苏氨酸操纵子的克隆表达及天冬氨酸激酶的测活

依据途径工程的基本原理，为构建大肠杆菌苏氨酸高产菌林，需强化表达苏氨酸生物合成途径中的关键基因。以Ecoli MC4100染色体DNA为模板，用PCR扩增了天冬氨酸激酶基因thrA部分片段，以之为探针从基因文库中克隆得到thr操纵子，包括启动子、结构基因(thrA、thrB、thrC)、终止子。将结构基因装载于pET-lla质粒上的17启动子下游，得到了表达质粒pTHlla-08。转化E.coli BL21(DE)，经异丙基硫代半乳糖苷(IPTG)诱导，获得了高效的表达。含表达质粒的菌体胞内粗提液经酶活分析表明，天冬氨酸激酶的活性为含载体质粒pET-lla的对照菌的100倍。

Cloning and Expression of the thr Operon and the Detection of Aspartokinase I Activity

In order to construct a Escherochia coli strain with high production of threonine, we improve the expression of some key enzymes in the biosynthetic pathway of threonine through genetic engineering technology. In this experiment, part of thr A expressing aspartokinase I (AKI) was cloned by PCR technology with the template of MC4100 DNA. The thr A operon consisting promoter, structure gene ( thr A, thr B, thr C), and terminator was cloned by shot gun technology from the gene bank. pTH lla 08 express cosmid was gotten by subcloning the structure gene to the down stream of T7 promoter of pET lla. Then transform this recombinant cosmid into BL21 (DE3) strain to express effectively after the induce by IPTG. The result, gotten by the analysis of the sonic extracts of the Escherochia coli containing the express cosmid, shows 100 higher the enzyme activity of the express cosmid to pET lla.