Citation

R. F. J. Vet. Res. 2007; 61(4): 357-361.

Copyright

(Copyright © 2007)

DOI

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PMID

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Abstract

To construct a specific mutation in E.coli O157:H7. Allelic exchange approach for producing stx[2]-negative mutant. 1-Obtaining PCR product from stx[2] gene.2-Cloning this product to T-vector.3-Inseting a gentamicin resistance cassette.4-Transfering the stx[2] gene with GMR cassette to suicide vector.5-Mating donor [SM10 [+pRE107 - stx[2]: GM] and recipient [Str[R]EC960904].6-Selection of single-cross-overs.7-Counter-selection of double cross-overs. Selecting Gm[R]/Str[R] and Amps colonies.8-Confirmation of mutant by PCR and cytotoxigenicity assay on vero cells. In this study, after mating between the donor [SM10[+pRE107 -Stx[2]: GM] and the recipient cells [Str R-EC960904 wild type], colonies were resistant to gentamycin and streptomycin but sensitive to ampicillin. The result of PCR showed that the mutant was the stx[2] negative strain. However, the mutant strain was unable to lyse the vero cells compare to wild type strain. Hutant strain was produced by a suitable suicide vector

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