中国科学院生态环境研究中心机构知识库
Advanced  
RCEES OpenIR  > 中国科学院环境生物技术重点实验室  > 学位论文
题名: ArsR蛋白质的定向进化提高砷全细胞传感器金属特异性
作者: 赵文娟
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: 齐鸿雁
关键词: 全细胞传感器 ; 定向进化 ; 易错PCR ; 高通量筛选 ; 同源建模
中文摘要:       随着工业化的不断推进,人为活动引起的重金属等污染物的危害日益严重,建立一个快速高效的重金属监测体系成为人们迫切的需要。传统的化学监测方法虽然准确且灵敏度高,但是昂贵的测试费用和复杂的操作技术使其应用受到了很大的局限。微生物全细胞传感器具有对单一污染物或多种污染物的感应作用,当目标污染物存在时会诱导体内的报告基因表达产生可测量的信号,从而达到检测的目的,具有低成本、原位检测、可实现生物有效性检测等优点,但也存在着一些如灵敏度较低、对检测底物特异性不强等缺点。 本论文选取了大肠杆菌砷全细胞传感器做为研究对象,针对砷全细胞传感器对同主族元素锑也有一定响应的问题,对其调控蛋白ArsR进行了定向进化以提高对砷检测的特异性。为了在不影响菌体活性的前提下进行流式细胞仪的高通量筛选,首先将质粒载体的报告基因虫荧光素酶Luc换成了发光稳定且不需破坏细胞结构就可以实现检测的增强型绿色蛋白EGFP,又采用易错PCR技术在arsR基因中引入低频的突变,构建突变文库。筛选策略采用流式细胞仪结合全波长多功能酶标仪进行高通量筛选。首先利用流式细胞仪将砷诱导细胞正向筛选,即选出正常响应菌株,再将正向筛选出的菌株用锑诱导进行反向筛选,即选出对锑响应弱的菌株。接着采用多功能全波长酶标仪对筛选出的菌株做进一步的验证,这种筛选的新方法为全细胞传感器的定向进化提供了新的平台和思路。 经过筛选得到一株正向突变M1,其对砷的特异性响应较野生型有了提高。为了从分子水平上解释特异性的提高,利用了同源建模技术对蛋白的三级结构进行了初步的模拟,同时结合文献和实验的结果在模拟的三级结构上对关键的高度保守位点和突变位点进行了定位,并做了尝试性的解释。为以后的理性设计打下基础,然而由于ArsR蛋白的X衍射和核磁共振成像结构还未见报道,故该蛋白的结构和功能的联系还有待于进一步研究。
英文摘要:       With continuous advance of industrialization, pollution of heavy metals and other pollutants caused by human activities are worsening.So it has become a pressing need to build a fast and efficient monitoring system. Traditional chemical monitoring methods are accurate and sensitive, but the high cost and complexity of operation make their application much restricted. Whole-cell biosensors have the ability to respond to single pollutant or multiple pollutants.The target contaminants existing in vivo will induce the expression of the reporter gene and then the whole-cell biosensor produces a measurable signal, thus achieving the purpose of detection.The new technology is low cost ,convenient ,and also can realize the bio-availability test, however, there are some disadvantages too, such as low sensitivity, low substrate specificity and so on. This thesis chose E. coli arsenic whole-cell biosensor for research, for it can also respond to antimony ,which is in the same main group of arsenic. ArsR regulator is key of metal selectivity , because ArsR protein can recogonize arsenic and enable the expression of reporter. So we decided to make directed evolution to this protein to improve selectivity of arsenic whole-cell biosensor In order to keep the activity of bacterial and make it also suitable for high-throughput screening with flow cytometry, the original reporter luciferase Luc was replaced by enhanced green fluorescence protein(EGFP). Error-prone PCR has brought some mutant sites to wild gene of arsR to construct a mutant library,and the mutation frequency was controlled low. We chose flow cytometry for the first round high-throughput screening and multi-wavelength microplate reader for sencond screening. Mutants responded to arsenic as the wild type were selected by flow cytometry in first round positive screening.Then such mutants were induced by antimony and the ones could not emit fluorescence or emit low fluorescence were selected in the second reverse screening. Multi-wavelength microplate reader was used for further verification, and the mutants with higher selectivity were selected and identified finally. This new screening method provides a new platform and ideas for the directed evolution of whole-cell sensor. M1, a mutant with improved selectivity, was identified. In order to explain the relationship between specific construction on molecular level and metal selectivity ,the tertiary structure of ArsR monomer was obtained by homology modeling. Highly conserved points and mutation sites were localized, some tentative explanation has also been given ,which may lay the foundation for the future rational design and research. However, no x-ray or NMR structures of ArsR have been reported to date. Furthermore,only some putative and designed models are reported.So the regulatory mechanism on the molecular level was not clear and needed further research.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/35014
Appears in Collections:中科院环境生物技术重点实验室_学位论文

Files in This Item:
File Name/ File Size Content Type Version Access License
ArsR蛋白质的定向进化提高砷全细胞传感器金属特异性.pdf(1540KB)学位论文--限制开放 联系获取全文

Recommended Citation:
赵文娟. ArsR蛋白质的定向进化提高砷全细胞传感器金属特异性[D]. 北京. 中国科学院研究生院. 2010.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[赵文娟]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[赵文娟]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2018  中国科学院生态环境研究中心 - Feedback
Powered by CSpace