Cloning of genes interrupted by Tn10 derivatives using antibiotic-resistance-carrying M13mp bacteriophages

Abstract

We have developed a system for rapidly cloning chromosomal DNA that flanks the site of insertion of Tn10 derivatives. A central portion of the tetracycline-resistance gene (tet) from Tn10 was cloned into a recombinant M13mp vector that carries a cat marker. Infection of a strain that contains a Tn10 derivative (mini-tet) leads to homologous recombination between the chromosomal tet gene and the cloned segment on the bacteriophage. Correct M13 lysogens can be identified by the inactivation of the tet gene and the gain of the cat gene. Digestion, ligation and transformation of chromosomal DNA from an M13 lysogen produces phage which carry a portion of the Tn10 as well as adjoining chromosomal DNA. The phage can be sequenced directly and are very useful for probing libraries for the wild type gene. Recombinant M13 clones have also been developed for the cloning of sequences adjacent to a Tn10 derivative which confers kanamycin resistance (mini-kan).