Chiang Chung-Jen, Chen Po Ting, Chen Shan-Yu, Chao Yun-Peng
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan.
Methods Mol Biol. 2011;765:113-23. doi: 10.1007/978-1-61779-197-0_8.
Genetic manipulation of Escherichia coli strains for desired traits is the most applied strain engineering approach in industrial applications. For chromosomal insertion of genes and controlled expression of genomic genes in E. coli, the replicon-free and markerless method is described based on a series of conditional-replication plasmids called phage-integration vectors. They mainly carry the multiple cloning site and the prophage attachment site, which are sandwiched by two FRT sites. With the aid of the phage integrase from conditional-replication helper plasmids, the passenger genes of either foreign or native type incorporated into the integration vectors can be specifically integrated into bacterial genome at the prophage attachment site. Finally, the inserted DNA containing replicon and/or selective markers in integrants can be eliminated by the act of the FLP recombinase provided from a conditional-replication helper plasmid.
为获得所需特性而对大肠杆菌菌株进行基因操作是工业应用中最常用的菌株工程方法。对于在大肠杆菌中进行基因的染色体插入和基因组基因的可控表达，基于一系列称为噬菌体整合载体的条件复制质粒，描述了无复制子和无标记方法。它们主要携带多克隆位点和原噬菌体附着位点，这两个位点被两个FRT位点夹在中间。借助来自条件复制辅助质粒的噬菌体整合酶，整合到整合载体中的外源或内源类型的过客基因可以在原噬菌体附着位点特异性整合到细菌基因组中。最后，整合体中含有复制子和/或选择标记的插入DNA可以通过条件复制辅助质粒提供的FLP重组酶的作用而消除。
