title: Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli.
abstract: 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.
  
  
  
  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 %.
  
  
  
  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.
authors.affiliation: Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, 62210, Cuernavaca, Morelos, Mexico.
authors.forename: Rogelio
authors.initials: R
authors.lastname: Hernández-Tamayo
authors.affiliation: Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, 62210, Cuernavaca, Morelos, Mexico.|Departamento de Ciencias Biológicas, Instituto de Biotecnología y Biología Molecular, UNLP, CCT-La Plata-CONICET, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina.
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Torres-Tejerizo
authors.affiliation: Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, 62210, Cuernavaca, Morelos, Mexico.
authors.forename: Susana
authors.initials: S
authors.lastname: Brom
authors.affiliation: Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apartado Postal 565-A, 62210, Cuernavaca, Morelos, Mexico. dromero@ccg.unam.mx.
authors.forename: David
authors.initials: D
authors.lastname: Romero
mesh_terms: D002876:Chromosomes, Bacterial; D003227:Conjugation, Genetic; D004247:DNA; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D007763:Lac Operon; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D042163:Rhizobium etli / Q000201:enzymology* / Q000235:genetics*
chemical_list: D049452:Green Fluorescent Proteins; D004247:DNA; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C077570:Site-specific recombinase
keywords: Chromosomal integration; Site-specific recombination; Tyrosine recombinase