title: Actinophage R4 integrase-based site-specific chromosomal integration of non-replicative closed circular DNA.
abstract: The actinophage R4 integrase (Sre)-based molecular genetic engineering system was developed for the chromosomal integration of multiple genes in Escherichia coli. A cloned DNA fragment containing two attP sites, green fluorescent protein (gfp) as a first transgene, and an antibiotic resistance gene as a selection marker was self-ligated to generate non-replicative closed circular DNA (nrccDNA) for integration. nrccDNA was introduced into attB-inserted E. coli cells harboring the plasmid expressing Sre by electroporation. The expressed Sre catalyzed site-specific integration between one of the two attP sites on nrccDNA and the attB site on the E. coli chromosome. The integration frequency was affected by the chromosomal location of the target site. A second nrccDNA containing two attB sites, lacZα encoding the alpha fragment of β-galactosidase as a transgene, and another antibiotic resistance gene was integrated into the residual attP site on the gfp-integrated E. coli chromosome via one of the two attB sites according to reiterating site-specific recombination. The integrants clearly exhibited β-galactosidase activity and green fluorescence, suggesting the simultaneous expression of multiple recombinant proteins in E. coli. The results of the present study showed that a step-by-step integration procedure using nrccDNA achieved the chromosomal integration of multiple genes.
authors: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.|Department of Bioresource Science, Ibaraki University College of Agriculture, Ibaraki, Japan.
authors: Takamasa
authors: T
authors: Miura
authors: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.|Department of Bioresource Science, Ibaraki University College of Agriculture, Ibaraki, Japan.
authors: Akito
authors: A
authors: Nishizawa
authors: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.|Department of Bioresource Science, Ibaraki University College of Agriculture, Ibaraki, Japan.
authors: Tomoyasu
authors: T
authors: Nishizawa
authors: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.|Department of Bioresource Science, Ibaraki University College of Agriculture, Ibaraki, Japan.
authors: Munehiko
authors: M
authors: Asayama
authors: United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Tokyo, Japan.|Department of Bioresource Science, Ibaraki University College of Agriculture, Ibaraki, Japan.
authors: Makoto
authors: M
authors: Shirai
mesh_terms: D001287:Attachment Sites, Microbiological / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D007763:Lac Operon / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D016662:Virus Integration / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D004270:DNA, Circular; D049452:Green Fluorescent Proteins; D019426:Integrases; D001616:beta-Galactosidase
keywords: Actinophage R4; Genome engineering; Integrase; Non-replicative closed circular DNA; Site-specific recombination