( ...)Borrelia burgdorferi

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.506 12/2001 Microorganism Borrelia burgdorferi B31 Cell type Bacteria, gram negative Molecules injected Linear DNA (in water) Growth medium Barbour-Stoenner-Kelly (BSK) II medium without gelatin Washing solution PBS, without divalent cations; EPS (272 mM sucrose, 15% glycerol) Electroporation solution EPS (272 mM sucrose, 15% glycerol) Outgrowth medium BSK II medium Cuvette 2 mm gap width Reference Samuels D.S., et al 1994 Journal of Bacteriology 176 6045-6049

Making electrocompetent cells:

1. Grow cells in 500 ml BSK II at 32 C to a cell density of 3-7 x 10⁷ cells/ml.


2. Harvest by centrifugation (4,000 x g, 20 min., 4 C). Wash twice in 60 ml cold PBS and pellet by centrifugation (3,000 x g, 10 min., 4 C).


3. Wash three times in 20 ml cold EPS and pellet by centrifugation (2,000 x g, 10 min., 4 C).


4. Resuspend the cells in 0.6 ml cold EPS.

Electroporation of cells:

1. Add 0.3 to 1 g DNA to 50 l of electrocompetent cells on ice. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette.


2. Wipe moisture from the cuvette and insert the cuvette into the device.


3. Electroporation:


Mode Prokaryotes O Voltage (V) 2,500 V Time constant (T) 5 ms

4. Immediately add 1 ml BSK II medium, transfer cells to a 15 ml tube and add another 9 ml of BSK II. Incubate for 24-48 hours at 32 C with shaking.
5. Plate 0.1 ml of culture on solid BSK II medium. Pellet the remaining 9.9 ml (4,000 x g, 10 min.), resuspend in 1 ml of supernatant and plate on selective plates. Incubate for 14 days, at 32 C in a humidified 5% CO₂ chamber.

Expected results:

Transformation efficiency up to 10³ transformants/g of DNA.

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1. Borrelia burgdorferi