[Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in XXXX ].

@entity6586 @entity6760 @entity15 @entity7913
OBJECTIVE: To construct double gene deletions at the htrA and clpP loci on the chromosome of @entity6586 (Sm) and to remove the antibiotic resistance markers with the Cre-loxP(*) site-specific recombination system. METHODS: The htrA gene was cloned into the pGEM-T-Easy TA cloning vector and then inactivated via the insertion of a @entity7913 resistance cassette (lox71-Km-lox66), yielding pGEM-T-/\htrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-/\clpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-/\htrA/Km in Sm, the homologous recombination event was selected. One such mutant was transformed with a cre expression plasmid (pCrePA). The @entity7913 resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain ( @entity15 /\htrA) carrying a deletion at the htrA loci without a selectable marker. This mutant was verified by PCR and DNA sequencing. Then, the clpP and @entity6760 resistance gene were deleted from @entity15 /\htrA, yielding markerless mutant strain lacking clpP and htrA. RESULTS: The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing. CONCLUSIONS: A mutant of Sm was constructed successfully which contained a deletion of the htrA and clpP gene without selectable marker. The Cre-loxP(*) system can be applied to Sm, which provides experimental evidence for generating markerless multiple gene deletion mutants.
