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Microbe mutagenizing atmospheric cold plasma method

A technology of cold plasma and atmospheric pressure, applied in biochemical equipment and methods, microorganisms, treatment of microorganisms with electricity/wave energy, etc., can solve the problems of low mutagenesis efficiency, polluted environment, long mutagenesis cycle, etc., and achieve shortened mutagenesis The effect of changing time, simple experimental operation, and improving mutagenesis efficiency

Inactive Publication Date: 2007-01-03
DALIAN UNIV OF TECH
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  • Summary
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

It solves the problems of high equipment cost, environmental pollution, damage to human health, low mutagenesis efficiency, long mutagenesis cycle, and high cost in the traditional physical and chemical mutagenesis processes, and is expected to provide a feasible and efficient method for industrial microbial breeding. new technology of microbial mutagenesis

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment one: bacterial suspension mutagenesis Klebsiella

[0019] 1) The experimental strain is Klebsiella pneumoniae, which is a facultative anaerobic bacterium. Purchased from China General Microorganism Culture Collection Center (CGMCC), the culture preservation number is 1.1736.

[0020] The medium composition is as follows (1L):

[0021] Glycerin: 20g K 2 HPO 4 ·3H 2 O: 4.454g

[0022] K H 2 PO 4 : 1.3g (NH 4 ) 2 SO 4 : 2.0g

[0023] MgSO 4 ·7H 2 O: 0.2g Yeast powder: 1.0g

[0024] CaCO 3 : 2.0g Trace elements: 2mL

[0025] 2% CaCl 2 Solution: 1 mL 0.5% FeSO 4 Solution: 1mL

[0026] Trace element composition (1L):

[0027] Saturated hydrochloric acid: 0.9mL ZnCl 2 : 70mg

[0028] MnCl 2 4H 2 O: 100mgH 3 BO 3 : 60mg

[0029] CoCl 2 ·6H 2 O: 200mg NiCl 2 ·6H 2 O: 25mg

[0030] NaMoO 4 2H 2 O: 35mg CuCl 2 2H 2 O: 20mg

[0031] 2) Strain pretreatment: the strain is inserted into the seed culture solution with 1% inoculum amount, c...

Embodiment 2

[0036] Embodiment two: plate mutagenesis Klebsiella

[0037] 1) The experimental strains, fermentation process and culture medium are the same as in Example 1.

[0038] 2) Strain pretreatment: After the high-yield strain of Example 1 was subjected to plasma secondary mutagenesis for 4 minutes, it was inserted into the seed culture solution with 1% inoculum, and cultured on a shaking table at 37°C and 150r / min until the For several phases, the OD value was measured to be 2.79, and the bacterial solution was diluted with 0.05M, pH 7.0 potassium phosphate buffer solution for 10 -5 After doubling, 50 μL of bacterial suspension was evenly spread on solid medium containing 8% glycerol, and the diameter of the plate was 6 cm.

[0039] 3) Strain mutagenesis: the distance between the two plate electrodes of the atmospheric pressure dielectric barrier discharge cold plasma is 3mm, the discharge gas is air, the applied voltage is 8kV, the frequency is 6kHz, and the mutagenesis time is 3...

Embodiment 3

[0044] Example 3: Bacterial Suspension Mutagenesis of Saccharomyces cerevisiae

[0045] 1) The experimental strain is Saccharomyces cerevisiae, which belongs to fungi, and was purchased from China Industrial Microorganism Culture Collection Center (CICC), and the strain preservation number is CICC 1001.

[0046] The medium composition is as follows (1L):

[0047] Glucose: 30g; Peptone: 3g; Yeast powder: 3.85g

[0048] 2) Strain pretreatment: The strain is first activated once, that is, under aseptic operation, the yeast is inoculated on the solid medium of glucose-peptone-yeast powder slant, and cultured in an incubator at 28°C for 24 hours; To activate, take one ring of activated yeast and put it into a 250mL Erlenmeyer flask, which contains 100mL of glucose-peptone-yeast powder liquid medium, and cultivate it at 28°C and 150r / min for 24h; min, 0°C, centrifuged for 30 min, the precipitate was washed twice with 0.05M, pH 7.0 potassium phosphate buffer, and then centrifuged a...

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Abstract

The atmospheric cold plasma microbe mutagenesis method belongs to the field of microbe mutagenesis technology, and features the cold plasma mutagenesis of microbe strain to denature. The microbe strain may be pronucleus microbe or eukaryotic microbe; the atmospheric cold plasma may have discharging medium of air, nitrogen or helium with discharge period of 10 s to 10 min, applied voltage of 1-100 kV and frequency of 0-15 MHz; and mutagenesed microbe may be in suspension or plate. Compared with other physical and chemical mutagenesis method, the present invention has the advantages of simple apparatus, low cost, operation safety and high mutagenesis effect, and may be applied widely in industrial microbe breeding.

Description

technical field [0001] The invention belongs to the technical field of microbial mutagenesis, in particular to a method for mutagenizing microorganisms by using atmospheric pressure cooling plasma. Background technique [0002] Microbial breeding has played an extremely important role in promoting the development of the microbial industry. A large number of studies and production practices have shown that mutation breeding can overcome the shortcomings of conventional breeding, such as long cycle, slow process, and difficulty in obtaining mutant varieties, and can make breakthroughs in creating new microbial strains, new materials and solving some special problems in breeding work and rapid development. The achievements of microbial breeding will lead to a leap forward in the development of microbial industry. [0003] So far, the most important method of microbial breeding is to use chemical mutagenesis, physical mutagenesis or the combination of the two mutagenesis. Che...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00
Inventor 修志龙董晓宇李爽侯英敏于红张代佳任春生
Owner DALIAN UNIV OF TECH
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