Application of Cre-loxP* system in constructing the markerless double-gene-deletion strain in Streptococcus mutans
10.3760/cma.j.issn.1002-0098.2011.02.009
- VernacularTitle:应用Cre-loxP*系统构建无标记的变形链球菌双基因缺陷株
- Author:
Dan-Ni YU
1
;
Wen-Juan ZHANG
;
Cheng PENG
;
Yu-Zhi HAN
;
Zhi-Ming REN
Author Information
1. 天津医科大学附属第二医院
- Keywords:
Streptococcus mutans;
Gene deletion;
Cre-loxP system
- From:
Chinese Journal of Stomatology
2011;46(2):102-106
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct double gene deletions at the htrA and clpP loci on the chromosome of Streptococcus mutans(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 kanamycin 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 kanamycin resistance gene was then excised. The pCrePA was then easily eliminated at nonpermissive temperatures, resulting in a mutant strain (MS △ 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 spectinomycin resistance gene were deleted from MS △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.
