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Propionibacterium freudenreichii

Multiporator / Electroporator 2510 Transformation Protocol Protocol No. 4308 915.527 04/2002 Microorganism Propionibacterium freudenreichii Cell type Bacteria, gram positive Molecules injected Plasmid DNA Growth medium SLB medium Washing solution Ice-cold 0.5 M sucrose Electroporation solution Ice-cold 0.5 M sucrose buffered with 1 mM potassium acetate (pH 5.5) Outgrowth medium SLB medium with 0.5 M sucrose Cuvette 1 mm gap width Reference Jore, J. P M. et al 2001 Applied and Environmental Microbiology 67, No. 2 499-503 Making electrocompetent cells:

1. Cultivate cells anaerobically at 30 C in SLB medium to the stationary growth phase. Dilute 1:50 in fresh SLB medium and incubate again for about 20 h. 2. Harvest in the exponential growth phase by centrifugation and wash extensively with ice-cold 0.5 M sucrose. 3. Wash once in ice-cold electroporation solution. 4. Resuspend in ice-cold electroporation solution (abo ut 1/100 of the original culture volume).

Electroporation of cells:

  1. Add plasmid DNA to 80-100 l of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into prechilled cuvette.
  2. Wipe moisture from the cuvette and insert the cuvette into the device.
  3. Electroporation:

    Mode Prokaryotes O Voltage (V) 2,000 V Time constant (T) 5 ms
  4. Immediately add 900 l cold outgrowth medium and incubate for 2.5-3 h at 30 C.
  5. Plate cells onto selective SLB plates; incubate 5-7 days at 30 C under anaerobic conditions.
Expected Results: Transformation efficiency up to 1 x 108 transformants/g of DNA.



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