CuteChlamyPreparation of Gamete Autolysin

An improved method, contributed by Sabine Waffenschmidt, May 2005

Preparation of g-lysin (autolysin) from plates

1. Inoculate 1-2 ml of liquid culture of each mating type separately on regular TAP or Sager-Granick agar plates (we use the R3+ and NO- strain, originally isolated by Ursula Goodenough and selected for high mating efficiency; available from the Chlamydomonas Resource Center as CC-620 and CC-621) and let them grow under continuous light for 5 days.2. Flood plates with 5 ml SG-N (can be TAP-N or distilled water as well) and keep plates under light for another hour. Then wash cells off of by using up to 5 ml of SG-N.

3. Pour cells into a beaker (prefer a wider one so that the height of volume is not exceeding 2-3 cm, gametes don’t like it, don’t know why) and shake them under light until flagella have developed (might take another hour, check aliquot under the microscope).

4. Mix mating type plus and minus cells (control mating under the microscope) and let the mating reaction proceed for not more than 30 minutes (otherwise g-lysin activity is reduced, the enzyme most probably adsorbs to cell walls).

5. Spin quadriflagellated and non-mated cells down (5000 g for 10 minutes). The supernatant is ready for use as crude g-lysin, but can be ammonium sulfate precipitated (60 % saturation) to concentrate and frozen after dialysis (we do this with 10 mM Tris pH 7, 0.1 mM Ca2+, 1 mM Mg2+).