Enhancing DNT Detection by a Bacterial Bioreporter: Directed Evolution of the Transcriptional Activator YhaJ

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.

Structural basis of transcription factor YhaJ for DNT detection

“…In the apo structures, the LS1 of the β5-β6 loop forms interactions with regions of the central β-sheet including strand β8, strand β4, and strand β7. The LS1 also interacts with the β3-β4 loop including Met154 which was implicated in the sensitivity-enhancing mutagenesis studies ( Figure 4 C) 16 , 19 (see below). Upon effector binding, the LS1 moves away from the cavity ( Figures 4 A, 4B, and 4D), which is seen in all eight CCU monomers.…”

“… 11 , 12 However, current explosives bioreporters utilizing YhaJ require sensitivity-enhancement engineering to overcome its low detection limit. 3 , 15 , 16 Although there has been progress in improving YhaJ bioreporters by using directed evolution, 16 , 17 , 18 , 19 rational approaches are needed for the development of YhaJ variants that are sensitive enough to detect trace amounts of explosives.…”

Introduction of quorum sensing elements into bacterial bioreporter circuits enhances explosives’ detection capabilities

“…An additional attempt at improving the bioreporter's performance was conducted by introducing into the same vector an enhanced version of the yhaJ gene, previously found to act as an activator of DNT‐related genes [ 12 , 24 ], enhanced by two rounds of directed evolution [ 13 ]. Cloning was performed by PCR amplification of yhaJ (G2) using primers 186_yhaJ_SphI_F and 187_yhaJ_SalI_R (Table S1 ).…”

Controlling the sensitivity and selectivity for the detection of nitro-based explosives by modulating the electronic substituents on the ligand of AIPE-active cyclometalated iridium(iii) complexes

“…Both DNT and TNT are present in the vapour phase in the soil where landmines are present. 19 These vapours migrate to the surface soil and hence cause contamination of soil and water. 20 Several analytical techniques, such as high-performance liquid chromatography (HPLC), 21 Raman spectroscopy, 22,23 X-ray analysis, 24 cyclic voltammetry, 25 and electrochemistry, 26,27 have been used to detect nitro-based explosive compounds.…”

“…Both DNT and TNT are present in the vapour phase in the soil where landmines are present. 19 These vapours migrate to the surface soil and hence cause contamination of soil and water. 20…”