RESUMO
The use of bacterial bioreporters for the detection of buried landmines and other explosive devices has been promoted for over 25 years, and several bacterial sensor strains capable of detecting traces of either 2,4,6-trinitrotoluene (2,4,6-TNT) or 2,4-dinitrotoluene (2,4-DNT) have since been reported. In all cases, however, detection sensitivity failed to reach the levels required to reliably sense the minute concentrations of 2,4-DNT vapors expected to exist in the soil above buried landmines. Here, we report on the application of a directed evolution process to enhance the performance of a previously described E. coli-based bioreporter harboring a plasmid-borne genetic fusion between the yqjF gene promoter and either luxCDABE or gfp genes, generating bioluminescent or fluorescent signals, respectively, in the presence of either 2,4,6-TNT or its main "signature" compound, 2,4-DNT. We have performed four sequential rounds of random mutagenesis to the yqjF promoter region, yielding a fourth-generation sensor that displayed significantly improved 2,4-DNT detection characteristics compared to the wild-type and to previous generations. Luminescence intensity in the presence of aqueous 2,4-DNT increased over 3000-fold, response ratios were improved over 50-fold, detection threshold was reduced by 75 %, and response time was cut down to half. These features were manifested also upon exposure to 2,4-DNT vapors or to 2,4-DNT and 2,4,6-TNT buried in sand. An analysis of the point mutations accumulated in the course of this process indicated that the major contributors to these effects were manipulations of the -35 element of the yqjF gene promoter.
