[Creation of a null mutation in the STX[2] gene of Escherichia coli o[157]: H[7]]
Journal of Veterinary Research. 2007; 61 (4): 357-361
in Persian
| IMEMR
| ID: emr-123131
ABSTRACT
To construct a specific mutation in E.coli O157H7. 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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Index:
IMEMR (Eastern Mediterranean)
Main subject:
Polymerase Chain Reaction
/
Mutagenesis, Insertional
/
Crossing Over, Genetic
/
Genetic Vectors
/
Mutation
Language:
Persian
Journal:
J. Vet. Res.
Year:
2007
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