A novel approach for T7 bacteriophage genome integration of exogenous DNA.

ABSTRACT: Background:The comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ?C31 integrase is one of the most robust site-specific recombination systems. ?C31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ?C31 integrase. Methods:Plasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7?G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7?G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7?G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths. Results:T7?G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ?C31 integrase (attB) was confirmed to be present in the T7?G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7?G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7?G10G11-attB was explored, and it's found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably. Conclusion:We advanced and established a novel approach for gene knock-in into the T7 genome using ?C31 integrase.

SUBMITTER: Liu Y 

PROVIDER: S-EPMC6966851 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature