Optimizing Gateway™ technology (Invitrogen) to construct Rhizobium leguminosarum deletion mutants

Abstract

The study of the role of different genes in Rhizobium leguminosarum requires the generation of mutants by homologous recombination. In this communication we describe a novel approach to obtain deletion mutants of genes in Rhizobium using Gateway TM Cloning technology (Invitrogen) and a new vector (pK18-attR), both conjugative and Rhizobium specific, that carries the recombination tails of Gateway system. This tool is a new alternative to the classic approach based on cloning using restriction enzymes. The first step consists of designing directed oligonucleotides with specific tails for isolating recombination fragments and a resistance marker cassette to an antibiotic by PCR. The three inserts are cloned by homologous recombination in three specific vectors, in a single step. The last step consists of multisite-directed recombination of the three donor vectors to the pK18-attR destination vector. After recombination, this vector loses the ccdB gene, whose expression results in synthesis of a DNA gyrase that is lethal to carrier cells and thus guarantees the effectiveness in obtaining clones that carry the homologous construction to the subsequent recombination in Rhizobium