Genome Integration and Excision by a New Streptomyces Bacteriophage, ϕJoe

Bacteriophages are the source of many valuable tools for molecular biology and genetic manipulation. In , most DNA cloning vectors are based on serine integrase site-specific DNA recombination systems derived from phage. Because of their efficiency and simplicity, serine integrases are also used for diverse synthetic biology applications. Here, we present the genome of a new phage, ϕJoe, and investigate the conditions for integration and excision of the ϕJoe genome. ϕJoe belongs to the largest phage cluster (R4-like) and encodes a serine integrase. The site from was used efficiently by an integrating plasmid, pCMF92, constructed using the ϕJoe locus. The site for ϕJoe integrase was occupied in several genomes, including that of , by a mobile element that varies in gene content and size between host species. Serine integrases require a phage-encoded recombination directionality factor (RDF) to activate the excision reaction. The ϕJoe RDF was identified, and its function was confirmed Both the integrase and RDF were active in recombination assays. The ϕJoe site-specific recombination system is likely to be an important addition to the synthetic biology and genome engineering toolbox. spp. are prolific producers of secondary metabolites, including many clinically useful antibiotics. Bacteriophage-derived integrases are important tools for genetic engineering, as they enable integration of heterologous DNA into the chromosome with ease and high efficiency. Recently, researchers have been applying phage integrases for a variety of applications in synthetic biology, including rapid assembly of novel combinations of genes, biosensors, and biocomputing. An important requirement for optimal experimental design and predictability when using integrases, however, is the need for multiple enzymes with different specificities for their integration sites. In order to provide a broad platform of integrases, we identified and validated the integrase from a newly isolated phage, ϕJoe. ϕJoe integrase is active and The specific recognition site for integration is present in a wide range of different actinobacteria, including , an emerging model bacterium in research.