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 the remote discovery of such devices, current detection practices still require the risky presence of personnel in the minefield. We have recently described bacterial sensor strains capable of reporting the existence of 2,4-dinitrotoluene (DNT) vapors in the soil above 2,4,6-trinitrotoluene (TNT)-based landmines, by generating a bioluminescent or a fluorescent signal. This may allow the identification of landmine location by remote imaging of an area over which the bacteria have been spread. In the study reported herein, we have improved the DNT-detection capabilities of these sensor strains by combining two DNT-responsive Escherichia coli gene promoters, yqjF and azoR, and subjecting them to three cycles of random mutagenesis by error-prone PCR, combined with segmentation and rearrangement ("DNA shuffling"). The activity of selected modified promoters was evaluated with the Aliivibrio fischeri and Photobacterium leiognathi luxCDABEG gene cassettes as the bioluminescent reporters, exhibiting a ten-fold background reduction that has led to a threefold decrease in detection threshold. Signal intensity was further enhanced by modifying the ribosomal binding site of the yqjF gene promoter. The superior DNT detection capabilities on a solid matrix by the improved sensor strain were demonstrated.
Key points• Performance of microbial sensor strains for buried explosives was molecularly enhanced.• Manipulations included random mutagenesis, "DNA shuffling," and RBS reprogramming.• The re-engineered constructs exhibited superior detection of trace explosives.
A possible solution for the standoff detection of buried landmines is based on the use of microbial bioreporters, genetically engineered to emit a remotely detectable optical signal in response to trace amounts of explosives’ signature chemicals, mostly 2,4‐dinitrotoluene (DNT). Previously developed DNT sensor strains were based on the fusion of a DNT‐inducible gene promoter to a reporting element, either a fluorescent protein gene or a bacterial bioluminescence gene cassette. In the present study, a different approach was used: the DNT‐inducible promoter activates, in Escherichia coli, the quorum‐sensing luxI and luxR genes of Aliivibrio fischeri. N‐Acyl homoserine lactone (AHL), synthesized by LuxI, combines with LuxR and activates the bioluminescence reporter genes. The resulting bioreporter displayed a dose‐dependent luminescent signal in the presence of DNT. Performance of the sensor strain was further enhanced by manipulation of the sensing element (combining the E. coli DNT‐inducible azoR and yqjF gene promoters), by replacing the luminescence gene cassette of Photorhabdus luminescens luxCDABE with A. fischeri luxCDABEG, and by introducing two mutations, eutE and ygdD, into the host strain. DNT detection sensitivity of the final bioreporter was over 340‐fold higher than the original construct.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.