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Mutation screening method and application of streptomyces strain SPC6-50-1

A technology of mutagenesis screening and Streptomyces, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as small-scale excision, frameshift mutation, etc., and achieve short cycle, high efficiency, and simple operation. Effect

Active Publication Date: 2014-06-25
COLD & ARID REGIONS ENVIRONMENTAL & ENG RES INST CHINESE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

NTG-induced mutations are mainly GC-AT transitions, in addition to small excisions, frameshift mutations, and loss of GC pairs

Method used

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  • Mutation screening method and application of streptomyces strain SPC6-50-1
  • Mutation screening method and application of streptomyces strain SPC6-50-1
  • Mutation screening method and application of streptomyces strain SPC6-50-1

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Mutagenesis of Streptomyces by chemical mutagen NTG, such as figure 1 Shown:

[0029] 1. Treatment method of NTG mutagenic strains:

[0030] 1. Preparation of the medium and the solution used:

[0031] TSBY (1L): 30g of Oxoid tryptone bean soup powder (TSB); 340g of sucrose; 5g of Oxoid yeast extract; distilled water to 1000ml.

[0032] YEME (1L): Oxoid Yeast Extract 3g; Oxoid Peptone Tryptone 5g; Malt Extract 3g; Glucose 10g; Sucrose 340g; MgCl 2 -6H 2 O (sterilized alone); distilled water to 1000ml.

[0033] pH6.0 Phosphate buffer: K 2 HPO 4 2g / L, KH 2 PO 4 8g / L, 0.1MPa sterilization for 20min.

[0034] Preparation of NTG mother liquor: Weigh 0.1g NTG plus 1mL of co-solvent formamide or acetone, then add 9mL of 0.1mol / L pH6.0 phosphate buffer to prepare 10mg / mL NTG mother liquor.

[0035] 2. Strain preparation:

[0036] The experimental strains were Streptomyces coelicolor A3(2) and Streptomyces sp.Lzsg6 (isolated from the sandy reed rhizosphere soil in Lin...

Embodiment 2

[0044] Screening of strains with mutations in the transcriptional system by rifampicin:

[0045] Spread the NTG-treated bacterial suspension evenly on Gao’s No. 1 medium plate containing 5 μg / ml rifampicin, culture at 30°C for 7-15 days, and inoculate the grown single colonies into 5 mL of liquid medium In the test tube of YEME or TSBY, and then prepare a mutagenized bacterial suspension for NTG mutagenesis, evenly spread the treated bacterial suspension on the Gaoshi No. 1 medium plate containing 10 μg / ml rifampicin, so Repeatedly, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, 50 μg / ml rifampicin screening was carried out to obtain rifampicin-insensitive strains.

Embodiment 3

[0047] Strains with mutations in the translation system by screening with streptomycin:

[0048] The strains screened by rifampicin were then subjected to NTG mutagenesis in the same way as above, and the treated bacterial suspension was evenly spread on the Gaoshi No. 1 medium plate containing 5 μg / mL streptomycin and 50 μg / mL rifampicin. Cultivate at 30°C for 7-15 days, inoculate the grown single colonies into test tubes containing 5mL of liquid medium YEME or TSBY, and then prepare a mutagenized bacterial suspension for NTG mutagenesis. The solution was evenly spread on the Gaoshi No. 1 medium plate containing 10 μg / mL streptomycin and 50 μg / mL rifampicin, so repeatedly, the concentration of rifampicin was 50 μg / mL, the concentration of streptomycin was 15 μg / ml, and the concentration of 20 μg / ml, 30μg / ml, 40μg / ml, 50μg / ml, 60μg / ml, 70μg / ml, 80μg / ml, 90μg / ml, 100μg / ml are increased in sequence to obtain strains insensitive to streptomycin and rifampin .

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Abstract

The invention discloses a mutation screening method of a streptomyces strain SPC6-50-1. The mutation screening method comprises the steps of inducing mutation of streptomyces coelicolor and streptomyces Lzsg6 through nitrosoguanidine to obtain a streptomyces mutant library, and screening the mutant library through rifampicin and streptomycin to obtain a strain with a transcription system and a translation system in mutation. The invention also discloses a streptomyces strain SPC6-50-1 and application thereof. The streptomyces strain SPC6-50-1 disclosed by the invention has the beneficial effects that the strain contains substances having bacteriostatic activities, and the method of nitrosoguanidine (NTG) inducing as well as rifampicin and streptomycin screening can activate silent genes of the streptomyces to obtain a new metabolite, thus obtaining a new antibiotic; meanwhile, the mutation screening method has the advantages of simple operation, short cycle and high efficiency, and is not only applicable to streptomyces but also suitable for such microorganisms as myxobacteria rich in secondary metabolites; the mutation screening method has good popularization and application values.

Description

technical field [0001] The invention relates to the field of antibiotic production, in particular to a mutagenesis screening method for Streptomyces strain SPC6-50-1, Streptomyces strain SPC6-50-1 and applications thereof. Background technique [0002] Since 1928, when the antibiotic penicillin was discovered for the first time, in the past few decades, the antibiotic industry has developed rapidly and made great contributions to human health. However, due to the irrational use of antibiotics, especially in my country, the problem of antibiotic resistance has gradually broken out. To solve this problem, in addition to paying attention to the rational use of antibiotics, it is also very important to develop new antibiotics that have inhibitory activity against drug-resistant bacteria. [0003] Streptomyces is a prokaryotic, Gram-positive bacterium that widely exists in nature, especially in soil, and belongs to the phylum Actinomycetes. Streptomyces has complex morphologica...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N1/20C12P1/06C12R1/465
Inventor 张昺林陈熙明张威张满效陈拓刘光琇
Owner COLD & ARID REGIONS ENVIRONMENTAL & ENG RES INST CHINESE
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