A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 74

that confer autonomous replication (ARS) were identified using BLAST (13)
to search for a genomic fragment that was similar to the open reading frames
(ORFs) of the B. burgdorferi shuttle vector pBSV2 (14) .
3. The selectable markers in plasmids pTABhFlgB-Kan and pTABhFlaB-Gent
( see Fig. 2 ) have been employed for genetic manipulation of three genes
in infectious B. hermsii . The following describes the construction of these
plasmids. Construction of pTABhflgB-Kan : A sequence contig containing the
B. hermsii DAH flagellar rod promoter, flgB-P , was identified ( see Note 2 ).
Oligonucleotides 5´ BhflgB+NgoMIV and 3´ BhflgB+NdeI were designed to
generate a 135-bp PCR amplicon containing flgB - P from genomic DNA of
DAH clone 2E7. The specified restriction endonuclease sites were included in
Fig. 2. The Borrelia hermsii shuttle vector and selectable marker plasmids. Plasmids
pTABhFlgB-Kan and pTABhFlaB-Gent contain the selectable markers used in the
B. hermsii genetic manipulation constructs. The vtp inactivation plasmid, pOKvtpKO
( see Fig. 1 ), utilizes the PflgB::kan-r selectable marker from pTABhFlgB-Kan. The
PflaB::gent-r cassette in pTABhFlaB-Gent was used in the vtp genetic reconstitution
plasmid, pOKvtpRECON ( see Fig. 1 ). The B. hermsii shuttle vector, pBhSV-2, was
constructed following the design of pBSV2 (14) . Restriction endonuclease sites are
shown. Primer-binding sites are specified by small arrows and correspond to sequences
listed in Subheading 2.2.2 . The source of open reading frames is indicated by shading:
antibiotic resistance gene (light gray), B. hermsii promoter (dark gray), autonomous
replication sequence (ARS) (black), and cloning vector (white). Details relevant to the
construction of these plasmids are provided in the text ( see Note 3 ).