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which is transmitted among humans by their body lice (
Pediculus humanus
corporis
). LBRF often occurs in epidemics where crowded and unsanitary
conditions promote body louse infestations and their spread among people
(3,4)
.
The
v
ariable
t
ick
p
rotein (Vtp = Vsp33) is a major outer-surface lipoprotein
produced by
B. hermsii
during its persistent infection in tick salivary glands
(5,6)
.
A number of studies suggest that Vtp, and its ortholog OspC in the Lyme disease
spirochetes
Borrelia burgdorferi
sensu lato, are involved in tick transmission of
the spirochetes or initial infection of mammals
(6,7,8,9,10)
. To study the role of
Vtp in the transmission of
B. hermsii,
we developed a system for site-specific
mutagenesis and genetic reconstitution. This chapter describes methods for the
construction of isogenic strains of
B. hermsii
in an infectious background. The
example used is the inactivation of the
vtp
gene; however, the method has been
applied to other loci and the techniques described should be applicable to any gene
of interest.
2. Materials
2.1. Spirochete Culture and Transformation Components
1. Modified Barbour-Stoenner-Kelly medium (mBSK) (
see
Note 1
).
2. Electroporation Solution (EPS): 0.27
M
sucrose (Fisher, Fair Lawn, NJ, Cat.
BP220-1), 15% (v/v) glycerol (MP Biomedicals, Solon, OH, Cat. 800688) solution
in dH
2
O. Filter-sterilize and store at 4°C.
3. Kanamycin sulfate (1 mg/mL, Sigma-Aldrich, St. Louis, MO, Cat. K-1377) and
gentamicin sulfate (200 μg/mL, Sigma, Cat. G3632). Prepare in dH
2
O and sterilize
by passage through a 0.22-μm filter. Store at 4°C.
4. Gene Pulser (BioRad, Hercules, CA) or other electroporation device capable of
delivering a pulse of 2.5 kV, 25 μF, and 200 .
5. 0.2-cm electroporation cuvettes (Harvard Apparatus, Holliston, MA, Cat. 45-0125).
6. 3
M
sodium acetate (Fisher, Cat. BP334-1), adjust pH to 5.0 with HCl.
7. 100% ethanol (Aaper, Shelbyville, KY).
8. Reverse-osmosis/deionized water (resistivity
18.0 M/cm). This water is used
for all solutions and media, and will be referred to simply as dH
2
O in this text.
9. 25% glycerol (MP Biomedicals) (v/v) in dH
2
O. Sterilize by autoclave.
10. 96-well round-bottom tissue culture plates (Nunc, Rochester, NY).
∼
2.2. Nucleic Acids
2.2.1. Genomic Sequence (see Note 2)
1. Endogenous promoters:
B. hermsii
flagellinprotein (
flaB-P
) and flagellar rodprotein
(
flgB-P
).
2. Target sequence: variable tick protein gene (
vtp
) described here, but any gene of
interest can be targeted.
3. Autonomous replication sequences (ARS).
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