染色体上大片段DNA的直接克隆及移植

Direct Cloning and Transplanting of Large DNA Fragments from Escherichia coli Chromosome

应用Red重组体内直接克隆染色体上大于10 kb的DNA序列非常困难.利用一种抗性拆分-融合策略成功地从供体菌染色体上直接克隆了约50 kb的DNA序列,并将其移植到受体菌的染色体上.首先将抗性基因(如cat)的互补片段cmb定向整合到染色体上拟克隆区域的一侧,然后将含有抗性基因另一互补片段cma的线性质粒载体电转到细胞内进行定向捕获,通过抗性筛选获得克隆.使用该方法,成功地将供体菌DH1-Z基因组上约48kb的大片段DNA捕获至质粒载体上,捕获阳性率达80%.然后,将该质粒转化到受体菌DH36,利用双链断裂促进同源重组策略,替换受体菌染色体上对应区域,一次敲除基因组上4个非必需区,获得重组菌DH40.该方法也可用于微生物天然产物生物合成途径的异源表达、基因组组装和大片段DNA的功能研究.

We applied a resistance split-fusion strategy to increase the in vivo direct cloning efficiency mediated by Red recombination. The cat cassette was divided into two parts： cma and cmb（has a homologous sequence with cma）. Each of them has no resistance separately unless the two parts are fused together. The cmb fragment was first integrated into a side of the target cloning region, then a linear cma-containing plasmid vector was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned a DNA fragment about 48 kb from E. coli DH1-Z chromosome with positive frequency of about 80%. Then, combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to replace the corresponding region of E. coli DH36 chromosome and knock out four nonessential genomic regions at one time. This strategy provides a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways, for genome assembly and for the function study of large DNA sequence.