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Method for breeding microorganisms by plasma-induced mutation

A technology of plasma and microorganisms, applied in the field of plasma mutagenesis of microorganisms, can solve the problems of low mutagenesis efficiency and achieve the effects of high mutagenesis efficiency, rich variety, low equipment and experiment costs

Inactive Publication Date: 2010-01-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patent CN1888063A reports microbial mutagenesis with atmospheric pressure cooling plasma, but the plasma generator used in this patent is a conventional dielectric barrier discharge plasma generator with a high working voltage (1-100kv), and the sample needs to be placed on the electrode The treatment between the plates is mainly due to the ultraviolet rays and charged particles generated during the plasma excitation process. After treatment, it must be incubated in the dark, and the main effect of this technology is the ultraviolet effect, so the mutagenesis efficiency is relatively low

Method used

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  • Method for breeding microorganisms by plasma-induced mutation
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  • Method for breeding microorganisms by plasma-induced mutation

Examples

Experimental program
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Effect test

preparation example Construction

[0036] 1. Preparation of sample slides:

[0037] 1) Preparation of bacterial suspension or spore suspension:

[0038] Cultivate the bacteria to the logarithmic growth phase, then take the culture solution and centrifuge at a speed of 5000-10000r / min for 2 minutes, collect the precipitate and wash it 2-3 times with buffer, sterile water or saline, and dilute it to 10 1 -10 9 The bacterial suspension of cfu / ml, preferably 10 6 cfu / ml to prepare the bacterial suspension.

[0039] Under sterile conditions, pick the spore colony on the solid plate into a test tube filled with 0.5-5ml of buffer solution, sterile water or physiological saline, preferably 2ml, stir with a glass rod, and oscillate to disperse the spores evenly to obtain spores Suspension; or add 0.5-5ml buffer solution, sterile water or physiological saline to the inclined test tube, preferably 2ml, use the inoculation shovel to scrape off the spores on the surface of the medium, then stir with a glass rod, and shak...

Embodiment 1

[0055] Embodiment 1, utilizing plasma to process DNA

[0056] The DNA used in this example is as follows:

[0057] pUC119, 3162bp, was purchased from TaKaRa Bio (Catalog No: D3318 or D3319). Concrete experimental steps are as follows in this embodiment:

[0058] 1) Sample slide preparation:

[0059] Dilute the pUC119 or pUC118 plasmid with sterile water, and drop 5 μL of the solution onto a clean slide for later use.

[0060] 2) Sample processing:

[0061] a. Wipe the slide placement area on the stage with 75% alcohol, and then close it;

[0062] b. Place the prepared sample slide on the stage so that the distance between the slide and the jet outlet is 2-4 mm;

[0063] c. Open the working gas valve. The working gas, that is, the discharge gas is helium;

[0064] d. Turn on the external power supply, and the external voltage is 200V, 13.56MHz RF voltage;

[0065] e. At this time, the jet temperature reaches 30±3°C;

[0066] f. The sample is irradiated. The irradiatio...

Embodiment 2

[0070] Embodiment 2, utilizing plasma mutagenesis Streptomyces avermitilis (Streptomyces avermitilis)

[0071] The medium used in this embodiment is as follows:

[0072] Solid medium: Gord's I medium. Autoclave at 121°C for 15 minutes.

[0073] Seed medium: Each liter medium contains 30g of potato starch, 2g of malt extract (Beijing Laibo Institute of Biological Experimental Materials, 20070610), 2g of soybean peptone (Beijing Shuangxuan Microbial Culture Medium Products Factory, 02-31), 2g of CoCl 2 ·6H 2 O 5mg, the balance is water; the pH of the seed medium is 7.0-7.2. Autoclave at 121°C for 15 minutes.

[0074] Fermentation medium: Each liter of medium contains 50g of potato starch, 12g of yeast powder (Beijing Aoboxing Biotechnology Co., Ltd., 01-14), MgSO 4 ·7H 2 O 0.5g, K 2 HPO 4 ·3H 2 O 0.5g, KCl 4g, CaCO 3 2g, CoCl 2 ·6H 2 O 5mg, the balance is water; the pH of the fermentation medium is 7.0-7.2. Autoclave at 121°C for 15 minutes.

[0075] Concrete experim...

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Abstract

The invention discloses a method for breeding microorganisms by plasma-induced mutation, which radiates microorganisms to be induced to mutate with a plasma jet flow that uses neutral active particles as acting particles to obtain mutated microorganisms. The method in which the neutral active particles in the plasma jet flow play a main induction role has the advantages of: ensuring the high activity of forward mutant strains and the high capability and genetic stability of produced target products, reducing mutagenesis period and improving mutagenesis efficiency, inducing a great variety of microorganisms such as prokaryotic microorganisms, eukaryotic microorganisms and archaebacteria in form of microbial community, bacterial suspension or spore suspension, simplifying experimental operation by avoiding culturing the processed microorganisms in the dark, having the characteristics of simple, safe and pollution-free whole operation, low equipment and experiment cost and the like and having great potential role in the field of industrial microorganism induced mutation breeding.

Description

technical field [0001] The invention relates to a method for mutation breeding of microorganisms, in particular to a method for using plasma to induce mutations in microorganisms. Background technique [0002] Microbial strains are the foundation and key of the fermentation industry. The key to improving the efficiency of the fermentation industry and reducing product costs lies in whether high-quality microbial strains can be bred. Therefore, microbial breeding is becoming more and more important. With the continuous progress of research, many different levels of microbial breeding technologies have emerged and gradually matured. Among them, mutation breeding has made rapid progress in the fields of transforming microbial strains, solving some special problems in breeding work, and developing new products. disadvantages, and compared with genetic engineering, mutation breeding is simple to operate, especially for transforming those strains with complex metabolic networks, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N15/01C12R1/465C12R1/01
Inventor 邢新会王立言赵洪新吴昊黄子亮缑仲轩丁楠李和平李果包成玉王森孙文廷
Owner TSINGHUA UNIV
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