Development of genetically engineered Escherichia coli for the detection of TNT and DNT

Abstract

2,4-Dinitrotoluene (2,4-DNT) is one of the most widely distributed explosives in soil with buried landmines. DNT can serve as a potential indicator for buried landmines, because this compound permeates slowly through mine casing and diffuse the soil. An accurate and safe method of detecting landmines is needed, but no method has been developed to effectively detect various kinds of mines. Biosensors are considered as an important tool to evolutionarily engineer some kinds of enzymes of metabolic pathway and identify desirable candidates from large gene libraries. To construct a DNT biosensor having 2,4-DNT inducible GFP expression system, a yqjF promoter (below PyqjF) was used by fusing it with gfp+ (a green fluorescent protein gene) on plasmid. The promoter was known to be activated by tri-hydroxy toluene (THT), one of derivatives of DNT. Because the actual inducer of PyqjF was already reported, it is open to figure out genes related the DNT degradation pathway using this promoter. Subsequently, the deletion of yhaK related to degradation of real inducer, THT, leads to enhanced performance of the biosensor. The induction ratio of GFP at 8 h after DNT treatment was about 8-fold higher than the control background strain.