Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus mutans / 中华口腔医学杂志
Chinese Journal of Stomatology
; (12): 102-106, 2011.
Article
em Zh
| WPRIM
| ID: wpr-339796
Biblioteca responsável:
WPRO
ABSTRACT
<p><b>OBJECTIVE</b>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.</p><p><b>METHODS</b>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-ΔhtrA/Km for deleting the htrA gene. Using the same method, the pGEM-T-ΔclpP/Sp was constructed for deleting the clpP gene. Following the transformation of pGEM-T-ΔhtrA/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 (MSΔhtrA) 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 MSΔhtrA, yielding markerless mutant strain lacking clpP and htrA.</p><p><b>RESULTS</b>The deletion of htrA, clpP and antibiotic resistance markers were confirmed by PCR analysis and DNA sequencing.</p><p><b>CONCLUSIONS</b>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.</p>
Texto completo:
1
Índice:
WPRIM
Assunto principal:
Plasmídeos
/
Streptococcus mutans
/
Resistência Microbiana a Medicamentos
/
Regulação Bacteriana da Expressão Gênica
/
Deleção de Genes
/
Integrases
/
Genes Bacterianos
/
Vetores Genéticos
/
Genética
Idioma:
Zh
Revista:
Chinese Journal of Stomatology
Ano de publicação:
2011
Tipo de documento:
Article