Accession Number ADA570108
Title Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli cells.
Publication Date Sep 2012
Media Count 37p
Personal Author M. E. Davidson M. O. Stone N. Kelley-Loughnane S. V. Harbaugh Y. G. Chushak
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.
Keywords Aptamers
Dinitrotoluene
Escherichia coli
Explosives detection
Fret(Fluorescence resonance energy transfer)
Peptide hydrolases
Ribonucleic acids
Riboswitches
Tev(Tobacco etch virus)
Tnt


 
Source Agency Non Paid ADAS
NTIS Subject Category 57B - Biochemistry
57F - Cytology, Genetics, & Molecular Biology
63F - Optical Detection
79A - Ammunition, Explosives, & Pyrotechnics
Corporate Author Universal Energy Systems, Inc., Dayton, OH.
Document Type Technical report
Title Note Final rept. 19 feb 2008-1 Sep 2012.
NTIS Issue Number 1314
Contract Number N/A

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