A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 77

Fig. 3. Restriction fragment length polymorphism (RFLP) analysis of wild-type, vtp
mutant, and reconstituted strains. This ethidium bromide-stained agarose gel verifies
the genomic structure of the Borrelia hermsii vtp mutant and isogenic reconstituted
strain relative to wild type. Amplicons were generated from wild-type, mutant, and
reconstituted clones using primers 17 and 18 ( see Fig. 1 ). These products were digested
with Eco RV and the resulting restriction fragments were separated by agarose gel
electrophoresis. The banding pattern ( f ragment l ength p olymorphism) of each strain
(loaded in duplicate) corresponds with the expected results: wild type (2795, 1675,
1644 bp), mutant (2795, 2289, 1644 bp), and reconstituted (2795, 1644, 1499, 582,
202 bp). Molecular size standards (MSS) are indicated to the right in base pairs.
flgB -promoted kanamycin resistance cassette ( see Fig. 2 ). Following transfor-
mation of TOP10 E. coli , clones were isolated on LB plates supplemented
with 50 μg/mL kanamycin. The resulting 7541-bp construct, pOKvtpKO, was