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=R2= | =R2= | ||
[[File:SCOR2.jpg | ''Streptomyces coelicolor'' A3(2) growing on a cellophane disk on R2 agar | thumb]] | |||
R2 is a medium developed for actinorhodin production from ''Streptomyces coelicolor'', note the blue pigmentation in the right image of ''Streptomyces coelicolor'' A3(2). This blue colour is due to the overproduction of redox active antibiotic actinorhodin. | |||
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==Preparation== | ==Preparation== | ||
Make up the following solution: | Make up the following solution: | ||
*103 g Sucrose | |||
*0.25 g K<sub>2</sub>SO<sub>4</sub> | |||
*10.12 g MgCl<sub>2</sub>.6H<sub>2</sub>O | |||
*10 g Glucose | |||
*0.1 g Difco Casaminoacids | |||
*800 ml dH<sub>2</sub>O | |||
Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar. | Pour 80 ml of the solution into 250 ml Erlenmeyer flasks each containing 2.2 g Difco Bacto agar. | ||
Sterilise by autoclaving | Sterilise by autoclaving. | ||
At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed: | At the time of use, remelt the medium and add to each flask the following autoclaved solutions in the order listed: | ||
*1 ml KH<sub>2</sub>PO<sub>4</sub> (0.5%) | *1 ml KH<sub>2</sub>PO<sub>4</sub> (0.5%) | ||
| Line 19: | Line 26: | ||
*1.5 ml L-proline (20%) | *1.5 ml L-proline (20%) | ||
*10 ml TES buffer (5.73%, adjusted to pH7.2) | *10 ml TES buffer (5.73%, adjusted to pH7.2) | ||
*0.2 ml | *0.2 ml [[Trace element solution]] | ||
*0.5 ml NaOH (1N) (unsterilised is okay) | *0.5 ml NaOH (1N) (unsterilised is okay) | ||
*0.75 ml Required growth factors for auxotrophs | *0.75 ml Required growth factors for auxotrophs | ||
==Uses== | ==Uses== | ||
*Protoplasts regeneration | |||
==Notes== | ==Notes== | ||
''[[Streptomyces clavuligerus]]'' does not utilise glucose and | ''[[Streptomyces clavuligerus]]'' does not utilise glucose and it must be substituted by glycerol. | ||
Latest revision as of 15:31, 26 September 2019
R2
R2 is a medium developed for actinorhodin production from Streptomyces coelicolor, note the blue pigmentation in the right image of Streptomyces coelicolor A3(2). This blue colour is due to the overproduction of redox active antibiotic actinorhodin.
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 Trace element solution
- 0.5 ml NaOH (1N) (unsterilised is okay)
- 0.75 ml Required growth factors for auxotrophs
Uses
- Protoplasts regeneration
Notes
Streptomyces clavuligerus does not utilise glucose and 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
