改造thrL前导调控元件获取不同灵敏度苏氨酸感应器  

Transform thr L leading regulatory elements for different threonine sensor

作　　者：刘仲祥[1,2] 郑阳阳[2] 陈涛[1] 马红武[2] 

机构地区：[1]天津大学化工学院,天津300072 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《生物技术》2016年第4期372-379,共8页Biotechnology

基　　金：科技部973项目("基于网络分析的DNA模块设计与组装";No.2012CB725203);天津市重点专项("数据和模型相结合的生物产品代谢途径设计的研究";No.14ZCZDSY00060)

摘　　要：[目的]构建不同灵敏度的苏氨酸感应器,以报告基因的表达表征苏氨酸的变化。[方法]通过克隆苏氨酸操纵子thrL前导调控元件,并与红色荧光蛋白基因rfp偶联构建在高拷贝质粒p LX07上,转化进大肠杆菌DH5α;之后又通过设计引物进行PCR改变thrL前导调控元件上苏氨酸密码子的编码数量;最后进行荧光检测实验确定不同突变菌感应环境中苏氨酸特性的不同。[结果]得到了以红色荧光蛋白基因为报告基因而能感应苏氨酸的大肠杆菌以及分别编码4个和10个苏氨酸密码子的thrL突变菌。三种菌株单位OD下红色荧光蛋白的相对表达量均跨2个浓度梯度而负相关于苏氨酸浓度,同一浓度下正相关于苏氨酸密码子编码数量。[结论]通过构建感应苏氨酸大肠杆菌并改造其thrL前导调控元件获得了负相关于苏氨酸浓度且灵敏度不同的苏氨酸感应器。[ Objective] This study was to build different sensitivity of sensor response to threonine, to report the change of threonine by reporter gene expression. [ Methods ] thrL leading regulatory elements cloned from Escherichia coli was coupled with the red fluorescent protein gene rfp and was both constructed on a high copy plasmid pLX07. It was transformed into Escherichia coli DHSα, and then the encoding number of Thr codon on the thrL leading regulatory element was changed by designing prim- ers for PCR. The different characteristics of sensing threonine in environment between different mutant strains was finally detec- ted by fluorescence detection experiment. [ Results ] The Escherichia coli sensing threonine was constructed while red fluorescent protein gene was report gene. And the mutant strains of which thrL element encoded respectively four and ten thr codon were al- so constructed. Three strains of the relative expression of red fluorescence protein per OD were in negative correlation with con- centration of threonine all across two concentration gradient and positively related to the encoding number of thr codon under the same concentration. [ Conclusion ] The threonine sensors which were in negative correlation with threonine concentration and has different sensitivity were derived by constructing Escherichia coli could sense threonine and transforming its thrL leader reg- ulatory elements.

关 键 词：苏氨酸 thrL前导调控元件 密码子 红色荧光蛋白 灵敏度 

分 类 号：Q789[生物学—分子生物学]