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Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli
Authors:Tuntufye Huruma N  Goddeeris Bruno M
Institution:Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Heverlee, Belgium. huruma.tuntufye@biw.kuleuven.be
Abstract:Avian pathogenic Escherichia coli (APEC) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda Red recombination is used to introduce a LoxP cassette (loxP-rpsL-neo-loxP) containing the rpsL gene for streptomycin sensitivity and the neo gene for kanamycin/neomycin resistance into the APEC genome, with attendant deletion of a desired chromosomal gene. The loxP sites are incorporated into primers used to amplify the rpsL-neo marker during the construction of the LoxP cassette, making the method rapid and efficient. The cassette is specifically integrated into the fiu gene or intergenic region 2051-52, and the Cre/lox system is used to remove the marker, hence deletion of the drug-resistance genes. The results demonstrate that the Cre/lox system can successfully be used to generate markerless deletions in APEC, and rpsL counter-selection can be used to select the deletions so that one does not have to pick and test to find the desired product.
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