Recombineering mediated by bacteriophage recombinases

Abstract:

DNA manipulation tools derived from bacteriophage, such as site-specific recombinases, cosmid libraries, and homologous recombinases, play an important role in genetic engineering. The homologous recombinases Redα/Redβ from Lambda phage or RecE/RecT from Rac prophage can efficiently mediate the recombination between homologous arms as short as 35-50 bp. Recombineering based on phage homologous recombinases Redα/Redβ and RecE/RecT can be used to precisely and efficiently modify target DNA molecules without limitation of endonuclease recognition sites and the size of DNA molecule, which has been developed into a new genetic engineering technology. This article reviews the phage homologous recombinases and their functional mechanism, their application and development in Escherichia coli and other bacteria. The broad use of recombineering has been demonstrated in the mining of microbial secondary metabolites, animal and plant transgenes, and viral genome cloning and modifications. In situ activation of a silent gene cluster accomplished by promoter engineering requires host specific recombineering system. The initial step of heterologous expression of secondary metabolites is direct cloning of large DNA fragment, which is based on RecET. Construction of large transgenic vector for both animal and plant mainly rely on Red recombineering. Generation and modification of infectious clone in E. coli mediated by RecET direct cloning and Red recombineering is the most efficient method for functional research of viral genome and vector vaccine development.

Key words:bacteriophage homologous recombinases;recombineering;red system;RecET system;secondary metabolites;animal and plant transgenesis;virus reverse genetics