Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli

Background: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 cat...

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Detalles Bibliográficos
Autores principales: Hernández Tamayo, R., Torres Tejerizo, Gonzalo Arturo, Brom, S., Romero, D.
Formato: Articulo
Lenguaje:Inglés
Publicado: 2016
Materias:
Biología
Chromosomal integration
Site-specific recombination
Tyrosine recombinase
Acceso en línea:http://sedici.unlp.edu.ar/handle/10915/86770
Aporte de:
SEDICI (UNLP) de Universidad Nacional de La Plata
id I19-R120-10915-86770
record_format dspace
institution Universidad Nacional de La Plata
institution_str I-19
repository_str R-120
collection SEDICI (UNLP)
language Inglés
topic Biología
Chromosomal integration
Site-specific recombination
Tyrosine recombinase
spellingShingle Biología
Chromosomal integration
Site-specific recombination
Tyrosine recombinase
Hernández Tamayo, R.
Torres Tejerizo, Gonzalo Arturo
Brom, S.
Romero, D.
Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
topic_facet Biología
Chromosomal integration
Site-specific recombination
Tyrosine recombinase
description Background: The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system. Results: To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a "landing pad" (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %. Conclusions: Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.
format Articulo
Articulo
author Hernández Tamayo, R.
Torres Tejerizo, Gonzalo Arturo
Brom, S.
Romero, D.
author_facet Hernández Tamayo, R.
Torres Tejerizo, Gonzalo Arturo
Brom, S.
Romero, D.
author_sort Hernández Tamayo, R.
title Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
title_short Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
title_full Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
title_fullStr Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
title_full_unstemmed Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
title_sort site-specific bacterial chromosome engineering mediated by inta integrase from rhizobium etli
publishDate 2016
url http://sedici.unlp.edu.ar/handle/10915/86770
work_keys_str_mv AT hernandeztamayor sitespecificbacterialchromosomeengineeringmediatedbyintaintegrasefromrhizobiumetli
AT torrestejerizogonzaloarturo sitespecificbacterialchromosomeengineeringmediatedbyintaintegrasefromrhizobiumetli
AT broms sitespecificbacterialchromosomeengineeringmediatedbyintaintegrasefromrhizobiumetli
AT romerod sitespecificbacterialchromosomeengineeringmediatedbyintaintegrasefromrhizobiumetli
bdutipo_str Repositorios
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