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题名: 基于yfp报告基因的砷抗性全细胞传感器的构建及表达
作者: 李丽
学位类别: 硕士
答辩日期: 2010
授予单位: 中国科学院研究生院
授予地点: 北京
导师: 庄国强
关键词: ; 报告基因 ; 黄色荧光蛋白(yfp) ; 全细胞传感器 ; 地下水
中文摘要:       环境中砷污染物的预测和评估是开展砷污染治理、环境保护、人类健康风险评估的重要前提。应用聚合酶链式反应(PCR)从大肠杆菌(Escherichia coli)DH5α基因组扩增得到砷抗性启动子及其调控蛋白arsR,并首次将一种新型黄色荧光蛋白yfp作为报告基因引入原核细菌构建出砷抗性全细胞传感器DH5α(ars-puc18-yfp),进而对工程菌的特性和表达特点进行研究。最后,对地下水组分于DH5α(ars-puc18-yfp)荧光表达的影响作了初步探索。研究得到了以下几点重要结论: 1. 采用As(Ⅲ)和As(Ⅴ)对工程菌株进行诱导。结果表明,DH5α(ars-puc18-yfp)能够很好的对砷盐应答,表达的黄色荧光呈现时间依赖性和剂量依赖性特点,并且经5 h诱导后,工程菌在0~0.8 μM的As(Ⅲ)标准溶液中呈线性应答。 2. 构建出砷抗性全细胞传感器DH5α (ars-puc18-gfp),并且比较了yfp和gfp这两种报告基因的时间依赖性表达。实验结果显示,yfp除了保留有gfp原有的优点(热稳定)外,还较好的改进了gfp信噪比低的缺点。 3. 通过比较基于两种不同SD序列(shine-delgarno sequence)的砷抗性全细胞传感器黄色荧光的时间依赖性表达,发现SD序列AAGGAG的应用可明显提高外源蛋白表达的信噪比。 4.分析了地下水样组分对DH5α(ars-puc18-gfp)检测砷的影响。结果表明,磷酸盐及硅酸盐对于工程菌检测砷污染物基本没有影响;而地下中含量较多的铁离子则会造成荧光产量的下降,因此,需要采用合适的络合剂来消除或最大限度的减轻重金属铁离子的干扰。此外,为保护地下水样组分稳定,在取样及保存样品时,应采用HNO3调节样本pH至2。
英文摘要:      Prediction and assessment about environmental arsenic pollution were the important preconditions of advocating to environmental protection and human health risk assessment. In this study, an arsenic-resistant promoter and the regulatory gene arsR were obtained by PCR from the genome of Escherichia coli DH5α, and yfp was introduced into E.coli DH5α as a reporter gene to construct an arsenic resistant whole cell biosensor DH5α (ars-puc18-yfp) in which phiYFP was expressed well for the first time. And then, the object narrowed down to the expression of the engineered construct. Finally, an introductory experiment was done to survey the effect of groundwater composition on the expression of DH5α (ars-puc18-yfp). Some key results were summarized as follows: 1. The induction of the sensor strain with As(Ⅲ) and As(Ⅴ) was evaluated. The results indicated that the sensor strain could clearly respond to arsenic, and the induction of yellow fluorescence of the DH5α (ars-puc18-yfp) strain showed time-dependent and dose-dependent. Moreover, the fluorescence production of DH5α (ars-puc18-yfp) was proportional to the arsenite concentration between 0 and 0.8 μM for 5 h induction. 2. The research was down to construct DH5α (ars-puc18-gfp) and compare the time-dependent expression of yfp and gfp. Our results demonstrated that yfp aiso could better improve the drawback of low signal-to-noise ratio apart from retaining the inherent merits of gfp (heat stability). 3. The fluorescence production of two SD sequence-based arsnic resistant biosensor strain was measured to investigate the effect of SD sequence on reporter gene. The results showed that the application of SD sequence AAGGAG could clearly increase the signal-to-noise ratio of heterologous protein for high expression. 4. Analysis the effect of groundwater composition on the expression of DH5α (ars-puc18-yfp). It was found that phosphate and silicate at concentrations naturally occurring in arsenic-contaminated groundwater had no effect arsenic measurement with DH5α (ars-puc18-yfp). However, iron generally reduced the sensor’s response to arsnite. Hence, to eliminate the negative influence of the disturbance of iron, some complexing agents were needed. Additionally, the groundwater samples should be acidified to a pH of about 2 by using HNO3, which could preserve the sample composition.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35031
Appears in Collections:中科院环境生物技术重点实验室_学位论文

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Recommended Citation:
李丽. 基于yfp报告基因的砷抗性全细胞传感器的构建及表达[D]. 北京. 中国科学院研究生院. 2010.
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