Search our Database of Scientific Publications and Authors

I’m looking for a
    An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.
    BMC Biotechnol 2017 Jul 14;17(1):62. Epub 2017 Jul 14.
    Unité Plasticité du Génome Bactérien, Département Génomes et Génétique, Institut Pasteur, 75015, Paris, France.
    Background: Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla).

    Results: The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested.

    Conclusions: The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

    Similar Publications

    PhiC31 recombination system demonstrates heritable germinal transmission of site-specific excision from the Arabidopsis genome.
    BMC Biotechnol 2010 Feb 23;10:17. Epub 2010 Feb 23.
    Crop Improvement and Utilization Research Unit, Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA.
    Background: The large serine recombinase phiC31 from broad host range Streptomyces temperate phage, catalyzes the site-specific recombination of two recognition sites that differ in sequence, typically known as attachment sites attB and attP. Previously, we characterized the phiC31 catalytic activity and modes of action in the fission yeast Schizosaccharomyces pombe.

    Results: In this work, the phiC31 recombinase gene was placed under the control of the Arabidopsis OXS3 promoter and introduced into Arabidopsis harboring a chromosomally integrated attB and attP-flanked target sequence. Read More
    Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31.
    Mol Microbiol 2000 Oct;38(2):232-41
    Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
    The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Read More
    In vivo site-specific integration of transgene in silkworm via PhiC31 integrase-mediated cassette exchange.
    Insect Biochem Mol Biol 2013 Nov 22;43(11):997-1008. Epub 2013 Aug 22.
    State Key Laboratory of Silkworm Genome Biology, Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, BeiBei, Chongqing 400716, China.
    Current techniques for genetic engineering of the silkworm Bombyx mori genome utilize transposable elements, which result in positional effects and insertional mutagenesis through random insertion of exogenous DNA. New methods for introducing transgenes at specific positions are therefore needed to overcome the limitations of transposon-based strategies. Although site-specific recombination systems have proven powerful tools for genome manipulation in many organisms, their use has not yet been well established for the integration of transgenes in the silkworm. Read More
    Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by Streptomyces phage phiC31 integrase.
    BMC Mol Biol 2013 Sep 8;14:20. Epub 2013 Sep 8.
    Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Science and Veterinary Medicine, Hubei Academy of Agricultural Science, Wuhan 430064, China.
    Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. Read More