1988 Fiscal Year Final Research Report Summary
Construction of genetic analysis system in Nocardia brasiliensis
| Project/Area Number |
62571000
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Toho University |
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Principal Investigator |
KOYAMA Yasumasa School of Pharmaceutical Science, Toho University, 薬学部, 教授 (10104142)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Michiyasu School of Pharmaceutical Science, Toho University, 薬学部, 助手 (60163581)
KATO Fumio School of Pharmaceutical Science, Toho University, 薬学部, 助教授 (50057767)
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| Project Period (FY) |
1987 – 1988
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| Keywords | Nocardia brasiliensis / Plasmid / Protoplast / プロトプラスト / 再生 |
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| Research Abstract |
1. Conjugation system: There could not find any recombinant by conjugation between various auxotrophic mutants derived from 4 strains of N. brasiliensis.
2. Sensitization of N. brasiliensis to lysozyme: N. brasiliensis shows high resistance to lysozyme. But the incubation at 32C with glycine changed the strains to lysozyme sensitive.
3. Isolation of plsmid: Mycelia prepared from the incubation at 32C with glycine were lysed. Five of 7 strains harboured plasmids (pNB1,2,3 and 4).
4. Preparation and regeneration of protoplasts: Protoplasts were prepared from the mycelia. Yields of portoplasts varied from strain to strain (10^6-10^8/50ml culture).Regeneration of the protoplast occured on the regeneration medium containing 0.4M mannitol and small amount of bovine serum albumin, or horse serum. No regeneration was observed on the media containg sucrose or Na-succinate.
6. Developement of host strain: Temperature sensitive mutants from IFM0236 and carotenoid non producing mutant from IFM15 were isolated. The latter mutant gave good protoplast yield (10^9/50ml culture).
7. Effect of PEG on the regeneration of protoplast: Treatment with 20-25% PEG 1000 or 4000 for 1 min decreased the regeneration freg. to under 1/2. It would be nedessary to examine other transformation methods.
8. Vector plasmid: As a marker gene of vector, carotenoidd gene (car) was selected. First, car of streptomycetes was cloned in streptomyces setonii. Hybridisation between restriction enzyme dijested chromosomal DNA of N. brasiliensis and car DNA of S. setonii gave strong bands. The DNA fragment would have car of N. brasiliensis will be ligated with plasmid DNA of N. brasiliensis.
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