Transposon Mutagenesis of the Lyme Disease Agent Borrelia burgdorferi - Bacterial Pathogenesis: Methods and Protocols - page 85

Himar1 to be functional in a wide range of prokaryotes (3,11,12,13,14) . The
characteristics and promiscuity of Himar1 make it a useful choice for rapidly
generating a large number of gene knockouts.
The utility of Himar1 in diverse organisms prompted us to adapt the
Himar1 element to generate a saturated library of random Borrelia burgdorferi
mutants (8) . The transposon vectors, pMarGent ( see Fig. 1A ) and the more
stable pGKT ( see Fig. 1B )( see Note 1 ), have several advantageous features
including random insertions in both the linear and the circular DNA molecules
present in the B. burgdorferi genome, and a high transposition frequency that
yields saturating levels of mutants from a single transformation.
The overall strategy for transposon mutagenesis ( see Fig. 2 ) is similar to that
used to obtain allelic exchange mutants. That is, B. burgdorferi -competent cells
are prepared and transformed with pMarGent or pGKT plasmid DNA isolated
from Escherichia coli . After an overnight recovery period, cells are plated in the
presence of selective antibiotic(s). Colonies that arise are transferred to liquid
growth medium and genomic DNA isolated. Digestion of the genomic DNA,
followed by ligation and transformation into competent E. coli cells, allows
the recovery and characterization of the B. burgdorferi DNA flanking the site
in which the transposon inserted. Subsequently, Himar1 -based systems have
been used successfully in mutagenesis systems for various spirochetes and are
readily adaptable to other microorganisms with limited genetic transformation
systems (5,6,15) .
2. Materials
2.1. Preparation of Plasmid DNA
1. Gentamicin and kanamycin are available from various sources. Gentamicin
selection for E. coli : 5 μg/mL; kanamycin selection for E. coli : 50 μg/mL.
2. We routinely use TOP10 chemically competent E. coli cells (Invitrogen, Carlsbad,
CA); however, other strains, such as DH5, should work.
3. Plasmid DNA may be isolated by various techniques or commercial kits, as long
as they produce highly pure DNA. We routinely use the Hispeed Maxi Plasmid
Kit (Qiagen, Valencia, CA).
2.2. Competent Cell Preparation
1. B. burgdorferi liquid cultures are grown in Barbour-Stoenner-Kelly (BSK)-H
medium (Sigma, St. Louis, MD).
2. Petroff-Hausser chambers may be purchased from Electron Microscopy Sciences,
Hatfield, PA.
3. Electroporation solution (EPS): 0.27 M sucrose, 15% (v/v) glycerol. Sterilize by
filtration and store at room temperature.