Generation of a
mutant. A. An IPTG-regulatable bb0028 mutant was generated by inserting a streptomycin resistance cassette followed by the flacp promoter immediately upstream of bb0028. Red arrows indicate primer-binding areas used to confirm insertion of the streptomycin cassette and flacp promoter into the borrelial genome. B. PCR amplification resulted in a 1.2 kb amplicon in the wildtype strain (WT), as expected, and a 3.0 kb amplicon in the BB0028 mutant (flacp::0028) indicating the streptomycin cassette and flacp promoter were inserted in the mutant. A PCR reaction with no DNA template (Neg) was included as a negative control. DNA sizes, in kilobase pairs, are indicated at left. C. Whole-cell lysate from the BB0028 mutant (flacp::0028) strain propagated with (+) or without (−) 1 mM IPTG, as well as from the parental, wildtype strain (WT), were subjected to immunoblot analysis using BB0028 specific antibodies (top panel) or FlaB antibodies (bottom panel). FlaB reactivity was used to ensure all lanes were loaded equally.