A New gcrR-Deficient Streptococcus mutans Mutant for Replacement Therapy of Dental Caries
Figure 1
(a) To confirm that gcrR was deleted, chromosomal DNA was isolated from the selected transformants as well as UA159 progenitor and used as a template for PCR amplification with the P1/P2 and RT-gcrRF/RT-gcrRR primers. The resulting amplicons (the gcrR upstream-kanamycin resistance (KanR) cassette junction and the part of gcrR gene) were analyzed via agarose gel electrophoresis. Nucleotide sequencing of the purified amplicons with the same primer set was conducted to confirm the gcrR knockout and KanR cassette insertion. (b) Lane 1: the resulting amplicons (chromosomal DNA of MS-gcrR-def as template and RT-gcrRF/RT-gcrRR as primers); lane 2: the resulting amplicons (chromosomal DNA of UA159 as template and RT-gcrRF/RT-gcrRR as primers); lane 3: 1 kb marker (Fermentas). (c) Lanes 1, 2, 3, and 4: the resulting amplicons (chromosomal DNA of MS-gcrR-def as template and P1/P2 as primers); Lane 5, 1 kb marker (Fermentas); Lane 6, the resulting amplicons (chromosomal DNA of UA159 as template and P1/P2 as primers).
