Transposon Mutagenesis of the Lyme Disease Agent Borrelia burgdorferi - Bacterial Pathogenesis: Methods and Protocols - page 89

(inverted terminal repeats, origin of replication, and the gentamicin marker) be
retained. We have found pGKT appears to be more stable than pMarGent and
produces more plasmid preparations with a higher transposition frequency.
2. The bovine serum albumin and rabbit serum are critical components of this
medium, and the ability to support B. burgdorferi growth to high cell densities
varies by source and from lot to lot. Typically, we reserve large batches from the
sources cited and test small samples.
3. Elias et al. tested a variety of growth stages and electroporation conditions
and concluded that mid- to late-exponential growth phase was optimal for
B. burgdorferi transformation and yielded the maximum number of competent
cells (23) . Transposition frequencies for strains lacking lp25 and lp56 and prepared
in this manner are 5×10 − 5 (8) .

4. The objective of the washes is to remove medium components from the cells that
might cause arcing during the electroporation, while concentrating the bacterium
in electroporation buffer. The number of washes and volumes of EPS used
were empirically determined and are a balance between efficient removal of
the medium, yet limiting mechanical damage to B. burgdorferi cells caused
by centrifugation and resuspension. The final volume that competent cells are
resuspended in varies, but typically ranges between a 500-fold and a 1000-fold
concentration of cells. Under darkfield microscopy, an ideal competence prepa-
ration should look like a continuous flowing sheet of spirochetes. Problems arise
when the cells clump (fewer spirochetes accessible to the DNA during transfor-
mation) or if the cells are too concentrated (which might result in arcing). If
clumping occurs, try moderate vortexing and repeated pipetting. If the cells are
too concentrated, then add more EPS until the cells begin to flow when viewed
under darkfield microscopy.
5. Generally, 10-20 μg of transposon plasmid DNA is alcohol-precipitated, washed
with 70% ethanol to remove salts that may cause arcing during electroporation,
and resuspended in a small volume (e.g., 5 μL) of sterile H 2 O. The final volume
of DNA must be small relative to the volume of competent cells, again to avoid
arcing.
6. Transformed cells are generally allowed to recover overnight before selection is
imposed. The length of the recovery period can be shortened to 8 h, which allows
transformation and plating to occur in the same day.
7. Isolating genomic DNA from small culture volumes (5-15 mL) produces
relatively low yields (a few micrograms). Harvest cells in late-exponential
phase to obtain the highest cell numbers and maximize DNA yields. However,
allowing cells to enter stationary phase usually results in lower yields and poor
quality DNA.
8. This procedure was empirically determined and may be optimized. The restriction
enzyme Hind III was chosen because the recognition sequence is absent from the
transposon but is relatively frequent in B. burgdorferi DNA.

∼