In vivo plasmid construction by integrative recombination within a 156 bp sequence homology

Abstract

pHG165, a pBR322 copy number derivative of pUC8, has a 38-bp polylinker multiple cloning site located at the 5′ end of lacα. A further 156 bp, 3′ to the multiple cloning site, completes the coding sequence for the production of the β galactosidase α peptide. We describe the use of in vivo plasmid-chromosone cointegrates as a construction method that in this instance has enabled us to cross out the pHG165 multiple cloning site to obtain a wild-type β-galactosidase sequence for the α peptide. Because the DNA sequence available for homologous recombination was only 156 bp in length, the frequency of crosses that removed the multiple cloning site was less than 1 × 10⁻⁹. These crosses were easily obtained, however, after amplification by ampicillin selection.