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


Full Text : https://apps.dtic.mil/dtic/tr/fulltext/u2/a570108.pdf


Report Date : Sep 2012


Pagination or Media Count : 37


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