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the primers and were utilized for subsequent gene fusions.
NdeI
allows for the
inframe fusion with the ATG start site. This product was cloned into pCR2.1-
TOPO (Invitrogen) resulting in pCR2.1+BhFlgB-P, which was then sequenced
using M13 Reverse and M13 Forward (-20) primers (Invitrogen) to confirm
the sequence and orientation of the insert. pTAkan-A
(15)
was used as the
source of the Tn903-derived kanamycin phosphotransferase gene,
kan-r
. The
925-bp
Nde
I/
Bam
HI fragment of pTAkan-A containing
kan-r
was ligated to
the
Nde
I/
Bam
HI-digested pCR2.1+BhFlgB-P, resulting in the 4919-bp plasmid,
pTABhFlgBP-Kan (
see
Fig. 2
). Following transformation of
E. coli
(as in
step
10, Subheading 3.1.1.
), this plasmid construct was verified by RFLP, PCR,
and sequence analysis. The
B. hermsii flgB
-promoted
kan-r
was used as the
selectable marker for site-specific inactivation of
vtp
(
see
Fig. 1
).
Construction
of pTABhflaB-Gent
: A fragment containing the DNA sequence for the
B. hermsii
DAH flagellin promoter
flaB-P
was identified (
see
Note 2
). Oligonucleotides
5´ BhflaB+
Ngo
MIV and 3´ BhflaB+
Nde
I were designed to PCR-amplify a 301-
bp fragment containing
flaB-P
. This product was cleaned (QIAquick), cloned
into pCR2.1-TOPO resulting in pCR2.1+BhFlaB-P, and sequenced to confirm
the integrity and orientation of the insert. pTAGmA
(16)
was used as the
source of the
aacC1
gene, which encodes a gentamicin acetyltransferase, here
termed
gent-r
. The 600-bp
Nde
I/
Bam
HI fragment of pTAGmA containing
gent-r
was fused to the
Nde
I/
Bam
HI digested pCR2.1+BhflaB-P resulting in the 4759-
bp plasmid construct, pTABhflaB-Gent. Following transformation of
E. coli
(as
in
step 10, Subheading 3.1.1.
), this plasmid construct was verified by RFLP,
PCR, and sequence analysis. The
B. hermsii flaB
-promoted
gent-r
is flanked
by a number of common restriction endonuclease sites and was used as the
selectable marker for genetic reconstitution of
vtp
(
see
Fig. 2
).
Construction
of pBhSV-2:
This plasmid was not directly used in the manipulation of
vtp.
However, as autonomously replicating plasmids are much more efficient at
transformation, this plasmid was frequently employed as a positive control for
electro-transformation of
B. hermsii
. The
B. burgdorferi
shuttle vector, pBSV2
(14)
was modified first by replacing the
Not
I
/Nde
I fragment containing the
B. burgdorferi flaB-P
with the 162-bp
NotI/NdeI
fragment of pTABhflgB-Kan
containing
B. hermsii flgB-P
, resulting in pBhSV-1. All attempts to transform
B. hermsii
by electroporation with pBSV2 and pBhSV-1 failed to generate
kanamycin-resistant spirochetes; thus another construct was made
.
Plasmid
pBhSV-1 was then modified by replacing the three
B. burgdorferi
ORFs shown
to confer autonomous replication, with the
B. hermsii
ARS loci (
see
Note 2
).
First, primers BhOriF+NotI and BhOriR+SphI were used to amplify 2643-bp
DNA from
B. hermsii
DAH 2E7 containing the ARS. The
Not
I and
Sph
I primer
tags were used to fuse the ARS amplicon to the 2384-bp
Not
I
/Nsp
I fragment
from pBhSV-1, resulting in pBhSV-2. Transformation of
E. coli
, selection, and
analysis were done as described in
steps 10-12, Subheading 3.1.1
. Introduction
of pBhSV-2 into
B. hermsii
by electro-transformation (
see
Subheading 3.2.
)
generated kanamycin-resistant spirochetes. Plasmid rescue
(14)
was used to
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