A System for Site-Specific Genetic Manipulation of the Relapsing Fever Spirochete Borrelia hermsii - Bacterial Pathogenesis: Methods and Protocols - page 72

resistance cassette. The resistance cassette utilized in the inactivation plasmid
must be different from the cassette used for the reconstitution plasmid.
2. Prepare pOK- target locus plasmid from E. coli using QIAprep Spin Kit (Qiagen),
or another plasmid DNA isolation method.
3. Generate amplicons from the pOK- target locus plasmid with the inactivation or
reconstitution endonuclease-tagged primer sets using the Expand Long Template
PCR System Kit (Roche Diagnostics) per the manufacturer's instructions.
4. Analyze products by agarose gel electrophoresis ( see Note 7 ). Adjust the annealing
temperature and extension time if needed to reduce non-specific background
products that could interfere with the cloning.
5. Analyze, clean, and digest the amplicons as described in steps 5-8, Sub-

heading 3.1.1 .
6. Ligate the amplicons to digested resistance cassettes with T4 DNA ligase at
16°C overnight, and transform into TOP10 E. coli as described in step 10, Sub-
heading 3.1.1 .
7. For cloning the Kan-r cassette, selection is accomplished on LB plates supple-
mented with 50 μg/mL kanamycin sulfate. Use gentamicin sulfate at a final
concentration of 10 μg/mL for cloning the Gent-r cassette in E. coli . Analyze
clones by PCR, RFLP, and sequence analysis.

3.2. Electro-Transformation (see Note 10)
3.2.1. Preparation of Electrocompetent B. hermsii DAH 2E7 ( see
Note 11).
1. Grow infectious B. hermsii in 500 mL mBSK-c at 34°C to a density of approxi-
mately 5-7 × 10 7 spirochetes/mL.
2. Wash the spirochetes three times in EPS (RT), decreasing the volume after each
centrifugation. First centrifuge two 250-mL cultures at 11,000 × g for 15 min at
4°C. Wash and resuspend pellets (gentle vortexing) with 50 mL EPS, centrifuge
for 15 min, combine pellets, resuspend in 5 mL EPS, centrifuge for 15 min, and
resuspend cells in 600 μL EPS. The purpose of these washes is to remove ions
contained in mBSK-c that can cause arcing during electroporation.
3. Place electrocompetent cells on ice and electroporate immediately.

3.2.2. Preparation of Genetic Manipulation Plasmid
1. Grow a 125-500 mL culture of the E. coli strain harboring the plasmid of interest.
2. Purify plasmid DNA using the QIAprep Plasmid Maxi Kits (Qiagen) per the
manufacturer's instructions.
3. Precipitate and wash plasmid DNA to concentrate and remove salts ( see Note 12 ).
4. Resuspend the pellet in dH 2 O to a final concentration of 5 μg/μL.
5. Place concentrated plasmid DNA on ice for use the same day. Alternatively, store
at -20°C for several days.