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Updated: Jun 3, 2026
Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis
Published on: January 30, 2020
[Application of Cre-loxP(*) system in constructing the markerless double-gene-deletion strain in Streptococcus
Dan-ni Yu1, Wen-juan Zhang, Cheng Peng
1Department of Stomatology, The Second Hospital of Tianjin Medical University, Tianjin 300211, China. danni2468@eyou.com
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-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.
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.
