A novel one-step method for targeted multiplication of DNA fragments from the Escherichia coli chromosome mediated by coordinated functioning of λ and φ80 bacteriophage recombination systems

Abstract

A novel technique for targeted stable multiplication of a specific long E. coli chromosome fragment was developed. The method is based on the coordinated functioning of λ and φ80 bacteriophage site-specific recombination and integration systems. In vivo cloning and targeted insertion of a chosen chromosomal region is accomplished by a simple one-step experiment. The method does not require PCR amplification of an inserted fragment, which makes it especially convenient for manipulation of long-length DNA. For this purpose, we constructed a pKDAH vector that can perform both λRed recombineering and φ80-integrase-mediated integration. Using this technique, the chromosome region is cloned via λRed recombination and immediately inserted into another chromosome locus by φ80-integrase. The method was effectively used for targeted chromosomal integration of additional copies of an individual gene (alaE), a short-length operon (kbl-tdh) and long-length DNA fragments harboring the E. coli atpIBEFHAGDC or nuoABCEFGHIJKLMN operons (7.5 and 15 kb, respectively), thus confirming the utility of the technique. Moreover, duplication of the target genes with simultaneous modification of the regulatory region was performed.