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

Lanza Lucio, Monica; Alborno, Marcelo; Rey Navarro, Luis y Imperial Ródenas, Juan (2010). Optimizing Gateway™ technology (Invitrogen) to construct Rhizobium leguminosarum deletion mutants. En: "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.

Descripción

Título: Optimizing Gateway™ technology (Invitrogen) to construct Rhizobium leguminosarum deletion mutants
Autor/es:
  • Lanza Lucio, Monica
  • Alborno, Marcelo
  • Rey Navarro, Luis
  • Imperial Ródenas, Juan
Tipo de Documento: Ponencia en Congreso o Jornada (Póster)
Título del Evento: XIII National Meeting of the Spanish Society of Nitrogen Fixation. II Portuguese-Spanish Congress on Nitrogen Fixation
Fechas del Evento: 15/06/2011 - 18/06/2011
Lugar del Evento: Zaragoza, España
Título del Libro: Biological Nitrogen Fixation and Plant-Associated Microorganisms
Fecha: Junio 2010
Materias:
Escuela: E.T.S.I. Agrónomos (UPM) [antigua denominación]
Departamento: Biotecnologia [hasta 2014]
Licencias Creative Commons: Reconocimiento - Sin obra derivada - No comercial

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Resumen

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

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ID de Registro: 9006
Identificador DC: http://oa.upm.es/9006/
Identificador OAI: oai:oa.upm.es:9006
Depositado por: Memoria Investigacion
Depositado el: 05 Oct 2011 09:04
Ultima Modificación: 20 Abr 2016 17:35
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