Open AccessUse of lambda R ed‐mediated recombineering and C re/ lox for generation of markerless chromosomal deletions in avian pathogenic E scherichia coli
Author(s) -
Tuntufye Huruma N.,
Goddeeris Bruno M.
Publication year - 2011
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2011.02421.x
Subject(s) - recombineering , biology , gene cassette , genetics , escherichia coli , gene , genome , homologous recombination , kanamycin , integron
Avian pathogenic E scherichia coli ( APEC ) are bacteria associated with extraintestinal diseases in poultry. A method to generate markerless deletions of APEC genome is described. Lambda R ed 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 C re/ lox system is used to remove the marker, hence deletion of the drug‐resistance genes. The results demonstrate that the C re/ 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.