CN103911363A - Method and device for mutagenizing biomaterial - Google Patents
Method and device for mutagenizing biomaterial Download PDFInfo
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- CN103911363A CN103911363A CN201310749658.8A CN201310749658A CN103911363A CN 103911363 A CN103911363 A CN 103911363A CN 201310749658 A CN201310749658 A CN 201310749658A CN 103911363 A CN103911363 A CN 103911363A
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Abstract
The invention discloses a method and a device for mutagenizing a biomaterial and belongs to the field of biotechnology. The method comprises the following steps of 1, coating a solid medium with cells to be treated, and 2, mutagenizing the coating cells which comprise microbial cells and suspension cells of animals and plants. The method can substantially improve mutagenesis efficiency and screening efficiency and is conducive to mutagenesis breeding adopting automatic high-flux experimental equipment.
Description
Technical field
The present invention relates to biological test method, particularly a kind of high-throughput is processed method and the device of biomaterial.
Background technology
Current most laboratory to biomaterial particularly microorganism carry out the method for mutagenic treatment, its treating processes is mostly as follows: (1) mutagenic treatment: by the method such as physical mutagenesis or chemomorphosis, the microbiological specimens being contained in container is carried out to mutagenic treatment; (2) spread plate: after mutagenic treatment finishes, bacterium liquid is coated on solid medium and cultivates and grow single bacterium colony; (3) analysis list bacterium colony screening mutant strain.
Above-mentioned existing methodical shortcoming is, is contained in the microorganism under the liquid state in container, in the time accepting mutagenic treatment, especially, while accepting physical method processing, owing to mutually blocking between thalline, or liquid is for treatment dosage weakening effect, can reduce treatment effect, cause efficiency of inducing mutation low.
Above-mentioned treatment process is also unfavorable for high-throughput and automated operation.High efficiency microorganism mutagenesis, selection and technology are the study hotspots of current biological technical field, and have the very large market requirement; Coordinate High Throughput Screening Assay to develop efficient mutation method necessary.
Summary of the invention
The demand that the present invention exists according to above-mentioned field, provides a kind of method of mutagenic treatment biomaterial and installs accordingly.Technical scheme is as follows:
A method for mutagenic treatment biomaterial, is characterized in that adopting following steps:
(1) on solid medium, be evenly coated with pending cell,
(2) cell of coating is carried out to mutagenic treatment,
Described cell refers to microorganism cells.
Described mutagenic treatment refers to adopt physical method, and as ultraviolet ray, gamma-rays, plasma body, electromagnetic radiation etc., irradiation is coated the cell on solid medium.
Described mutagenic treatment is coated with chemical mutagen after referring to be coated with pending cell again, then cultivates.
After mutagenic treatment finishes, also comprise cultivation, described biomaterial cell refers to microorganism, and described cultivation comprises that cultivation grows up to bacterium colony.
Described cultivation also comprises expands numerous one-tenth bacterium liquid by the bacterium colony growing up to.
In described substratum, be added with chemical mutagen.
A method for mutagenic treatment biomaterial, is characterized in that:
(1) on the solid medium that is added with chemical mutagen, be coated with pending cell,
(2) cultivate the cell that is coated on solid medium.
After being coated with pending cell, the pending cell of coating is carried out to Physics Irradiation processing, described Physics Irradiation processing refers to employing ultraviolet ray, gamma-rays, plasma body, electromagnetic radiation etc., and irradiation is coated the cell on solid medium.
The cell that described coating is pending, coating concentration is: the density that makes the cell through growing at solid culture primary surface after mutagenic treatment is every square centimeter and is less than and equals 50.
The invention provides a kind of mutagenic treatment method of microorganism, difference from prior art is: the cell with mutagenic treatment is first evenly coated on nutrition flat board, then carried out mutagenic treatment.After mutagenic treatment, cultivate again seed selection mutant strain in single bacterium colony of turning out.The outstanding advantages of method of the present invention embodies both ways: can significantly improve efficiency of inducing mutation on the one hand.Be uniformly coated on the mutagenic treatment dosage that cell on flat board can both receive equality strength, the bacterium colony ratio of undergoing mutation in cultivating the bacterium colony growing after processing finishes increases substantially, and has effectively improved mutagenesis and screening efficiency; On the other hand, method of the present invention is easier to realize mutagenic treatment and screening operation in the high-throughput treatment facility that adopts automatization.
In method of the present invention, mutagenic treatment can refer to physical mutagenesis, comprises ultraviolet, gamma-rays, Cement Composite Treated by Plasma etc.; Also can be chemomorphosis processing, be included in and in plate culture medium, add chemomorphosis reagent and/or at plate culture medium surface coated chemical mutagen.Chemical mutagen can be any mutagenesis reagent of reporting in state of the art.
In a preferred embodiment of the present invention, both adopted chemomorphosis also to adopt physical mutagenesis, in substratum, added chemical mutagen, and be coated with pending bacterium liquid and accept Cement Composite Treated by Plasma afterwards.
In method of the present invention, for be coated with cell on plate culture medium, coating density should guarantee that every square centimeter is no more than 100; Or the mortality ratio that depends on cell after mutagenic treatment, the standard of the concentration of coating is: no more than 5 of the colony number that the density of the bacterium colony growing after mutagenic treatment grows on being every square centimeter.After mutagenic treatment, the mortality ratio of cell depends on processing mode (physics or chemistry, the dosage in physical treatment, treatment time, dosage, the treatment agent kind etc. in chemical treatment all can affect the mortality ratio of pending cell).In order to obtain the precise number of every kind of cell mortality under mutagenic treatment mode, before every batch of mutagenic treatment experiment, can do a preliminary experiment that obtains mortality ratio data.In two specific embodiments of the present invention, coating biomaterial is microorganism (milk-acid bacteria and yeast), and coating suitable concentration is 10
2～10
3individual/ml, each dressing plate is coated with 100 μ l.
The experimental data of the embodiment of the present invention 1 and 2 proves, microorganism mutagenic treatment method of the present invention is effective
The bacterial strain of having evaded change because of air-dry, osmotic pressure, the impact of objective factor such as splash of slide glass landing in treating processes, bacterium liquid, has effectively improved efficiency of inducing mutation.
Term defines:
Term used herein " coating ", it is an action, can find out from context of the present invention, its object is to make pending biomass cells on flat board, to disperse to a certain extent, and the cell count for example, growing after the mutagenic treatment that the present invention limits is no more than the density of 50/every square centimeter.
Accompanying drawing explanation
Fig. 1 adopts method mutagenic treatment milk-acid bacteria simultaneous test of the present invention,
Fig. 2 adopts method mutagenic treatment yeast simultaneous test of the present invention,
Wherein X-coordinate is the treatment time, and ordinate zou is lethality rate.
Embodiment
Embodiment 1 adopts method mutagenic treatment milk-acid bacteria of the present invention
Equipment: ARTP(atmospheric pressure at room plasma body) selection by mutation system
Reagent: 0.9% physiological saline, MRS solid culture based formulas: peptone 10g/L, beef powder 5g/L, yeast powder 4g/L, glucose 20g/L, sodium-acetate 5g/L, dibasic ammonium citrate 2g/L, tween 80 1mL, dipotassium hydrogen phosphate (7H
2o) 2g/L, sodium acetate (3H
2o) 5g/L, Triammonium citrate 2g/L, magnesium sulfate (7H
2o) 0.2g/L, manganous sulfate (4H
2o) 0.05g/L, agar 15g/L.
Pending bacterium liquid: lactobacterium acidophilus (Lactobacillus acidophilus) ACCC10637, derives from Chinese agriculture microbial strains preservation administrative center
Solid medium: MRS solid medium
Controlled trial step:
(1) prepare bacteria suspension: by milk-acid bacteria original strain MRS solid medium activation culture, 35 ℃ of culture temperature, incubation time 24h, scraping thalline, in physiological saline, obtains the bacterium liquid of growing vigorous, thalline is sturdy;
(2) prepare specimen slides: the bacterium liquid of getting fresh preparation is diluted to cell concn OD
600=0.5～1, drip 10～20uL to the cooled slide glass of sterilizing;
(3) mutagenic treatment: take 10SLM as gas flow, take 0～90s as the treatment time, specimen slides is carried out to plasma body mutagenesis;
(4), after mutagenesis, by the mycoderm wash-out on slide glass, after corresponding dilution, coating MRS solid medium is cultivated 24h, 35 ℃, calculates lethality rate.
(5) experimental result is as shown in accompanying drawing 1 control group, and wherein, X-coordinate is the treatment time, and ordinate zou is lethality rate.
The step of method of the present invention:
(1) prepare bacteria suspension: by milk-acid bacteria original strain MRS solid medium activation culture, 35 ℃ of culture temperature, incubation time 24h, scraping thalline, in physiological saline, obtains the bacterium liquid of growing vigorous, thalline is sturdy;
(2) spread plate: the bacterium liquid of getting fresh preparation is diluted to cell concn and is about 10
2～10
3individual/mL, gets 100uL spread plate;
(3) mutagenic treatment: take 10SLM as gas flow, take 0～90s as the treatment time, flat board is carried out to plasma body mutagenesis;
(4), after mutagenesis, by the mycoderm wash-out on slide glass, after corresponding dilution, coating MRS solid medium is cultivated 24h, 35 ℃, calculates lethality rate.
(5) as shown in as dull and stereotyped in accompanying drawing 1 group of experimental result, wherein, X-coordinate is the treatment time, and ordinate zou is lethality rate.
As shown in Figure 1, compared with control group, although the lethality rate that utilizes dull and stereotyped processing to obtain is lower than control group, but lethality rate curve smoothing is stable, fluctuating range is less, effectively got rid of bacterial strain and changed because of air-dry, osmotic pressure, the impact of objective factor such as splash of slide glass landing in treating processes, bacterium liquid, has effectively improved efficiency of inducing mutation.
Embodiment 2 adopts method mutagenic treatment yeast of the present invention
Equipment: ARTP(atmospheric pressure at room plasma body) selection by mutation system
Reagent: 0.9% physiological saline, PDA solid culture based formulas: potato is soaked powder 5g/L, glucose 20g/L, agar 20g/L, paraxin 0.1g/L.
Pending bacterium liquid: pichia spp (Pichia pastoris) 21003, purchased from deriving from Chinese agriculture microbial strains preservation administrative center
Solid medium: PDA solid medium
Controlled trial step:
(1) prepare bacteria suspension: by yeast original strain PDA solid medium activation culture, 28 ℃ of culture temperature, incubation time 48h, scraping thalline, in physiological saline, obtains the bacterium liquid of growing vigorous, thalline is sturdy;
(2) prepare specimen slides: the bacterium liquid of getting fresh preparation is diluted to cell concn OD
600=0.5～1, drip 10～20uL to the cooled slide glass of sterilizing;
(3) mutagenic treatment: take 10SLM as gas flow, take 0～120s as the treatment time, specimen slides is carried out to plasma body mutagenesis;
(4), after mutagenesis, by the mycoderm wash-out on slide glass, after corresponding dilution, coating PDA solid medium is cultivated 48h, 28 ℃, calculates lethality rate.Experimental result is as shown in accompanying drawing 2 control groups, and wherein, X-coordinate is the treatment time, and ordinate zou is lethality rate.
The Step By Condition of method of the present invention:
(1) prepare bacteria suspension: by milk-acid bacteria original strain PDA solid medium activation culture, 28 ℃ of culture temperature, incubation time 48h, scraping thalline, in physiological saline, obtains the bacterium liquid of growing vigorous, thalline is sturdy;
(2) spread plate: the bacterium liquid of getting fresh preparation is diluted to cell concn and is about 10
2～10
3individual/mL, gets 100uL spread plate;
(3) mutagenic treatment: take 10SLM as gas flow, take 0～120s as the treatment time, flat board is carried out to plasma body mutagenesis;
(4), after mutagenesis, by the mycoderm wash-out on slide glass, after corresponding dilution, coating PDA solid medium is cultivated 48h, 28 ℃, calculates lethality rate.As shown in as dull and stereotyped in accompanying drawing 2 group of experimental result, wherein, X-coordinate is the treatment time, and ordinate zou is lethality rate.
As shown in Figure 2, compared with control group, although the lethality rate that utilizes dull and stereotyped processing to obtain is lower than control group, but lethality rate curve smoothing is stable, fluctuating range is less, effectively got rid of bacterial strain and changed because of air-dry, osmotic pressure, the impact of objective factor such as splash of slide glass landing in treating processes, bacterium liquid, has effectively improved efficiency of inducing mutation.
Claims (9)
1. a method for mutagenic treatment biomaterial, is characterized in that: adopt following steps:
(1) on solid medium, be coated with pending cell,
(2) cell of coating is carried out to mutagenic treatment,
Described cell refers to microorganism cells.
2. method according to claim 1, described mutagenic treatment refers to adopt ultraviolet, Co 60 or atmospheric pressure at room plasma radiation to be coated on the cell on solid medium.
3. method according to claim 1, described mutagenic treatment is coated with chemical mutagen after referring to be coated with pending cell again, then cultivates.
4. method according to claim 1, also comprises cultivation after mutagenic treatment finishes, and described biomaterial cell refers to microorganism, and described cultivation comprises that cultivation grows up to bacterium colony.
5. method according to claim 5, described cultivation also comprises expands numerous one-tenth bacterium liquid by the bacterium colony growing up to.
6. according to the arbitrary described method of claim 1～5, in described substratum, be added with chemical mutagen.
7. a method for mutagenic treatment biomaterial, is characterized in that:
(1) on the solid medium that is added with chemical mutagen, be coated with pending cell,
(2) cultivate the cell that is coated on solid medium.
8. method according to claim 7, after it is characterized in that being coated with pending cell, carries out Physics Irradiation processing to the pending cell of coating, and described Physics Irradiation is processed and adopted ultraviolet, Co 60 or atmospheric pressure at room plasma body.
9. according to the arbitrary described method of claim 1～8, the cell that described coating is pending, coating concentration is: the density that makes the cell through growing at solid culture primary surface after mutagenic treatment is every square centimeter and is less than and equals 50.
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| CN201310749658.8A CN103911363A (en) | 2012-12-31 | 2013-12-31 | Method and device for mutagenizing biomaterial |
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| CN201210593443 | 2012-12-31 | ||
| CN201210593443.7 | 2012-12-31 | ||
| CN201310749658.8A CN103911363A (en) | 2012-12-31 | 2013-12-31 | Method and device for mutagenizing biomaterial |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212713A (en) * | 2014-09-25 | 2014-12-17 | 北京伟恩斯技术有限公司 | Multifunctional plasma mutation apparatus |
| CN104371994A (en) * | 2014-10-16 | 2015-02-25 | 江南大学 | Method for high-throughput screening of recombinase high-yield bacillus subtilis host in combination with normal pressure room temperature plasma mutation mode |
| CN109006693A (en) * | 2018-08-03 | 2018-12-18 | 中国水产科学研究院北戴河中心实验站 | A method of induction lefteye flounder gene mutation |
| CN109423489A (en) * | 2017-08-28 | 2019-03-05 | 江苏食品药品职业技术学院 | A kind of continous way microorganism physical mutagenesis breeding method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888063A (en) * | 2006-07-14 | 2007-01-03 | 大连理工大学 | Microbe mutagenizing atmospheric cold plasma method |
| CN101875929A (en) * | 2010-04-30 | 2010-11-03 | 大连理工大学 | Strain for generating phospholipase D with high and stable yield by utilizing physical and chemical mutation |
-
2013
- 2013-12-31 CN CN201310749658.8A patent/CN103911363A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888063A (en) * | 2006-07-14 | 2007-01-03 | 大连理工大学 | Microbe mutagenizing atmospheric cold plasma method |
| CN101875929A (en) * | 2010-04-30 | 2010-11-03 | 大连理工大学 | Strain for generating phospholipase D with high and stable yield by utilizing physical and chemical mutation |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104212713A (en) * | 2014-09-25 | 2014-12-17 | 北京伟恩斯技术有限公司 | Multifunctional plasma mutation apparatus |
| CN104371994A (en) * | 2014-10-16 | 2015-02-25 | 江南大学 | Method for high-throughput screening of recombinase high-yield bacillus subtilis host in combination with normal pressure room temperature plasma mutation mode |
| CN109423489A (en) * | 2017-08-28 | 2019-03-05 | 江苏食品药品职业技术学院 | A kind of continous way microorganism physical mutagenesis breeding method |
| CN109006693A (en) * | 2018-08-03 | 2018-12-18 | 中国水产科学研究院北戴河中心实验站 | A method of induction lefteye flounder gene mutation |
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Owner name: WUXI TMAXTREE BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: WUXI SIQINGYUAN BIOTECHNOLOGY CO., LTD. Effective date: 20150210 |
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Effective date of registration: 20150210 Address after: 214072 Jiangsu City, Wuxi province Binhu District West building, building A3, No. 777, layer 2 Applicant after: WUXI TMAXTREE BIOTECHNOLOGY CO., LTD. Address before: Room 214135 building E-308 Jiangsu science and Technology Park Business District of Wuxi city of Wuxi Province Institute of Pacific University Applicant before: Wuxi Siqingyuan Biotechnology Co., Ltd. |
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Application publication date: 20140709 |
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