A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 78

verified by PCR, RFLP, and sequence analysis and used for site-specific vtp
inactivation.
9. Construction of the vtp reconstitution plasmid : Oligonucleotides ReconF+XmaI
and ReconR+SpeI ( see Fig. 1 , primers 11 and 12) were used to amplify 6675 bp
of pOKvtp that includes all of pOKvtp with the exception of a 251-bp fragment
that is 282 bp upstream of the start codon of vtp . Oligonucleotides Gent5´+XmaI
and Gent3´+SpeI ( see Fig. 2 , primers 13 and 14) were used to generate an 862-bp
fragment from pTABhflaB-Gent that contains the B. hermsii flaB -promoted
gentamicin resistance cassette. The pOKvtp and pTABhflaB-Gent amplicons
were digested with Xma I and Spe I, cleaned, and ligated, resulting in the plasmid
pOKvtpRECON ( see Fig. 1 ). Following transformation of TOP10 E. coli , clones
were isolated on LB plates supplemented with 10 μg/mL gentamicin, and the
construct was verified by sequence analysis.
10. All procedures are done in a laminar flow hood using aerosol barrier pipette tips
and sterile technique. Methods used to transform B. hermsii by electroporation
are similar to those procedures previously described for B. burgdorferi (15,21,
22,23) . Spirochetes were cultivated and cloned by limiting dilution in liquid
mBSK-c.
11. B. hermsii DAH is a wild-type non-clonal isolate that originated from blood of a
human with relapsing fever (24) . A clonal population of this isolate, designated
DAH 2E7, was made by limiting dilution in liquid medium, and was infectious
in RML mice by intraperitoneal injection ( see Fig. 4 ).
12. Ethanol precipitation is used to purify and concentrate the genetic manipu-
lation plasmids prior to electroporation. The following procedure is performed
in micro-centrifuge tubes. Reagents should be prepared with sterile deionized
Fig. 4. Virulence of strains by mouse infection. Following in vitro manipulation,
strains were assessed for virulence by intraperitoneal injection of mice and subse-
quent microscopic detection of Giemsa-stained spirochetes in blood. Injection of
equal numbers of (A) electrocompetent Borrelia hermsii DAH 2E7, (B) electroporated
without a manipulation construct or antibiotics and cloned by limiting dilution, and (C)
the vtp mutant all produced comparable spirochetemias at 65 h post infection. Images
of infected blood were taken at 400×.