Use of lambda Red-mediated recombineering and Cre/lox for generation of markerless chromosomal deletions in avian pathogenic Escherichia coli.
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.
Department of Biosystems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Heverlee, Belgium. huruma.tuntufye@biw.kuleuven.be
Huruma N
HN
Tuntufye
Bruno M
BM
Goddeeris
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics*; D049109:Cell Proliferation; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000821:virology; D004927:Escherichia coli Infections / Q000382:microbiology; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D019426:Integrases / Q000235:genetics*; D009154:Mutation; D009876:Operon / Q000235:genetics; D016133:Polymerase Chain Reaction; D011201:Poultry Diseases / Q000382:microbiology; D011995:Recombination, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D000091243:Ribosomal Protein S9; D014764:Viral Proteins / Q000235:genetics
D029968:Escherichia coli Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases