Biology Reference
In-Depth Information
3.2.3. Electroporation and Selection
1. Place the concentrated DNA, electrocompetent cells, and cuvettes on ice.
2. Add 100 μL of electrocompetent cells (approximately 5 × 10
9
spirochetes) and
5 μL (25 μg) of plasmid DNA in a microfuge tube, mix gently, and transfer to
the bottom of a 0.2-cm electroporation cuvette. Avoid introducing air bubbles in
the sample that can cause arcing. Gently tap the cuvette to ensure that the sample
goes to the bottom.
3. Electroporate with a Gene Pulser (BioRad), or other electroporator capable of
delivering a similar pulse (2.5 kV, 25 μF, and 200)(
see
Note 13
).
4. Immediately after delivering the pulse, transfer the contents of the cuvette into 5
mL mBSK-c and incubate overnight at 34ºC.
5. Transfer the 5 mL overnight culture into 45 mL mBSK-c containing the appro-
priate antibiotic (200 μg/mL kanamycin or 40 μg/mL gentamicin).
6. Incubate the culture at 34°C and monitor daily by dark-field microscopy for the
presence of motile spirochetes. Typically 3-7 days of cultivation is required. Once
detected, cryopreserve a portion of the liquid culture by combining with an equal
volume of 25% glycerol and freeze at -80°C.
7. Isolate individual clones of antibiotic resistant spirochetes by limiting dilution
in 96-well tissue culture plates of mBSK-c (supplemented with the appropriate
antibiotic) (
see
Note 14
).
8. Inoculate clones in mBSK-c (supplemented with the appropriate antibiotic), grow
to mid-exponential phase, and cryopreserve at -80°C by combining with an equal
volume of 25% glycerol.
4. Notes
1. The mBSK-c formulation is based on fortified Kelly's media
(12)
. The
following recipe is for a 2× concentrate: 10× CMRL-1066 (US Biological,
Swampscott, MA, C5900-05) 19.4 g/L;
N
-acetylglucosamine (Sigma A-8625)
0.8 g/L; sodium citrate (Sigma C-7254) 1.4 g/L; sodium pyruvate (Sigma
P-5280) 1.6 g/L; sodium bicarbonate (Sigma S-5761) 4.4 g/L; HEPES (Sigma
H-9136) 12 g/L; BSA-Fraction V (Serologicals, Norcross, GA, 81-003-6) 100
g/L; Neopeptone (Difco, MI 0119-01) 10 g/L; Yeastolate (Difco, Detroit,
MI 5577-15-5) 4 g/L; d-Glucose (Sigma G-7021) 12 g/L. Adjust pH to 7.5
with NaOH. Sterilize with a 0.22-μm filter. Store 500 ml of filter-sterilized
2× concentrate in 1-L Pyrex bottles at -20°C. To obtain 1 L of complete mBSK
(mBSK-c), thawa frozen 500-mL aliquot and add 120mLof non-hemolyzed rabbit
serum (Sigma-Aldrich or PelFreez, Rogers, AR, Cat. 31125) and 380 mL sterile
dH
2
O. This results in a final concentration of rabbit serum of 12%. mBSK-c is
dispensed into sterilepolycarbonate culture tubes or pyrexbottles andused immedi-
ately or frozen at -20°C.
2. In our unpublished genome sequence database for
B. hermsii
, we identified DNA
sequences of the promoter for the flagellar rod protein (
flgB-P
), the promoter for
the flagellin protein (
flaB-P
), and the variable tick protein gene (
vtp
). Sequences
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