构建随机突变文库筛选敏感生物元件实现TNT的检测

发布时间：2017-12-27 18:41

本文关键词：构建随机突变文库筛选敏感生物元件实现TNT的检测　出处：《安徽大学》2017年硕士论文　论文类型：学位论文

【摘要】：合成生物学是21世纪新出现的一门交叉学科,它将工程化的思想运用到系统生物学研究中,为解决人类在医疗、环境及能源等方面面临的难题提供了新技术、新思路。目前基于合成生物学技术构建的生物传感器在环境污染物检测领域表现出了良好的发展势头。有特定感应功能的生物识别元件和可将信号进行放大输出的报告元件构成了生物传感器的核心。其中的生物识别元件通过感应并结合被测物质后,引发自身的生物学反应,进而导致下游报告元件的信号表达,信号强度代表生物元件对靶标化合物的感应强度。目前报告元件的研究相对成熟,而感应环境中靶物质的生物识别元件还有待大量挖掘、鉴定和优化,它是生物传感器选择性测定的基础。2,4,6-三硝基甲苯(TNT)是一种带苯环的有机化合物,因其良好的爆炸性能,常被用来制造地雷以及各种炸药。地雷的广泛使用以及TNT的长效性,导致战争遗留下来的地雷每年都在造成上百人的伤亡,严重威胁着人们的生命安全。同时泄露在环境中的TNT还是一种重要的环境污染物,不仅可以大面积地污染土壤和水域,产生极难清理和净化的"粉红水",而且TNT及其降解物还可以进入食物链,对生态环境和人类健康都造成了极大威胁,因此,研发高效、准确的检测TNT的工具,对战后雷场清除以及环境治理都有重要意义。目前的检测方法大都需要昂贵的仪器以及专业的操作流程,在实际应用中会受到很大的限制。由于生物传感器具有成本低、敏感度高、易携带及易操作等优点,因此可作为传统方法的辅助手段,用于TNT的检测。本文在合成生物学思想的指导下,大量挖掘、鉴定感应TNT的生物元件,为生物传感器的构建提供元件储备。研究内容主要包括三个方面:(1)构建严谨型的筛选平台根据差异荧光诱导技术的原理,进行生物元件的筛选,首先需要构建良好的筛选平台。pPROBE-TT是无启动子、带有绿色荧光蛋白(GFP)、进行高拷贝复制的载体,我们用大肠杆菌的低拷贝复制子pSC101 ori替换掉pPROBE-TT载体上的pBBR1 oriV的高拷贝复制子,构建低拷贝严谨型复制的筛选载体101-pPROBE。将用于指示TNT的生物元件插入GFP上游的多克隆(MCS)位点,GFP的荧光强度即可指征生物元件对TNT的感应强度。(2)构建随机启动子文库筛选DNA元件我们采用启动子非保守序列随机化的方法构建随机启动子文库,通过分析启动子的一致序列,保留-35区及-10区,其余部分进行完全随机合成,退火后连入构建好的严谨型低拷贝筛选载体101-pPROBE内。通过对文库进行筛选,获得了一个启动元件5M6A,其对TNT及其靶标化合物表现出了较高的感应强度、灵敏度及特异性。(3)构建Xy1R突变文库筛选蛋白元件恶臭假单胞mt-2菌株中的TOL质粒上携带的Xy1R-Pu是经典的甲苯代谢通路,在甲苯类化合物存在时,调控蛋白Xy1R可以特异性地激活Pu启动子,进而启动相应甲苯代谢通路的表达。本文基于合成生物学的思想,优化设计该通路并导入遗传背景清楚、操作简单的大肠杆菌中构建全细胞生物传感器,用于检测TNT。我们以pETDuet-1为载体骨架,构建了 Xy1R-Pu基因线路,并以GFP为报告分子,GFP的荧光值可以指征结合TNT后的Xy1R蛋白对Pu启动子的诱导强度,并在基因线路中加入四串联终止序列来降低背景值。最后对Xy1R蛋白的信号识别区进行连续易错PCR,构建随机突变文库。实验表明四串联终止序列可有效降低Xy1R-Pu通路的背景值,并从随机突变体文库中筛选出的蛋白元件eX0-4,对TNT表现出良好的感应强度、灵敏度以及特异性。综上所述,我们通过采用启动子非保守序列随机化和易错PCR的方法构建随机突变文库,获得了两个全新的对TNT及其衍生物有高感应强度、灵敏度及特异性的生物元件,而且其感应灵敏度均达到了国际上报道同类元件的领先水平。获得的元件为后期成型生物传感器的完整开发提供了良好的元件储备,我们构建的启动子文库也有望成为检测各类环境污染物的工具。
[Abstract]:Synthetic biology is a new interdisciplinary subject emerging in the twenty-first Century. It applies the idea of engineering to the research of systems biology, and provides new technologies and new ideas for solving the problems faced by human beings in the aspects of health, environment and energy. At present, biosensors based on synthetic biological technology have shown good momentum in the field of environmental pollutants detection. A biometric element with a specific induction function and a report element that can amplify the output of the signal constitute the core of the biosensor. The biometrics element triggering its own biological response after induction and binding with the tested substance, and then leads to the signal expression of downstream report components, and signal intensity represents the biometric strength of biological components. At present, the research of reporting components is relatively mature, and the biometric elements of target materials in the induction environment need to be excavated, identified and optimized. It is the basis for selective determination of biosensors. 2,4,6- three nitrotoluene (TNT) is a kind of organic compound with benzene ring. Because of its good explosive properties, it is often used to make mines and various explosives. The widespread use of landmines and the long-term effectiveness of TNT cause the landmines left by the war to cause hundreds of casualties and threaten people's lives and safety every year. At the same time, leakage in the environment of TNT is a kind of important environmental pollutants, not only a large area of contaminated soil and water, which is extremely difficult to clean and purify the "pink water", and TNT and its degradation products can also enter the food chain to the ecological environment and human health caused great threat, therefore, the detection of TNT development of an efficient and accurate tool, has the important meaning to the minefield clearance and environmental governance after the war. Most of the current detection methods need expensive instruments and professional operation processes, which will be greatly restricted in practical applications. Because the biosensor has the advantages of low cost, high sensitivity, easy to carry and easy to operate, it can be used as an auxiliary means of traditional methods for the detection of TNT. Under the guidance of the thought of synthetic biology, this paper excavate and identify the biological components of induction TNT, and provide component storage for the construction of biosensors. The research contents mainly include three aspects: (1) build a rigorous screening platform based on the principle of differential fluorescence induction, and select biological elements. First, we need to build a good screening platform. PPROBE-TT is a promoter, a green fluorescent protein (GFP), high copy vector, we use E.coli low copy replicon pSC101 ori replace pPROBE-TT vector pBBR1 oriV high copy replicon construct low copy screening stringent replication carrier 101-pPROBE. The biological components used to indicate TNT are inserted into the polyclonal (MCS) loci in the upstream of GFP, and the fluorescence intensity of GFP can indicate the induction intensity of biological elements to TNT. (2) the construction method of random promoter library screening DNA promoter element we use non conservative sequence randomization to construct a random promoter library, through sequence analysis of the promoter, retention of -35 and -10 regions, the rest were completely random synthesis, even after annealing in the constructed type strict low copy vector 101-pPROBE screening. Through screening the library, a promoter 5M6A was obtained, which showed high sensitivity, sensitivity and specificity to TNT and its target compounds. (3) to construct Xy1R mutant library screening with TOL plasmid protein components of Pseudomonas putida strain MT-2 in Xy1R-Pu on the metabolic pathway of toluene in the presence of the classic Methylbenzenes, regulatory protein Xy1R can specifically activate the Pu promoter, and then start the expression of the corresponding metabolic pathway of toluene. Based on the idea of synthetic biology, we optimize the design of this pathway and introduce a whole cell biosensor to detect TNT in Escherichia coli with clear background and simple operation. We constructed the Xy1R-Pu gene line with pETDuet-1 as the carrier skeleton, and took GFP as the reporting molecule. The fluorescence value of GFP could be combined with the Xy1R protein induced by TNT to induce the Pu promoter, and the four sequence termination sequence was added to the gene line to reduce the background value. At the end of the Xy1R protein signal recognition area for error prone PCR, random mutant library construction. Experiments show that four tandem termination sequences can effectively reduce the background value of the Xy1R-Pu pathway, and the protein component eX0-4 selected from the random mutant library shows a good sensitivity, sensitivity and specificity to TNT. In summary, we by promoter non conserved sequence of randomization and error prone PCR construct random mutant library, obtained two new to TNT and its derivatives with biological components with high induction strength, sensitivity and specificity, and the induction sensitivity has reached the international advanced level of reported similar elements. The acquired components provide good element storage for the complete development of post formed biosensors. Our promoter library is also expected to become a tool for detecting various environmental pollutants.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

【参考文献】