摘要
应用Gap-Repair新技术,以pBR322为载体,在λ噬菌体Red重组酶的作用下,通过同源重组直接从大肠杆菌DY330染色体上亚克隆了长度为6.7kb的包含Red重组酶基因的λ噬菌体左向操纵子基因序列。建立了一种能够随意在不同细菌宿主中转移的基于pBR32-2Red的重组工程系统。为了验证pBR32-2Red的生物功能,以大肠杆菌染色体上的galk基因为靶标,用Red介导的单链DNA重组技术敲入T→G单碱基突变,使galk基因内编码第145位氨基酸的密码子由TAT转变成TAG,产生了一个琥珀突变。确定了pBR322Red系统的重组功能。
Using lambda phage Red recombinase mediated in vivo homologous recombination system,a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique,and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria.To confirm the recombination functions of pBR322-Red,a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T→G mutation in galK gene on the bacterial chromosome.The result demonstrated that a new λ Red-mediated recombineering system based on pBR322-Red was successfully established.
基金
:军队"十五"医药卫生科学基金(编号:01MA089)~~
