Abstract
Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli-based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter’s molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated.
Highlights
The high risks involved in most prevalent methodologies for buried landmine detection, which require the on-site presence of personnel, have created an acute need for a standoff detection technology
We set out to improve the performance of yqjF-based bacterial bioreporters for landmine detection by the directed evolution of YhaJ, a transcriptional regulator of yqjF activation (Palevsky et al, 2016)
Using error-prone PCR, two yhaJ-targeted sequential mutagenesis rounds were performed, in which the yhaJ variants were incorporated into a plasmid (Figure 1A) that was subsequently introduced into a ΔyhaJ strain
Summary
The high risks involved in most prevalent methodologies for buried landmine detection, which require the on-site presence of personnel, have created an acute need for a standoff detection technology. The primary explosive found in most landmines is 2,4,6-trinitrotoluene (TNT), which is often accompanied by a 2,4-dinitrotoluene (DNT) manufacturing impurity. Vapors of both TNT and DNT have been reported to exist in soils above buried landmines (Sylvia et al, 2000; Jenkins et al, 2001). These vapors migrate to the surface through plastic components or from cracks in the casing, and their identification has served as a basis for diverse approaches for Abbreviations: CDS, coding sequence; DNT, 2,4-dinitrotoluene; LTTR, LysR type transcription regulator; TNT, 2,4,6trinitrotoluene. The relatively volatile and stable DNT is considered an excellent signature chemical for the presence of TNT-based explosive devices (Jenkins et al, 2001)
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