Development of a 2,4-Dinitrotoluene-Responsive
Synthetic
Riboswitch in <i>E. coli</i> Cells
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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 <i>E. coli</i> 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