Research Of Landmine Detection Methods Based On Synthetic Biology

Landmine is a common type of highly hidden defensive firearm containing high explosives. The use of landmines was recorded early in the year of 1130. Since high explosive 2,4,6-trinitrotoluene was discovered in the mid-19 th century, it has promoted the regularization and diversification of landmines. Landmines are still being widely used today as cheap and efficient defensive weapons.The application of landminesplayed a very important role in facilitating modern warfare evolution. The two advantageous characteristics of landmine such as being highly hidden and being highly destructive make it nearly favored by all the previous modern warfare, so how to locate the landmines on the battle field safely and effectively is the problem all the warring parties must solve. In addition, the killing power of landmines to effective strength does not merely exist in the course of the war, the long-lasting and destruction of landmines would not disappear with the end of the war as well. Many landmines were left behind in the battlefield undetonated or excluded, which still threaten the lives of local people. So the safe and effective detection method for landmines has been widely studied by military experts across the world.There are some traditional methods of mine detection, such as biological detection, metal detection, acoustic detection, radar, etc. All these traditional means have certain limitations. The biosensor detection is based on animal sensory response to odor. Due to animal’s uncontrollable thinking, this method is of much uncertainty; metal, acoustic and radar detection, based on subjective judgments, requires the operators to step into the minefield with great risk, and cannot detect some new landmine materials like ceramic, plastic. Based on these issues, mine detection method using synthetic biology emerged. This methods can target the explosion compound of mines to avoid the mines material issues, the use of bacteria colony detection can effectively avoid the impact of individual subjective differences in mine detection. It can be a complementary method for the traditional methods, thus it helps to improve the accuracy and safety of landmine detection.Synthetic biology, founded on life science, is a new multidisciplinary subject, which started to develop in the 20 th century. At present, synthetic biology can be divided into the following three levels: First, normalize and modulate the known components in nature and thenassemble them from top to bottom step by step to construct a new gene circuit and life system; second, with the development of gene synthesis technology, through artificial design and synthesis, assemble new genome in order to reconstruct the living body; the third level is to create a complete new life system, and even artificial life based on the first two methods mentioned above.The construction of the Whole-cell biosensors with the assembly of the sensing component and the report component develops from the first level of synthetic biology. We can acquire the information of the concentration and the location of the target compounds through analyzing the signal data of the report component.This study intends to construct a whole-cell mine detection biosensor, mainly composed of the sensing elements and the reporting components. Nowadays, the development of report element is relatively mature, which mainly includes lux CDABE, GFP, and lac Z, etc. Our main work is to find sensing elements existing in nature and then improve their promoter activity via some analysis and alteration, or build up some gene circuits with some reported TNT sensing elements, and then construct a module for bacteria self-cleavage. Finally we expect to achieve the construction of a whole biosensor.The major results of my study are as follows:(1)Explore the natural TNT sensing elementChoose Escherichia coli to start the exploration of TNT natural sensing element since the genetic manipulation of Escherichia coli is of the most clearness. In order to facilitate the promoter library construction and screening, we first constructed a platform named p TJH629, which can be used as promoter screening, with the introduction of a low copy of E. coli replicon, p SC101 ori.Through extracting and purifying E. coli K-12 MG1655 genome, and incompletely cutting them with SAU3 AI endonuclease, we successfully built up the E. coli genome promoter library with lux CDABE chemiluminescthence as report means. After two rounds of screening of both high and low concentrations of TNT in the library, five promoter elements with a certain sensitivity and specificity were acquired. We further analyzed the specificity, sensitivity, timeliness of the five elements and found out that they had an excellent promoter activity.Further analysis of the five components has been done to identify the functional sequences. We firstly found the location in the E.coli genome through BLAST with the genome sequence, and then we found the known promoters through the comparison with the E.coli database and discovered the potential promoters by utilizing the promoter online prediction. We used the laboratory methods to find whether the promoters sensing TNT are sensitive or not. Finally we identified two minimal functioning sequence top Ap4 and rec EA4. The minimal functioning sequence of other elements are still been studied.(2)Contruct and screen an artificial artificial promoter library based on the stress and injury response family.By screening TNT sensing elements in the genome promoter library, we found out that top Ap4 and rec EA4 belong to the bacterial SOS family. Combined with the literature research we discovered that the family emergency trauma of the bacteria may be subject to the induced reaction of TNT. Therefore, SOS family gene promoters were further investigated, and some of them were found to be induced by TNT significantly. The promoters obtained by screening from SOS family were analyzed for their sequence construction, from which the common sequence of the promoters(sul A, rec A, umu DC) were found. Then we extracted its conserved region sequences and conducted the random mutation of its variable region so as to construct the SOS promoter library of random mutations. We screened out 5 elements in the library, which may be subject to TNT induction. Further promoter activity study showed that these five elements have a significant improvement on its sensitivity on TNT compared to the wild-type SOS promoters with the same specificity and timeliness.(3)Construct a 2,4-DNT detection circuit Xyl R5-Pu-GFP in Pseudomonas putidaTNT can be used as a nitrogen source in Pseudomonas putida mt-2 nitro physiological metabolism, so there must be some regulatory elements induced by TNT in Pseudomonas putida. With the literature survey we found out that the key factors for its main function is the Xyl R-Pu regulatory circuit in Pseudomonas putida mt-2’s TOL plasmid. Xyl R protein, as the key regulatory factor in toluene metabolic pathways in P. putida, can be regulated by the toluene and its derivative compounds in order to activate the upstream promoter Pu. According to this regulation principle, we used the Xyl R mutant Xyl R5, reported as a 2,4-DNT specific mutant as the regulator, GFP as the reporter to construct Xyl R-Pu-GFP gene regulatory circuits. Then the recombinant engineering method was applied to convert the circuit into the genome of Pseudomonas putida, and knock out the resistance gene through Cre-lox P directional knockout technology. At the same time we established a plasmid platform p SN129. Therefore, we can achieve different function of the circuits through the replacements of the components in it.(4)Construct a arabinose-induced self-lysis moduleIn order to make the engineered bacteria cause no pollution to the environment and leave no residue, we also constructed a post-test self-lysis module. Through literature research we found out that the T4 phage perforin protein Holin and T4 lysozyme can be employed in conjunction to achieve the bacterial suicide. So based on this, we built up a p BAD-Holin-Lysozyme-Anti Holin suicide circuit. By introducing Holin antagonistic protein Anti Holin, we could make the strains containing suicide expression module grow normally, meanwhile we could control the expression intensity of the Anti-Holin by replacing the promoters to regulate the onset time and keep the speed of the suicide under control.In summary, in this study we used E.coli genome as a template to build the genomic promoter library, from which we screened out 5 TNT-induced natural promoters, whose promoter activity reached or exceeded the international reporting elements level. Combining the screened results with the literature research, we discovered some TNT-induced SOS promoters. Through the sequence analysis of these promoters, we constructed a SOS promoter artificial library, from which we screened out five completely new SOS promoters with a great improvement in sensitivity for TNT. With the application of the Xyl R-Pu circuits, we constructed a Xyl R5-Pu-GFP gene circuit which is introduced into P.putida genome, then we kicked out the resistance gene, and we also built up a genetic circuit platform p SN129, easy to change the function by replacing the components in it. By using the performing principle of Holin by T4 bacteriophage proteins and the perforin protein, a self-arabinose-induced bacterial lysis line was designed as a result. In conclusion, this study intends to construct a target for TNT mine detection biosensor and provide more sensing elements and modules, thus laying a solid foundation for the subsequent complete construction of the biosensor basis.