Accession Number:

ADA570108

Title:

Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli cells

Descriptive Note:

Final rept. 19 feb 2008-1 Sep 2012

Corporate Author:

UES INC DAYTON OH

Personal Author(s):

• Davidson, Molly E.
• Harbaugh, Svetlana V.
• Chushak, Yaroslav G.
• Stone, Morley O.
• Kelley-Loughnane, Nancy

Report Date:

2012-09-01

Pagination or Media Count:

37.0

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.

Descriptors:

• *EXPLOSIVES DETECTION
• *RIBONUCLEIC ACIDS
• ESCHERICHIA COLI
• PEPTIDE HYDROLASES
• TNT

Subject Categories:

• Biochemistry
• Genetic Engineering and Molecular Biology
• Miscellaneous Detection and Detectors
• Ammunition and Explosives

Distribution Statement:

APPROVED FOR PUBLIC RELEASE