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verify that the plasmid replicated autonomously. The genomic source of the
ARS in pBhSV-2 is presently unclear. This ARS is not the chromosomal origin.
BLAST analysis
(13)
demonstrates that this ARS is similar to a number of
B. hermsii
and
B. burgdorferi
plasmid sequences. Searches were performed
with the entire ARS sequence as well as with individual ORFs A, B, and
C´ [identified using ORF finder (NCBI) (
see
Fig. 2
)]. Although a contiguous
sequence containing this ARS was not found in GenBank,
orfA
and
orfB
are
most similar to members of the
B. burgdorferi
paralogous families 57 and 50,
respectively
(17,18)
. This ARS also contains one truncated ORF
orfC´
, which
is 67% identical to the carboxy-terminal portion of
B. hermsii
variable major
protein VlpC54silD
(19)
.
4. On the basis of the size of the amplicon, two different PCR
(20)
systems
were utilized for the construction of plasmids. Amplicons under 1000 bp
were generated using a GeneAmp Core Kit (Roche Molecular Systems), and
amplicons over 1000 bp were produced using an Expand Long Template PCR
System Kit (Roche Diagnostics), per the manufacturer's instructions.
5. BlueView Nucleic Acid Stain (Sigma-Aldrich) was used to visualize DNA
fragments over 1000 bp in agarose gels for excision and purification. This stain
is less sensitive than EtBr and requires a minimum of several micrograms of
DNA per well for visualization, but does not damage the samples. EtBr is
more efficient for visualization but is carcinogenic, requires ultraviolet light,
and prolonged exposure to UV light results in DNA damage that significantly
reduces cloning efficiency. Personal protective devices including UV-resistant
eyeglasses, gloves, and coat are required when working with EtBr.
6.
Construction of pOKvtp
: Primers VTP F2+
Avr
II and VTP B3+
Asc
I were
designed to PCR amplify a 5193-bp fragment from
B. hermsii
containing
vtp
with approximately 2 kbp of upstream and downstream flanking sequence.
The pOK12 amplicon consists of a 1720-bp portion of pOK12
(11)
that
includes the P15A origin of replication and a kanamycin resistance cassette,
and was generated by the Expand PCR Kit with primers pOK12F1+AscI and
pOK12B1+AvrII. Perform the cleanings and restriction digest (
see
steps 5-8,
Subheading 3.1.1.
) above to prepare the
vtp
and pOK12 amplicons for ligation.
Following transformation of TOP10
E. coli
, and selection on LB plates supple-
mented with 50 μg/mL kanamycin, clones were isolated and the construct was
verified by PCR, RFLP, and sequence analysis. The
vtp
inactivation and recon-
stitution plasmids were generated by modifying pOKvtp.
7. RFLP is the analysis of DNA by restriction digestion followed by agarose gel
electrophoresis to display the number and size of restriction fragments. An
example of RFLP is shown in
Fig. 3
.
8.
Construction of the
vtp
inactivation plasmid
: Primers vtpF1+ScaI and
vtpB1+SpeI generate 6470 bp from pOKvtp that includes all of pOKvtp
excluding a 456-bp internal region of the
vtp
gene (
see
Fig. 1
, primers 9
and 10). Following
Spe
I and
Sca
I digestion, this amplicon was ligated to the
1066-bp
Spe
I
/Eco
RV fragment of pTABhFlgB-Kan containing the
B. hermsii
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