Electroporation of P. aeruginosa

1. Preparation of Electrocompetent Cells

    

   1. Inoculate a 5-ml of L-broth with cells of the P . aeruginosa strain to be electrotransformed. Grow culture overnight at 37°C on a roller.

       

   2. Transfer 2 ml of the overnight culture to 200 ml of fresh L-broth in a 1-liter sidearm flask. Grow this culture at 37°C with shaking (200 rpm) until OD540 = 0.3-0.5.

       

   3. Aliquot 35-40 ml of culture to each of four sterile Oak Ridge centrifuge tubes.

       

   4. Pellet the cells by centrifugation at 7000 x g (8000 rpm in SS-34 rotor) at 4°C for 10 minutes. Discard supernatant.

       

   5. Resuspend and wash the cells in an equal volume (35-40 ml) of ice-cold sterile 300 mM sucrose. Repellet the cells by centrifuging as before. Discard supernatant.

       

   6. Resuspend and wash the cells once again in 0.5 volume (18-20 ml) ice-cold 300 mM sucrose. Centrifuge as before. Discard supernatant.

       

   7. Resuspend the pellet in 0.01 volumes (350-400 µl) ice-cold 300 mM sucrose. The cell density at this point should be around 1 x 10(exp)11 cfu/ml.

       

   8. Chill the cells on ice for 30 minutes. P . aeruginosa cells thus prepared are ready for electroporation. Unlike E. coli , these cells cannot be electroporated with high efficency after being frozen.

       

    

2. Electroporation of the cells.

    

    

   1. Set the electroporation apparatus to 1.6-2.5 kV, 25 µF. Set the pulse controller to 200 omega.

       

   2. Add 1-5 µl plasmid DNA to tubes containing 40 µl of electrocompetent cells on ice. Mix by swirling with pipette tip. Transfer the DNA and cells to a pre-chilled electroporation cuvette (0.2 cm electrode gap) using a narrow pipette tip. Wipe any ice or water from sides of cuvette using a Kimwipe. Place the cuvette into the sample chamber.

       

   3. Energize the electroporation apparatus and deliver the pulse by pushing in both charging buttons simultaneously and holding until a short beep is heard. Note the time constant of the pulse and the actual voltage delivered.

       

   4. Remove the cuvette from the sample chamber. Add 3 ml L-broth and transfer the cells to a sterile polypropylene culture tube using a glass Pasture pipette. Incubate cultures for 2 hours at 37°C on a roller or with moderate shaking to allow for plasmid expression.

       

   5. Plate aliquots of the electroporation mixture on L-agar plates supplemented with the appropriate antibiotics. Incubate plates at 37°C.

       

   300 mM Sucrose