Physarum DNA Transformation by Electroporation

Tim Burland (Last Update June 9, 1993)

1. Grow amoebae axenically

Keep amoebae growing exponentially in SDM - subculture to 2-5x10⁵/ml before cell density reaches 10⁷/ml.  Mean generation time should be 24 hours or less.

2. Harvest amoebae & prepare for electroporation

Determine how many amoebae you need.  Typically, 5x10⁷ amoebae are electroporated in one electrical discharge.

• For LUC expression, use 1-2x10⁷ amoebae per sample for assay
• For CAT expression, use 5x10⁷ amoebae per sample
• For Hygromycin selection, use ~5x10⁷ amoebae per plasmid

Harvest amoebae by centrifugation for 2min at 200 x g

Wash the amoebae once in the original volume of ZAPP at room temp.

Resuspend the washed amoebal pellet in room-temp ZAPP.

Electroporate at 5x10⁷ cells per 600ml

Transfer cells to refrigerator; DO NOT PUT ON ICE YET! Incubate 3hr in refrigerator

Transfer cells to ice, add 0.5-20 ug DNA

3. Electroporate amoebae

Immediately transfer amoebae - DNA mixtures in 600-uL portions to ice-cold BioRad 4-mm cuvettes (Cat# 165-2088)

Electroporate once with BioRad Gene Pulser at 0.8kV, 25uF with 800 ohm set on pulse controller

Immediately transfer cells to tubes at 30^o and let recover for 20 min. Survival should be 10-80%

4. Grow amoebae to allow expression of transforming DNA

For each 600ul cells electroporated, add 5ml SDM.

For short-term CAT & LUC  expression assays, cells need no further dilution; just incubate on shaker at 26^o for 2-10hr

For long-term expression (>12hr) and for selecting cells stably resistant to Hygromycin B, transfer cells to SDM containing 200ug/ml each Penicillin G and Streptomycin sulphate

For  selecting Hygromycin resistance, incubate cells on shaker at 26^o for 2 days

NOTE:

The cuvettes may vary, and will wear after repeated use, causing differences in electrical performance; use luciferase expression assay to check optimal electrical discharge for your cuvettes