Protoplasts

Protoplasts are cells that have lost their cellular wall, but are kept whole in medium. These protoplasts appear perfectly round when checked under the optical microscope.

Materials

• SCS (1.2M) buffer
• Cells in exponential phase
• Lallzyme MMX

Buffers

For the cell wall digestion, we will use certain buffers that we have made based on the protocol in Flor-Parra et al 2014, doi:10.1002/yea.2994:

• SCSa
  • 20mM Sodium Citrate
  • 1.2M D-sorbitol
• SCSb
  • 20mM Citric Acid
  • 1.2M D.sorbitol
• SCS: Mix equal amounts of SCSa and SCSb. Add SCSa (alkaline) or SCSb (acidic) and keep checking the pH until it reaches 5.8

Generating the protoplasts

1. Grow Sz. pombe cells in YES overnight (the cells need to be in exponencial phase for ideal results).
2. The next morning, centrifuge the cells at 1800xg for 3min.
3. Wash the cells with SCS buffer and centrifuge again
   1. While this is happening, prepare 4ml of SSC in a 15ml tube, adding 0.25g of Lallzyme MMX directly in the mix. This will need a fair amount of vortexing
4. When the MMX is ready, resuspend the cells in the mix and leave in the 32ºC incubator, slowly shaking
   1. Don't put directly in the shaker, as the protoplasts would be too fragile for this
5. Keep checking the cells every 5 minutes using the microscope. First you will see how the cells get a hole in their wall, and the cell escapes through that hole.
6. Once >95% of the cells are round (20-30min), centrifuge at 170xg for 3min, and resuspend in EMM + 1.2M sorbitol (depending on what you need the cells for).