Love Aaron M, Biggs Bradley W, Tyo Keith E J, Ajikumar Parayil Kumaran
Manus Bio, Cambridge, MA, USA.
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Methods Mol Biol. 2019;1927:37-45. doi: 10.1007/978-1-4939-9142-6_4.
Chemically inducible chromosomal evolution (CIChE) was developed for stable multicopy chromosomal integration of heterologous genes. In this technique, flanking an antibiotic selection marker and a gene of interest with identical regions of homology permits gene duplication via recA mediated homologous recombination. A strong selective pressure for gene duplication can be applied by increasing antibiotic concentration, and in a week's time one can create a set of strains with a wide range of cassette copy numbers (upward of 20×), which can be made stable by deletion of recA. Herein, we describe a generalized workflow for this methodology.
化学诱导染色体进化（CIChE）技术是为实现异源基因的稳定多拷贝染色体整合而开发的。在该技术中，利用同源性相同的区域将抗生素选择标记和目的基因两侧进行侧翼连接，可通过recA介导的同源重组实现基因复制。通过提高抗生素浓度可对基因复制施加强大的选择压力，在一周时间内就能创建一组具有广泛盒式拷贝数（超过20倍）的菌株，通过删除recA可使其稳定。在此，我们描述了该方法的通用工作流程。
