Ryu Young Shin, Biswas Rajesh Kumar, Shin Kwangsu, Parisutham Vinuselvi, Kim Suk Min, Lee Sung Kuk
School of Energy and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea.
School of Biological Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, Republic of Korea.
PLoS One. 2014 Apr 18;9(4):e94266. doi: 10.1371/journal.pone.0094266. eCollection 2014.
Multiplex genome engineering is a standalone recombineering tool for large-scale programming and accelerated evolution of cells. However, this advanced genome engineering technique has been limited to use in selected bacterial strains. We developed a simple and effective strain-independent method for effective genome engineering in Escherichia coli. The method involves introducing a suicide plasmid carrying the λ Red recombination system into the mutS gene. The suicide plasmid can be excised from the chromosome via selection in the absence of antibiotics, thus allowing transient inactivation of the mismatch repair system during genome engineering. In addition, we developed another suicide plasmid that enables integration of large DNA fragments into the lacZ genomic locus. These features enable this system to be applied in the exploitation of the benefits of genome engineering in synthetic biology, as well as the metabolic engineering of different strains of E. coli.
多重基因组工程是一种用于细胞大规模编程和加速进化的独立重组工程工具。然而，这种先进的基因组工程技术仅限于在选定的细菌菌株中使用。我们开发了一种简单有效的、不依赖菌株的方法，用于在大肠杆菌中进行有效的基因组工程。该方法包括将携带λ Red重组系统的自杀质粒导入mutS基因。通过在无抗生素条件下进行选择，自杀质粒可从染色体上切除，从而在基因组工程过程中使错配修复系统瞬时失活。此外，我们还开发了另一种自杀质粒，可使大DNA片段整合到lacZ基因组位点。这些特性使该系统能够应用于合成生物学中基因组工程优势的开发，以及不同大肠杆菌菌株的代谢工程。
