinitrotoluene-responsive synthetic riboswitch in E. coli cells.
abstract: Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered. A plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. Screening was performed by placing the riboswitch library upstream of the Tobacco Etch Virus (TEV) protease coding sequence in one plasmid; a second plasmid encoded a FRET-based construct linked with a peptide containing the TEV protease cleavage site. Addition of DNT to bacterial culture activated the riboswitch, initiating translation of TEV protease. In turn, the protease cleaved the linker in the FRET-based fusion protein, causing a change in fluorescence. This new riboswitch exhibited a 10-fold increase in fluorescence in the presence of 0.5 mM DNT compared to the system without target.
authors.affiliation: 711th Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Ohio 45433, United States.
authors.forename: Molly E
authors.initials: ME
authors.lastname: Davidson
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Harbaugh
authors.forename: Yaroslav G
authors.initials: YG
authors.lastname: Chushak
authors.forename: Morley O
authors.initials: MO
authors.lastname: Stone
authors.forename: Nancy
authors.initials: N
authors.lastname: Kelley-Loughnane
mesh_terms: D004136:Dinitrobenzenes / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli; D015723:Gene Library; D008958:Models, Molecular; D058928:Riboswitch / Q000502:physiology*; D013997:Time Factors; D015854:Up-Regulation
chemical_list: D004136:Dinitrobenzenes; D058928:Riboswitch; C016403:2,4-dinitrotoluene