NEW CLONING VECTORS FOR INTEGRATION INTO THE LAMBDA ATTACHMENT SITE ATTB OF THE ESCHERICHIA-COLI CHROMOSOME

A set of plasmid cloning vectors has been constructed, allowing the integration of any DNA fragment into the bacteriophage λ attachment site attB of the Escherichia coli chromosome. The system is based upon two components: (i) a number of cloning vectors containing the λ attachment site attP and (ii) a helper plasmid, bearing the λ int gene, transcribed from the λ PR promoter under the control of the temperature-sensitive repressor cI857. The DNA fragment of interest is cloned into the multicloning site of one of the attP-harboring plasmids. Subsequently, the origin of the plasmid, located on a cloning cassette, is cut out and the DNA becomes newly ligated, resulting in a circular DNA molecule without replication ability. The strain of choice, containing the int gene carrying helper plasmid, is transformed with this DNA molecule and incubated at 42 °C to induce int gene expression. Additionally, the temperature shift leads to the loss of the helper plasmid after a few cell generations, because the replication ability of its replicon is blocked at 42 °C. These vectors have been successfully used for integration of several promoter-lacZ fusions into the chromosome. The ratio between integration due to homologous recombination and Int protein-mediated integration has been determined. © 1992.