A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 70

Fig. 1. The target locus plasmid, inactivation and reconstitution constructs, and
genomic structures of developed strains. pOKvtp is the target locus plasmid and was
generated prior to the inactivation and reconstitution constructs. Primer-binding sites,
indicated by small arrows, are numbered corresponding to the list in Subheading 2.2.2 .
BLAST (13) analysis with amino acid sequences predicted by open reading frame
(ORF) Finder (NCBI) shows that truncated orfZ´ is most similar to Borrelia burgdorferi
thymidylate synthase (67% identical, accession NC_001857.1), where orfX and orfY
encode conserved hypothetical proteins. The amplicon generated with primers 9 and
10 from pOKvtp was fused to the Spe I /Eco RV fragment of pTABhFlgB-Kan, resulting
in pOKvtpKO, the vtp inactivation construct. Electro-transformation of pOKvtpKO
resulted in a double-crossover event that mutagenized vtp without affecting the flanking
ORFs of this locus. The structure of the wild-type and mutant loci are illustrated and
were verified by sequencing both strands of the amplicon generated from primers
(17 and 18) flanking the cloned target locus. EcoRV restriction sites are indicated ( E ) and
correspond to the restriction fragment length polymorphism (RFLP) shown in Fig. 3 .
Fusion of amplicons derived from pOKvtp (using primers 11 and 12) and pTABhFlaB-
Gent (using primers 13 and 14) resulted in the genetic reconstitution construct, pOKvt-
pRECON. Electro-transformation of the vtp mutant with pOKvtpRECON resulted in a
double homologous recombination event that replaced the mutated vtp with a wild-type
copy and simultaneously inserted the PflaB::Gent-r cassette at an intergenic location
just upstream from vtp . The structure of the reconstituted locus is illustrated and was
verified by sequencing both strands of the amplicon generated from primers (17 and 18)
flanking the cloned target locus. Details relevant to the construction of these plasmids
are provided in the text ( see Notes 6 , 8 , and 9 ).