R2

R2

Preparation

Make up the following solution:

• 103 g Sucrose
• 0.25 g K₂SO₄
• 10.12 g MgCl₂.6H₂O
• 10 g Glucose
• 0.1 g Difco Casaminoacids
• 800 ml dH₂O

Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar. Sterilise by autoclaving. At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed:

• 1 ml KH₂PO₄ (0.5%)
• 8 ml CaCl₂.2H₂O (3.68%)
• 1.5 ml L-proline (20%)
• 10 ml TES buffer (5.73%, adjusted to pH7.2)
• 0.2 ml ¹Trace element solution
• 0.5 ml NaOH (1N) (unsterilised is okay)
• 0.75 ml Required growth factors for auxotrophs

¹Trace element solution(l^-1):

• • 40 mg ZnCl₂
  • 200 mg FeCl₃.6H₂O
  • 10 mg CuCl₂.2H₂O
  • 10 mg MnCl₂.4H₂O
  • 10 mg Na₂B₄O₇.10H₂O
  • 10 mg (NH₄)₆Mo₇O₂₄.4H₂O

Uses

• Protoplasts regeneration

Notes

Streptomyces clavuligerus does not utilise glucose and so, it must be substituted by glycerol

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

Hopwood DA, Wright HM (1978) Bacterial protoplast fusion-. recombination in fused protoplasts of Streptomyces coelicolor. Mol.Gen.Genet., 162, 307-317

Okanishi M, Suzuki K, Umezawa H (1974) Formation and reversion of streptomycete protoplasts: cultural conditions and morphological study. J.Gen.Microbiol., 80 389-400