Disruption of the mutanocyclin synthesis gene cluster in S. mutans

An in-frame deletion mutant of the S. mutans mutanocyclin gene cluster was constructed by two rounds of homologous recombination. The 2.1-kb counter selection marker IFDC2 cassette was amplified using the primer pair ldhF1-BsaI/ermR1-BsaI with pIFDC2 as a template [³³]. A .7-kb and .8-kb fragment flanking the gene cluster were PCR amplified using primers 35C2-upF/35C2-upR-BsaI and 35C2-dnF-BsaI/35C2-dnR using the genomic DNA of S. mutans 35, a clinically isolated S. mutans strain, as a template [³⁴]. The two fragments and IFDC2 cassette were ligated using the Golden Gate cloning assay and transformed into strain S. mutans 35. Colonies with resistance to erythromycin on BHI plates were selected and PCR-verified with primers 35C2-upF1/35C2-dnR1. The IFDC2 cassette was then removed. The .7-kb fragments flanking the IFDC2 cassette were PCR-amplified with primers 35C2-upF/35C2-upR3-BsaI and 35C2-dnF3-BsaI/35C2-dnR, ligated using Golden Gate cloning, and transformed to remove the IFDC2 cassette. Colonies resistant to p-Cl-Phe on BHI plates were selected and PCR-verified.