Developing Biosensors For 2,4,6-Trinitrotoluene With New Sensing Elements Derived From Constructing A Promoter Library

The substance,2,4,6-trinitrotoluene (TNT) is a pale or colorless crystal nitrobenzene explosive material, which is an important raw chemical that is useful for the defense industry, mining, and infrastructures. In the areas of explosive manufacturing, battlefield sites, and military training, TNT pollution is a major environmental issue. TNT can also seep into the soil and water systems; thus, entering an ecological circulation. Furthermore, it can produce the "pink water" problem, which is extremely difficult and costly to decontaminate .In addition, there are reports that TNT has potential toxic effects on microorganisms, green algae, and animals including fish. More importantly, TNT and its degradation products can become integrated into the food chain ; this poses serious adverse effects on human health. Short-term exposure to a large amount of TNT can cause acute poisoning, which can result in respiratory failure and death. Long-term exposure to TNT can cause chronic poisoning that can damage the liver, eyes, hematologic system, and the nervous system. This can cause immune system dysfunction that can increased the risk of anemia and cancer.To overcome or avoid the potential toxic effects of TNT,UV, gas chromatography-mass spectrometry (GC-MS), surface-enhanced Raman laser spectroscopy (SERS), nuclear magnetic resonance, and ion mobility spectrometry [7,8], all of which require expensive and complex equipment and/or complicated sample preparation methods. Biosensors, an expanding research area in modern engineering technology, process the response signals when biological units (e.g., enzymes, antibodies, nucleic acids, and cells) respond to specific target(s) [9]. The reaction includes signal receiving, processing, conversion, and output; therefore, the target(s) can be detected qualitatively and quantitatively. For example, in the antibiotic industry, an immobilized enzymatic bio-analyzer allow us to monitor the production of glucose and other compounds in a real-time manner; similarly, the same method is used in the enzyme industry for rapid analysis of glucoamylase [10]. These advances in the biosensor field inspired us to develop a new detection method for TNT based on its biological activity.Currently, the biosensor technology, which is based on the biological activity of target(s), has been widely utilized for detecting toxic or environmental harmful compound [11-15]. The common sensing elements are found in promoter regions that are involved in the cellular responses. Chemiluminescence (LuxCDABE of Photorhabdusluminesce) [16] and fluorescence (green fluorescent protein, GFP) [17] are the most common reporter elements (reporter genes). Recent reports were focused on developing biosensors for detecting toxic heavy metal compounds. For example, DNase (deoxyribosylase) is highly sensitive and specific to heavy metal ions:Pb(Ⅱ), Cu(Ⅱ), and Zn(Ⅱ)). A modified DNase gene is labeled with fluorophore and quencher groups, and then conjugated with Pb(Ⅱ), Cu(Ⅱ) and Zn(Ⅱ). A DNase biosensor using this specific gene exhibits a detection limit down to at 10 nM for those heavy metal ions [18]. Liao et al. [19] successfully constructed the DH5α biosensor, in which a Cadherin gene was used as a regulatory gene and a GFP gene was used as a reporter gene to detect the heavy metal ions (0.1 nmol/L of Sb(Ⅲ) and Cd(Ⅱ),10 nmol/L of Pb(Ⅱ)) in soils and sediments. Such a biosensor has been highly sensitive and cost-efficiently; thus the prospects for real-world application is promising.Currently, the literature contains extremely limited reports regarding biosensors for the detection of TNT [20-22]. A decade ago, Eun-Mi Ho and colleagues [23] reported a new TNT detection method using TNT-induced stress shock proteins (SSPs) in the bacterial stain Stenotrophomonas sp. OK-5. In 2011, Behzadian et al. [24] reported on a biosensor system containing an E. coli stain to detect toluene and related compounds. Similarly, using an E. coli strain, Sharon Yagur-Kroll et al. [25] in 2014 discovered the promoters, yqjF and ybiJ, responding to TNT and its relative compounds,2,4-dinitrotoluene(2,4-DNT) and 1,3-dinitrobenzene(1,3-DNB). These were used to construct a biosensor for the detection of TNT. In addition, a Japanese group [26] studied the mechanism whereby TNT induced a bacterial injury response (or SOS response), and utilized the key genes in the SOS response as TNT-responding elements (umu Test) to detect TNT in soils. However, the detection limits of the above methods are relatively high, and the reaction time for response to the toxic substances is relatively slow, ranging from 1 to 3 hours. Therefore, there is a significant need to develop more sensitive and timesaving methods for detecting TNT.Previous studies have shown that the utilization of a bacterial SOS response is useful for detection of TNT [26], and TNT is able to stimulate certain physiological pathways in bacteria [26,27]. Therefore, we hypothesized that construction of a bacterial biosensor for detection of TNT is feasible. We first examined the possible responding elements in a bacterial SOS promoter family for TNT. Among all TNT-responding elements, we further analyzed the sequences and constructed the random promoter mutant library, which we termed the Mutsos promoter mutant library. We also used the method of random chopped digested E. coli K-12 MG1655 genome constructed a genomic promoter library.From this library, we selected a new batch of promoter elements that were responding to TNT and its relative compounds. Then, a GFP reporter gene was fused to these genes, which allowed us to examine factors such as the response time, sensitivity, and specificity.