Abstract
BackgroundThe 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.ResultsTo 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 %.ConclusionsIntegration 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.
Highlights
The bacterial chromosome may be used to stably maintain foreign deoxyribonucleic acid (DNA) in the mega-base range
Systems for chromosomal integration of foreign DNA mediated by tyrosine integrases were devised as early as the 1990s for Escherichia coli [3], Staphylococcus aureus [4] and Actinobacteria [5, 6]
For insertion of the landing pad” (LP) sector into the chromosome, a region was chosen, where insertion of additional sequences most likely does not interfere with essential activities of Rhizobium etli
Summary
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). 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. All of them employ special plasmid vectors harboring the corresponding recombinase recognition sequence (att or attachment site), where foreign DNA can be cloned. Upon introduction into target cells expressing the cognate integrase, site-specific integration occurs via the endogenous attachment site
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