Bacterial bioreporters for the detection of trace explosives: performance enhancement by DNA shuffling and random mutagenesis

Landmines and other explosive remnants of war pose a global humanitarian problem that claims numerous casualties long after the conflict has ended. As there are no acceptable methodologies for remote discovery such devices, current detection practices still require risky presence personnel in minefield. We have recently described bacterial sensor strains capable reporting existence 2,4-dinitrotoluene (DNT) vapors soil above 2,4,6-trinitrotoluene (TNT)-based landmines, by generating bioluminescent or fluorescent signal. This may allow identification landmine location imaging an area over which bacteria been spread. In study reported herein, we improved DNT-detection capabilities these combining two DNT-responsive Escherichia coli gene promoters, yqjF azoR, subjecting them to three cycles random mutagenesis error-prone PCR, combined with segmentation rearrangement (“DNA shuffling”). The activity selected modified promoters was evaluated Aliivibrio fischeri Photobacterium leiognathi luxCDABEG cassettes as reporters, exhibiting ten-fold background reduction led three-fold decrease threshold. Signal intensity further enhanced modifying ribosomal binding site promoter. superior DNT on solid matrix strain were demonstrated. • Performance microbial buried explosives molecularly enhanced. Manipulations included mutagenesis, “DNA shuffling,” RBS reprogramming. re-engineered constructs exhibited trace explosives.