Biology Reference
In-Depth Information
2.3. Transformation and Plating of B. burgdorferi Cells
1. Gentamicin selection for
B. burgdorferi
: 40 μg/mL; kanamycin selection for
B. burgdorferi
: 200 μg/mL.
2. Solid (plating) BSK medium: Weigh 69.37 g bovine serum albumin, fraction V
(Celliance, Kankakee, IL), 6.9 g Neopeptone, 8.3 g HEPES acid, 6.9 g glucose,
1.0 g sodium citrate, 1.1 g sodium pyruvate, 0.56 g
N
-acetylglucosamine, 6.4 g
sodium bicarbonate, 3.5 g Yeastolate, and 12.7 g powdered Connaught Medical
Research Laboratories (CMRL) without glutamine (US Biological, Swampscott,
MA). Solubilize in a final volume of1LH
2
O and stir 2-4 h. Adjust pH to 7.5
with 1
N
NaOH and sterilize by filtration. Add 12 mL rabbit serum (Pel-Freeze
Biologicals, Rogers, AK) and aliquot into 310-mL portions. Store frozen at -20 ºC
until ready to use. Thaw at
55 ºC and add 200 mL sterile, molten 1.7% agarose
(55 ºC) and antibiotic, if desired. Aliquots of transformed cells are mixed directly
with 30 mL medium per plate (
see
Note 2
).
3.
Borrelia burgdorferi
plates should be held in a CO
2
incubator (2.5%) at 35 ºC.
≥
2.4. Identification of Transposon Insertion Site
1. Gentamicin selection for
E. coli
: 5 μg/mL.
2. PCR reaction and cycling conditions: 1× PCR reaction conditions (20 μL
volume)—1× Taq Polymerase buffer, 0.2 m
M
each deoxynucleotide, 10 pmoles
each primer, and 0.5 U Taq polymerase. Cycling conditions are 94 ºC (1 min),
followed by 30 cycles of 94 ºC (45 s), 55 ºC (1 min), and 68 ºC (2 min).
3. Primers may be obtained from any vendor and, after resuspension in H
2
O, should
be stored at -20 ºC. Sequence of primers:
col: 5´-CAGCAACGCGGCCTTTTTACG
flg: 5´-GCTTAAGCTCTTAAGTTCAACC
Primers col and flg are used during sequencing (
see
Fig. 2
).
JK62 (Gent F): 5´-GGCAGTCGCCCTAAAACAAAGTT
JK61 (Gent RC): 5´-TCTCGGCTTGAACGAATTGTTAGGT
Primers JK61 and JK62, used for amplifying a fragment of the gentamicin-
resistance gene, produce a 520 base pair PCR product.
3. Methods
Borrelia burgdorferi
strain B31 contains a large complement of linear and
circular plasmids, two of which (lp25 and lp56) correlate with a reduced
transformation efficiency with some constructs, including shuttle vectors and
pMarGent
(8,16)
. The transformation barrier is presumably due to the presence
of restriction/modification enzymes encoded on these
B. burgdorferi
plasmids.
Therefore, generating transposon mutants requires the use of
B. burgdorferi
strains that lack these plasmids. However, lp25 and lp56 also encode factors
that enhance or are required for
B. burgdorferi
survival in the mammal and tick
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