Zhu Ying, Yang Yan, Den Pingping, Huang Yong, Ni Mengxiang, Fang Hongqing
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing, 100071, China.
Institute of Life Science and technology, China Pharmaceutical University, Nanjing, 210009, China.
Sci China Life Sci. 2016 Oct;59(10):1034-1041. doi: 10.1007/s11427-016-5100-z. Epub 2016 Oct 13.
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 (which has a homologous sequence with cmb) and cmb, each of which has no resistance separately unless the two parts are fused together. The cmb sequence was integrated into one flank of a target cloning region in the chromosome, and a linear vector containing the cma sequence was electroporated into the cells to directly capture the target region. Based on this strategy, we successfully cloned an approximately 48 kb DNA fragment from the E. coli DH1-Z chromosome with a positive frequency of approximately 80%. Combined with double-strand breakage-stimulated homologous recombination, we applied this strategy to successfully replace the corresponding region of the E. coli DH36 chromosome and knock out four non-essential genomic regions in one step. This strategy could provide a powerful tool for the heterologous expression of microbial natural product biosynthetic pathways for genome assembly and for the functional study of DNA sequences dozens of kilobases in length.
我们应用了一种抗性分裂-融合策略来提高由Red重组介导的体内直接克隆效率。氯霉素乙酰转移酶（cat）盒被分为两部分：cma（与cmb具有同源序列）和cmb，除非这两部分融合在一起，否则它们各自都没有抗性。cmb序列整合到染色体中目标克隆区域的一侧，将含有cma序列的线性载体电穿孔导入细胞以直接捕获目标区域。基于此策略，我们成功地从大肠杆菌DH1-Z染色体中克隆了一个约48 kb的DNA片段，阳性频率约为80%。结合双链断裂刺激的同源重组，我们应用此策略成功替换了大肠杆菌DH36染色体的相应区域，并一步敲除了四个非必需基因组区域。该策略可为微生物天然产物生物合成途径的异源表达、基因组组装以及数十千碱基长度的DNA序列的功能研究提供强大工具。
