Difference between revisions of "Protoplasts Formation, Regeneration and Transformation"

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(Instructions)
(Materials)
 
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*[[P buffer]]
 
*[[P buffer]]
 
*10.3% sucrose
 
*10.3% sucrose
*1 g polyethylene glycol 1000 (PEG)
 
 
*Lysozyme solution (1 mg/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.  
 
*Lysozyme solution (1 mg/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.  
 
*Autoclaved filter tubes/syringes plugged with cotton wool
 
*Autoclaved filter tubes/syringes plugged with cotton wool
*[[R5]] or [[R2YE]] plate
 
  
 
==Instructions==
 
==Instructions==

Latest revision as of 22:55, 24 March 2020

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 or 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. 109 spores/ml) or frozen stock in 20% glycerol
  • YEME medium
  • P buffer
  • 10.3% sucrose
  • Lysozyme solution (1 mg/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.
  • Autoclaved filter tubes/syringes plugged with cotton wool

Instructions

  1. Add 25 ml YEME medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. 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)
  2. Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
  3. Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
  4. Repeat step 3. The mycelial pellet, without added liquid, can be frozen at -20° before resuspension.
  5. 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.
  6. Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
  7. Add 5 ml P buffer. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
  8. Filter protoplasts through cotton wool and transfer to a fresh tube.
  9. Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
  10. Discard supernatant and suspend protoplasts in 1 ml P buffer.

Notes

  • Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
  • At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending P buffer (otherwise the protoplast pellet is difficult to disperse)
  • Avoid vortexing, which induces foaming and consequent lysis
  • To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
  • To thaw, shake the frozen tube under running warm water (i.e. 'freeze slowly, thaw quickly')
  • To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in P buffer and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

Further Manipulations of Protoplasts

Protoplast Fusion

Protoplast Transformation

Protoplast Transfection

Alternative Methods

References

Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK