| Abstract |
The primary explosive found in most land mines, 2,4,6-trinitrotoluene (2,4,6-TNT), is often accompanied by 2,4-dinitrotoluene (2,4-DNT) and 1,3-dinitrobenzene (1,3-DNB) impurities. The latter two compounds, being more volatile, have been reported to slowly leak through land mine covers and permeate the soil under which they are located, thus serving as potential indicators for buried land mines. We report on the construction of genetically engineered Escherichia coli bioreporter strains for the detection of these compounds, based on a genetic fusion between two gene promoters, yqjF and ybiJ, to either the green fluorescent protein gene GFPmut2 or to Photorhabdus luminescens bioluminescence luxCDABE genes. These two gene promoters were identified by exposing to 2,4-DNT a comprehensive library of about 2,000 E. coli reporter strains, each harboring a different E. coli gene promoter controlling a fluorescent protein reporter gene. Both reporter strains detected 2,4-DNT in an aqueous solution as well as in vapor form or when buried in soil. Performance of the yqjF-based sensor was significantly improved in terms of detection threshold, response time, and signal intensity, following two rounds of random mutagenesis in the promoter region. Both yqjF-based and ybiJ-based reporters were also induced by 2,4,6-TNT and 1,3-DNB. It was further demonstrated that both 2,4,6-TNT and 2,4-DNT are metabolized by E. coli and that the actual induction of both yqjF and ybiJ is caused by yet unidentified degradation products. This is the first demonstration of an E. coli whole-cell sensor strain for 2,4-DNT and 2,4,6-TNT, constructed using its own endogenous sensing elements.
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