NMMP: Difference between revisions
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=Minimal liquid medium (NMMP)= | =Minimal liquid medium (NMMP)= | ||
NMMP medium is used to obtain dispersed growth of ''Streptomyces'' sp. with specific carbon sources. | NMMP medium is used to obtain dispersed growth of ''Streptomyces'' sp. with specific carbon sources.<sup>[2]</sup> | ||
==Preparation== | ==Preparation== | ||
Make up the following solutions: | Make up the following solutions: | ||
Minor elements solution: (per litre) | '''Minor elements solution''': (per litre) | ||
*1g ZnSO<sub>4</sub>*7H<sub>2</sub>O | *1g ZnSO<sub>4</sub>*7H<sub>2</sub>O | ||
*1g FeSO<sub>4</sub>*7H<sub>2</sub>O | *1g FeSO<sub>4</sub>*7H<sub>2</sub>O | ||
| Line 12: | Line 12: | ||
*1g CaCl<sub>2</sub>, anhydrous | *1g CaCl<sub>2</sub>, anhydrous | ||
Phosphate buffer | '''Phosphate buffer''' | ||
*Mix 0.1M solutions of NaH<sub>2</sub>PO<sub>4</sub> and K<sub>2</sub>HPO<sub>4</sub> to give pH 6.8 | *Mix 0.1M solutions of NaH<sub>2</sub>PO<sub>4</sub> and K<sub>2</sub>HPO<sub>4</sub> to give pH 6.8 | ||
Carbon source | '''Carbon source''' | ||
*20% solution, sterilized separately | *20% solution, sterilized separately | ||
NMMP | '''NMMP''' | ||
*2 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> | *2 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub> | ||
*5 g Difco Casaminoacids | *5 g Difco Casaminoacids | ||
| Line 32: | Line 32: | ||
#Make up the phosphate buffer solution, carbon source, and any other required growth factors, and autoclave these separately | #Make up the phosphate buffer solution, carbon source, and any other required growth factors, and autoclave these separately | ||
#Just before using, add: | #Just before using, add: | ||
15 mL NaH<sub>2</sub>PO<sub>4</sub>/K<sub>2</sub>HPO<sub>4</sub> buffer (0.1M, pH 6.8) | |||
2.5 mL carbon source (20%) | |||
2.5 mL any other required growth factors (add 2.5 mL sterile water if none) | |||
==Uses== | ==Uses== | ||
| Line 41: | Line 41: | ||
==Notes== | ==Notes== | ||
*Using PEG-6000 and adding stainless steel springs in the culture flasks allows for good dispersed growth of many ''Streptomyces'' strains.<sup>[1]</sup> | |||
*Agar can be added (20 g/L) to make solid NMMP.<sup>[3]</sup> | |||
*A modified version of NMMP suitable for <sup>14</sub>N/<sup>15</sup>N labelling has been developed.<sup>[4]</sup> | |||
==References== | ==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 | [1] Hodgson (1982). Glucose Repression of Carbon Source Uptake and Metabolism in ''Streptomyces coelicolor'' A3(2) and its Perturbation in Mutants Resistant to 2-Deoxyglucose. Journal of General Microbiology 128: 2417-2430. | ||
[2] 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 | |||
[3] Rico, S., Yepes, A., Rodríguez, H., Santamaría, J., Antoraz, S., Krause, E. M., … Santamaría, R. I. (2014). Regulation of the AbrA1/A2 two-component system in ''Streptomyces coelicolor'' and the potential of its deletion strain as a heterologous host for antibiotic production. PloS one, 9(10), e109844. doi:10.1371/journal.pone.0109844 | |||
[4] Świątek, M. A., Gubbens, J., Bucca, G., Song, E., Yang, Y. H., Laing, E., … van Wezel, G. P. (2013). The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in ''Streptomyces coelicolor''. Journal of bacteriology, 195(6), 1236–1248. doi:10.1128/JB.02191-12 | |||
Revision as of 10:23, 29 July 2019
Minimal liquid medium (NMMP)
NMMP medium is used to obtain dispersed growth of Streptomyces sp. with specific carbon sources.[2]
Preparation
Make up the following solutions:
Minor elements solution: (per litre)
- 1g ZnSO4*7H2O
- 1g FeSO4*7H2O
- 1g MnCl2*4H2O
- 1g CaCl2, anhydrous
Phosphate buffer
- Mix 0.1M solutions of NaH2PO4 and K2HPO4 to give pH 6.8
Carbon source
- 20% solution, sterilized separately
NMMP
- 2 g (NH4)2SO4
- 5 g Difco Casaminoacids
- 0.6g MgSO4*7H2O
- 50g PEG 6000
- 1 mL minor elements solution
- 800 mL ddH2O
Instructions
- Make up the NMMP solution described above
- Dispense in 80 mL aliquots
- Autoclave
- Make up the phosphate buffer solution, carbon source, and any other required growth factors, and autoclave these separately
- Just before using, add:
15 mL NaH2PO4/K2HPO4 buffer (0.1M, pH 6.8) 2.5 mL carbon source (20%) 2.5 mL any other required growth factors (add 2.5 mL sterile water if none)
Uses
- Routine growth of Streptomyces sp. in liquid culture
Notes
- Using PEG-6000 and adding stainless steel springs in the culture flasks allows for good dispersed growth of many Streptomyces strains.[1]
- Agar can be added (20 g/L) to make solid NMMP.[3]
- A modified version of NMMP suitable for 14N/15N labelling has been developed.[4]
References
[1] Hodgson (1982). Glucose Repression of Carbon Source Uptake and Metabolism in Streptomyces coelicolor A3(2) and its Perturbation in Mutants Resistant to 2-Deoxyglucose. Journal of General Microbiology 128: 2417-2430.
[2] 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
[3] Rico, S., Yepes, A., Rodríguez, H., Santamaría, J., Antoraz, S., Krause, E. M., … Santamaría, R. I. (2014). Regulation of the AbrA1/A2 two-component system in Streptomyces coelicolor and the potential of its deletion strain as a heterologous host for antibiotic production. PloS one, 9(10), e109844. doi:10.1371/journal.pone.0109844
[4] Świątek, M. A., Gubbens, J., Bucca, G., Song, E., Yang, Y. H., Laing, E., … van Wezel, G. P. (2013). The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in Streptomyces coelicolor. Journal of bacteriology, 195(6), 1236–1248. doi:10.1128/JB.02191-12
