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Lanza Lucio, Monica, Alborno, Marcelo, Rey Navarro, Luis ORCID: https://orcid.org/0000-0003-3477-6942 and Imperial Ródenas, Juan
ORCID: https://orcid.org/0000-0002-5002-6458
(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.
Title: | Optimizing Gateway™ technology (Invitrogen) to construct Rhizobium leguminosarum deletion mutants |
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Author/s: |
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Item Type: | Presentation at Congress or Conference (Poster) |
Event Title: | XIII National Meeting of the Spanish Society of Nitrogen Fixation. II Portuguese-Spanish Congress on Nitrogen Fixation |
Event Dates: | 15/06/2011 - 18/06/2011 |
Event Location: | Zaragoza, España |
Title of Book: | Biological Nitrogen Fixation and Plant-Associated Microorganisms |
Date: | June 2010 |
Subjects: | |
Faculty: | E.T.S.I. Agrónomos (UPM) [antigua denominación] |
Department: | Biotecnologia [hasta 2014] |
Creative Commons Licenses: | Recognition - No derivative works - Non commercial |
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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
Item ID: | 9006 |
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DC Identifier: | https://oa.upm.es/9006/ |
OAI Identifier: | oai:oa.upm.es:9006 |
Deposited by: | Memoria Investigacion |
Deposited on: | 05 Oct 2011 09:04 |
Last Modified: | 20 Apr 2016 17:35 |