Biology Reference
In-Depth Information
hosts, and the loss of these plasmids reduces or eliminates the infectivity of
these strains
(17,18,19,20)
. An infectious strain, 5A18 NP1, has been engineered
that lacks the restriction/modification systems encoded on lp25 and lp56, and
has a high transformation frequency with shuttle vector DNA
(21)
. 5A18 NP1
provides a genetic background in which virulence factors may be assessed by
transposon mutagenesis
(22)
.
3.1. Preparation of Plasmid DNA
1. Transform pMarGent or pGKT (
see
Fig. 1
) into competent
E. coli
cells and plate
in the presence of the appropriate antibiotic (i.e., gentamicin for pMarGent or
gentamicin and kanamycin for pGKT).
2. Inoculate
E. coli
colonies into growth medium with antibiotic selection and isolate
plasmid DNA.
3.2. Competent Cell Preparation
1. Grow
B. burgdorferi
cultures to mid- to late-exponential phase (approximately
5-9 × 10
7
cells/mL as determined by darkfield microscopy using a Petroff-Hausser
chamber) (
see
Note 3
). Usually a 100 mL culture provides sufficient cell numbers
for about two transformations.
2. Pellet cells by centrifugation (6000 ×
g
, 10 min) and wash by resuspension in
0.25 volumes EPS, i.e., 25 mL EPS for a 100 mL culture (
see
Note 4
).
3. Pellet cells again (as in
step 2
) and resuspend in 0.1 volume EPS (i.e., 10 mL for
an original culture volume of 100 mL).
4. Pellet cells for a final resuspension in
0.001 volumes EPS, i.e., 100 μL EPS.
Cells may be used immediately or stored at -80 ºC.
∼
3.3. Transformation and Plating of B. burgdorferi Cells
1. Add transposon plasmid DNA (10-20 μg ina5μLvolume of sterilized H
2
O) to
50-100 μL of competent cells (
see
Note 5
).
2. Electroporate cell/DNA mix in a prechilled 0.2-cm gapped cuvette. Settings for
the electroporator are: 2.5 kV, 25 μF, and 200 .
3. Immediately after transformation, quickly resuspend cells in a final volume of
5 mL BSK medium and incubate overnight at 35 ºC (
see
Note 6
).
4. After overnight recovery, enumerate
B. burgdorferi
cells using a Petroff-Hausser
chamber and plate in solid BSK medium with appropriate antibiotic selection.
Subsurface colonies develop in
∼
6-10 days, depending on the strain.
3.4. Identification of Transposon Insertion Site
1. Confirm that colonies arising under selective pressure are transposon mutants (and
not spontaneously-arising antibiotic-resistant variants) by PCR for the presence
of the gentamicin-resistance marker. Stab the
B. burgdorferi
colony with a sterile
toothpick, then swirl the toothpick in a tube containing the PCR reaction mix.
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