To accomplish the mission of landmine detection, we have been striving to increase the feasibility of our design. Various detection methods have been reported in biology, which inspired us to develop our own design. Several parts previously used in the process of landmine detection had been identified. These parts combined with our newly created parts are all applied to our design.
The main idea of our project is detecting a substance, 2,4-Dinitrotoluene (DNT), from landmines with our engineered bacteria. With the promoter (yqjF) induced by DNT’s metabolite, 2,4,5-trihydroxytoluene (THT), the reporter gene downstream of the promoter will be activated and the presence of DNT becomes visible. The transcription factor regulating the yqjF promoter activity has also been added into the list for optimization. We also constructed the bacteria to improve the conversion from DNT to THT. The improvement and optimization of the parts in our project lead to greatly enhanced sensitivity of landmine detection.
Response of DNT
In our case, the first thing is to build a connection between our engineered bacteria and DNT, a substance released from landmines. Our goal is to employ bacteria to detect landmines in the field. The promoter of the yqjF gene in bacteria has been reported to be activated by DNT and became the core element to convert landmine-released chemicals to detectable signal . To make the response of our engineered bacteria visible, we compared reporting systems, luciferase and fluorescent protein, to evaluate which system could better suit our purpose. Once the yqjF promoter is activated by THT, the metabolite of DNT, the reporter gene expression will be triggered, leading to the accumulation of bioluminescent or fluorescent proteins that can be readily detected by us. A literature showd that the threshold of DNT detection by the yqjF promoter is 25 mg/L, which is much higher than what we expected. Therefore, how to reduce the detection threshold and increase the sensitivity of our device has become one of our major tasks. For this purpose, we employed the YhaJ, as introduced below.
Important regulator: YhaJ
As we described above, yqjF could be activated by THT. At the same time, researches show that once YhaJ knockout, yqjF completely lose the response to THT . Therefore, we speculate that YhaJ may form a complex with THT and then activate the yqjF promoter. Based on this hypothesis, we design an YhaJ overexpression modular in order to further improve the sensitivity and strength of the yqjF promoter. The design of plasmid construction is described below.
Previous studies indicated that the DNT concentration on the soil surface of landmines was about 17.5 ng/cm2 , much lower than the threshold of DNT concentration 25 mg/L needed to activate the unmodified yqjF promoter. To incorporate the engineered bacteria into our landmine detection device, we must reduce the threshold of DNT detection or increase the sensitivity of the yqjF promoter. In addition to yhaJ overexpression and yhaK knockout, we also employed error prone PCR and point mutations to generate different mutations of yqjF and yhaJ genes. Previous literature indicated that 2,4,5-trihydroxytoluene (THT) was a metabolite of DNT, and differential expression of the genes related to DNT metabolic pathway could impact the induction of the yqjF promoter . Thus, we proposed that THT could bind yhaJ to regulate the yqjF promoter cooperatively. In order to find the reasonable mutation sites of yhaJ, we mimicked the three-dimensional structure of THT binding site on the YhaJ by the Discovery Studio™, a professional molecular simulation software for life science research, to search for key binding sites.
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