Abstract
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.
Highlights
Red homologous recombination has been widely used in gene knockout, mutation and replacement strategies in molecular biology research (Datsenko and Wanner, 2000; Ellis et al, 2001; Murphy, 1998; Muyrers et al, 2000; Yu et al, 2000)
*Corresponding author **Corresponding author cular or linear, the direct cloning method can be divided into two categories (Fu et al, 2012): LCHR and LLHR
Target DNA regions of approximately 30 kb, 3 kb fragments from plasmid BACs and genomic DNA fragments can be cloned by LCHR assay (Zhang et al, 2000)
Summary
Direct cloning and transplanting of large DNA fragments from Escherichia coli chromosome Ying Zhu, Yan Yang, Pingping Den, Yong Huang, Mengxiang Ni2* & Hongqing Fang1,3**. 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.
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