Protoplasts Formation, Regeneration and Transformation

Protoplasts are used for fusion, transformation and transfection.

Protoplasts Preparation

Although this protocol was written for Streptomyces lividans and Streptomyces coelicolor, many Streptomyces species can be protoplasted and regenerated using this procedure written by Okanishi et al. (1974) and adapted by Bibb et al. (1978) and Thompson et al. (1982).

Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation/transfection frequencies in S. lividans are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of S. parvulus protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

Materials:

• Streptomyces spores, fresh suspension (c. 10[sup]9[/sup] spores/ml) or frozen stock in 20% glycerol
• YEME medium
• P buffer
• 10.3% sucrose
• Lysozyme solution (1mg/ml in P buffer, filter sterilised). For most purposes, lysozyme is dissolved in P buffer; use of L buffer gives more efficient transformation of S. lividans by plasmid DNA.
• Filter tubes plugged with cotton wool

Instructions:

1. Add 25 ml YEME medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of S. lividans and S. coelicolor are ready for harvesting when they start to produce red pigment)

1. Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant

1. Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant

1. Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.

1. Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for S. lividans and S. clavuligerus, whereas S. coelicolor requires 60 min.

1. Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.

1. Add 5 ml P buffer. Repeat step 6. This help to free protoplasts from the mycelium so that they will pass through the cotton wool.

1. Filter protoplasts through cotton wool and transfer to a fresh tube.

1. Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)

1. Discard supernatant and suspend protoplasts in 1 ml P buffer