Journal of Southern Medical University 2014;34(6):904-908

Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain.

Peng HAN 1 ; Qi SUN ; Suhui ZHAO ; Qiwei ZHANG ; Chengsong WAN

Affiliations

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Country

China

Language

Chinese

Abstract

OBJECTIVETo construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics.

METHODSThe gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5' and 3' ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining.

RESULTS AND CONCLUSIONWe established a ppk-deleted EHEC O157:H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.