Citation
Lanza Lucio, Monica and Alborno, Marcelo and Rey Navarro, Luis and Imperial Ródenas, Juan
(2010).
Optimizing Gateway™ technology (Invitrogen) to construct Rhizobium leguminosarum deletion mutants.
In: "XIII National Meeting of the Spanish Society of Nitrogen Fixation. II Portuguese-Spanish Congress on Nitrogen Fixation", 15/06/2011 - 18/06/2011, Zaragoza, España.
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