Transposon Mutagenesis of the Lyme Disease Agent Borrelia burgdorferi - Bacterial Pathogenesis: Methods and Protocols - page 88

2. Once confirmed as transposon mutants by PCR, aspirate colonies from the plate
using a sterile Pasteur pipet and transfer to liquid BSK medium (5-15 mL)
containing appropriate antibiotic(s). Incubate cultures at 35 ºC until cells reach
high densities (between5×10 7 and1×10 8 cells/mL) ( see Note 7 ).
3. Isolate genomic DNA from 5 mL cultures by pelleting the cells, removing the
supernatant, and resuspending in 500 μL sterile H 2 O. Lyse cells by the addition
of 25 μL of 10% SDS and 5 μL of a 10 mg/mL RNase solution, and incubate at
room temperature for 5 min. Phenol:chloroform extractions are repeated (usually
two to three times) to remove protein and purify genomic DNA. Precipitate DNA
with ethanol or isopropanol, wash with 70% ethanol, and resuspend in 50 μL
sterile H 2 O or TE. Alternatively, genomic DNA can be isolated from a larger 15
mL volume using the Wizard Genomic DNA Purification Kit (Promega, Madison,
WI), following manufacturer's recommendations for gram-negative bacteria.
4. To isolate the transposon and DNA flanking the insertion site ( see Fig. 2 ), digest
approximately 500 ng of genomic DNA overnight with the restriction enzyme
Hind III in a 15 μL volume ( see Note 8 ).
5. Remove about 8 μL of digested DNA and ligate in 10 μL volume at 14 ºC for 6

h (or overnight). Transform entire ligation into chemically competent E. coli cells
and plate about half of the transformed cells on LB plates containing gentamicin.
Incubate plates at 37 ºC overnight.
6. Isolate plasmid DNA from E. coli colonies.
7. Sequence the B. burgdorferi DNA flanking the transposon insertion from the
plasmid DNA ( see Fig. 2 ) using primers flg and col ( see step 3, Subheading
2.4. ). Identify the transposon insertion site by submitting the sequence results
to the TIGR-CMR BLAST and search against

∼

the B. burgdorferi genome

(http://tigrblast.tigr.org/cmr-blast/) ( see Note 9 ).

4. Notes
1. The Borrelia promoters used to drive expression of the Himar transposase, the
gentamicin-resistance marker, and the kanamycin-resistance marker on pMarGent
and pGKT ( see Fig. 1 ) are recognized and functional in E. coli . The transposase is
therefore active in E. coli and allows transposition of the gentamicin marker during
growth of the culture for plasmid DNA isolation, maintaining the gentamicin resis-
tance of the cells but resulting in an inactive variant of pMarGent ( see Fig. 1A) .
The plasmid preparations derived from different E. coli colonies, therefore, yield
a range of plasmid forms and transposase activities. We generally isolate plasmid
DNA from about six different E. coli clones and test each plasmid preparation
in B. burgdorferi to determine which have a high transposition frequency. To
reduce the chances of isolating inactive plasmid preparations from E. coli ,we
constructed the vector pGKT ( see Fig. 1B ) by cloning the kanamycin-resistance
marker adjacent to the transposase gene of pMarGent. Growth of E. coli colonies
harboring pGKT in the presence of both antibiotics requires that both the trans-
posase (linked to the kanamycin marker) and the transposon portion of the plasmid