A versatile method for integration of genes and gene fusions into the lambda attachment site of Escherichia coli

Abstract

We have developed a versatile method for integration of modified genes and gene fusions into the bacteriophage lambda attachment site (attB) of the Escherichia coli chromosome. The method relies on two components: (1) a DNA integration cassette, flanked by multiple restriction enzyme sites, which contains the lambda attP site and, as a selectable marker, the Tn5 aphA gene conferring kanamycin resistance (KmR); and (2) a plasmid with the lambda int gene transcribed from the tet promoter. A fragment carrying the gene in question is ligated to the integration cassette, resulting in a circular piece of DNA unable to replicate. The ligation product is then transformed into a strain that contains the int-carrying plasmid. Selection for KmR results in colonies with the cassette integrated into the attB site of the E. coli chromosome. This method was used for integration of several lacZ and phoA promoter fusions. The integration products were analyzed by Southern hybridization. In addition, we found, fortuitously, that the ligated DNA circles could also integrate by homologous recombination, although usually at a much lower frequency than the Int-mediated integration into attB.