本文關(guān)鍵詞：構(gòu)建隨機(jī)突變文庫篩選敏感生物元件實(shí)現(xiàn)TNT的檢測　出處：《安徽大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 合成生物學(xué) 2 4 6-三硝基甲苯 隨機(jī)突變文庫 生物元件 生物傳感器

【摘要】：合成生物學(xué)是21世紀(jì)新出現(xiàn)的一門交叉學(xué)科,它將工程化的思想運(yùn)用到系統(tǒng)生物學(xué)研究中,為解決人類在醫(yī)療、環(huán)境及能源等方面面臨的難題提供了新技術(shù)、新思路。目前基于合成生物學(xué)技術(shù)構(gòu)建的生物傳感器在環(huán)境污染物檢測領(lǐng)域表現(xiàn)出了良好的發(fā)展勢頭。有特定感應(yīng)功能的生物識別元件和可將信號進(jìn)行放大輸出的報(bào)告元件構(gòu)成了生物傳感器的核心。其中的生物識別元件通過感應(yīng)并結(jié)合被測物質(zhì)后,引發(fā)自身的生物學(xué)反應(yīng),進(jìn)而導(dǎo)致下游報(bào)告元件的信號表達(dá),信號強(qiáng)度代表生物元件對靶標(biāo)化合物的感應(yīng)強(qiáng)度。目前報(bào)告元件的研究相對成熟,而感應(yīng)環(huán)境中靶物質(zhì)的生物識別元件還有待大量挖掘、鑒定和優(yōu)化,它是生物傳感器選擇性測定的基礎(chǔ)。2,4,6-三硝基甲苯(TNT)是一種帶苯環(huán)的有機(jī)化合物,因其良好的爆炸性能,常被用來制造地雷以及各種炸藥。地雷的廣泛使用以及TNT的長效性,導(dǎo)致戰(zhàn)爭遺留下來的地雷每年都在造成上百人的傷亡,嚴(yán)重威脅著人們的生命安全。同時(shí)泄露在環(huán)境中的TNT還是一種重要的環(huán)境污染物,不僅可以大面積地污染土壤和水域,產(chǎn)生極難清理和凈化的"粉紅水",而且TNT及其降解物還可以進(jìn)入食物鏈,對生態(tài)環(huán)境和人類健康都造成了極大威脅,因此,研發(fā)高效、準(zhǔn)確的檢測TNT的工具,對戰(zhàn)后雷場清除以及環(huán)境治理都有重要意義。目前的檢測方法大都需要昂貴的儀器以及專業(yè)的操作流程,在實(shí)際應(yīng)用中會受到很大的限制。由于生物傳感器具有成本低、敏感度高、易攜帶及易操作等優(yōu)點(diǎn),因此可作為傳統(tǒng)方法的輔助手段,用于TNT的檢測。本文在合成生物學(xué)思想的指導(dǎo)下,大量挖掘、鑒定感應(yīng)TNT的生物元件,為生物傳感器的構(gòu)建提供元件儲備。研究內(nèi)容主要包括三個(gè)方面:(1)構(gòu)建嚴(yán)謹(jǐn)型的篩選平臺根據(jù)差異熒光誘導(dǎo)技術(shù)的原理,進(jìn)行生物元件的篩選,首先需要構(gòu)建良好的篩選平臺。pPROBE-TT是無啟動子、帶有綠色熒光蛋白(GFP)、進(jìn)行高拷貝復(fù)制的載體,我們用大腸桿菌的低拷貝復(fù)制子pSC101 ori替換掉pPROBE-TT載體上的pBBR1 oriV的高拷貝復(fù)制子,構(gòu)建低拷貝嚴(yán)謹(jǐn)型復(fù)制的篩選載體101-pPROBE。將用于指示TNT的生物元件插入GFP上游的多克隆(MCS)位點(diǎn),GFP的熒光強(qiáng)度即可指征生物元件對TNT的感應(yīng)強(qiáng)度。(2)構(gòu)建隨機(jī)啟動子文庫篩選DNA元件我們采用啟動子非保守序列隨機(jī)化的方法構(gòu)建隨機(jī)啟動子文庫,通過分析啟動子的一致序列,保留-35區(qū)及-10區(qū),其余部分進(jìn)行完全隨機(jī)合成,退火后連入構(gòu)建好的嚴(yán)謹(jǐn)型低拷貝篩選載體101-pPROBE內(nèi)。通過對文庫進(jìn)行篩選,獲得了一個(gè)啟動元件5M6A,其對TNT及其靶標(biāo)化合物表現(xiàn)出了較高的感應(yīng)強(qiáng)度、靈敏度及特異性。(3)構(gòu)建Xy1R突變文庫篩選蛋白元件惡臭假單胞mt-2菌株中的TOL質(zhì)粒上攜帶的Xy1R-Pu是經(jīng)典的甲苯代謝通路,在甲苯類化合物存在時(shí),調(diào)控蛋白Xy1R可以特異性地激活Pu啟動子,進(jìn)而啟動相應(yīng)甲苯代謝通路的表達(dá)。本文基于合成生物學(xué)的思想,優(yōu)化設(shè)計(jì)該通路并導(dǎo)入遺傳背景清楚、操作簡單的大腸桿菌中構(gòu)建全細(xì)胞生物傳感器,用于檢測TNT。我們以pETDuet-1為載體骨架,構(gòu)建了 Xy1R-Pu基因線路,并以GFP為報(bào)告分子,GFP的熒光值可以指征結(jié)合TNT后的Xy1R蛋白對Pu啟動子的誘導(dǎo)強(qiáng)度,并在基因線路中加入四串聯(lián)終止序列來降低背景值。最后對Xy1R蛋白的信號識別區(qū)進(jìn)行連續(xù)易錯(cuò)PCR,構(gòu)建隨機(jī)突變文庫。實(shí)驗(yàn)表明四串聯(lián)終止序列可有效降低Xy1R-Pu通路的背景值,并從隨機(jī)突變體文庫中篩選出的蛋白元件eX0-4,對TNT表現(xiàn)出良好的感應(yīng)強(qiáng)度、靈敏度以及特異性。綜上所述,我們通過采用啟動子非保守序列隨機(jī)化和易錯(cuò)PCR的方法構(gòu)建隨機(jī)突變文庫,獲得了兩個(gè)全新的對TNT及其衍生物有高感應(yīng)強(qiáng)度、靈敏度及特異性的生物元件,而且其感應(yīng)靈敏度均達(dá)到了國際上報(bào)道同類元件的領(lǐng)先水平。獲得的元件為后期成型生物傳感器的完整開發(fā)提供了良好的元件儲備,我們構(gòu)建的啟動子文庫也有望成為檢測各類環(huán)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.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78

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