CN106222162A - A kind of method of high flux screening avilamycin superior strain based on flow cytometry - Google Patents
A kind of method of high flux screening avilamycin superior strain based on flow cytometry Download PDFInfo
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- CN106222162A CN106222162A CN201610597287.XA CN201610597287A CN106222162A CN 106222162 A CN106222162 A CN 106222162A CN 201610597287 A CN201610597287 A CN 201610597287A CN 106222162 A CN106222162 A CN 106222162A
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Abstract
A kind of method that the invention discloses high flux screening avilamycin superior strain based on flow cytometry, belongs to High Throughput Screening Assay field.The present invention is by carrying out PI staining to the spore of different activities after mutation, FSC is optimized by regulation, SSC, the parameters such as voltage threshold is correctly distinguished activated spore region, is got rid of and bring, owing to the volume of spore own is too small, the interference mixed with background in flow cytomery.Spore alive after sorting is used the high flux culture scheme of solid-liquid combination, is effectively increased Avid kyowamycin orifice plate rate of growth.The present invention provides a kind of novel effective manner based on the activated Avid kyowamycin of selected by flow cytometry apoptosis, the high flux screening for Avid kyowamycin.
Description
Technical field
A kind of method that the present invention relates to high flux screening avilamycin superior strain based on flow cytometry, belongs to high
Throughtput screening technologies field.
Background technology
Avilamycin is as Avid kyowamycin secondary metabolite, and its metabolism exists complicated huge network.Although A Wei
Rhzomorph genome sequencing is complete, but its biosynthetic process is complicated, is regulated and controled by varying level, and metabolic pathway is the most not yet
Clearly illustrate.Costly, process is complicated for molecular breeding means.In order to accelerate selection-breeding speed, set up a simplicity, quickly and accurately
Detection method be a need for.High Throughput Screening Assay is on the basis of conventional screen selecting technology, by biochemistry, modern life
The new model of the new and high technology such as thing, computer, Automated condtrol organic assembling Cheng Yigegao automatization, with tradition random screening
Comparing, high flux screening cycle and investment are lower, and probalility of success is high.From fermentation engineering Microbial Breeding history, it can be seen that classical
Mutagenic breeding is topmost breeding technique, is also most basic breeding technique, and classical mutation combines high flux screening and still can make
For reliable method.
Current classical mutation combines high-throughput screening method, may lead owing to classical mutation can exist a large amount of dead cell
Causing follow-up cultivation cannot realize, simultaneously the bacterial strain after classical mutation is the most isolated and purified, just can enter after amplification culture
The row high flux screening such as follow-up ELISA Plate and there is the problems such as screening cycle length, previous work amount are big.
Summary of the invention
In order to solve the problems referred to above, the present invention combines classical mutation, flow cytometry, the screening of high flux orifice plate, solid-liquid connection
Close and cultivate, it is provided that a kind of method of high flux screening avilamycin superior strain based on flow cytometry.The present invention based on
Mass efficient bacterial strain, to Avid kyowamycin high flux screening, can be analyzed, thus sort by flow cytometry at short notice
Purpose spore alive, and then carry out high flux cultivation detection.
The method of the high flux screening avilamycin superior strain based on flow cytometry of the present invention, mainly includes step
As follows:
(1) the dead spore of multiple content of Avid kyowamycin is obtained at interior spore to be screened；
(2) utilize PI dyestuff that the spore to be screened of step (1) is dyeed；
(3) flow cytomery spore activity is used；
(4) utilize the active spore of selected by flow cytometry apoptosis, directly single activated spore is sorted into equipped with solid
In the high flux orifice plate of culture medium；
(5) treating to cultivate 3～4 days on the solid medium of high flux orifice plate, direct adding liquid seed culture medium continues training
Support and obtain seed liquor；Cultured seed liquor is transferred in the new high flux orifice plate containing fermentation medium, fermentation culture；
(6) configure the avilamycin standard substance of finite concentration gradient, detect by microplate reader, obtain in the range of linear concentration Ah
The relation of the light absorption value OD under dimension rhzomorph and 240nm；After fermentation culture terminates, absorption fermentation liquid, centrifuging and taking supernatant after extraction,
Suitably measure the light absorption value OD under 240nm by microplate reader under concentration, and then learn that bacterial strain to be screened produces avilamycin content
Height relatively；
(7) choose the many strains of bacterial strain that avilamycin content obtained in the previous step is of a relatively high, carry out multiple sieve.
In one embodiment of the invention, the spore to be screened of described step (1) can be the spore of different activities,
Including dead spore.
In one embodiment of the invention, the spore to be screened of described step (1) is to be lured by Avid kyowamycin
Obtain after change.
In one embodiment of the invention, in the spore to be screened of described step (1), dead spore content reaches 90%
Above, preferably reach more than 95% and more preferably reach more than 99%.
In one embodiment of the invention, described mutation, can be physical mutagenesis or chemomorphosis, such as normal pressure
Room-temperature plasma (ARTP) mutation, ultraviolet mutagenesis, ethylmethane sulfonate (EMS) mutation, nitrosoguanidine (NTG) mutation, sulphuric acid
Diethylester (DES) mutation etc..
In one embodiment of the invention, the dyeing of described step (2), specifically: spore to be screened is adjusted
To concentration 105～106The suspension of individual spore/mL, mixes equal-volume suspension and 10 μ g/mL PI stains, and lucifuge places 4
DEG C refrigerator hatches 15min.
In one embodiment of the invention, described step (3) and step (4) are specifically: not have labelling any glimmering
The sample of light element is negative control, the fully inactive sample being combined with PI stain is set to positive control, determines the moon
Property with positive spore colony；Take the Avid kyowamycin spore suspension after dyeing, again filter, dilute certain multiple, up flow type
Cytometric Analysis, detects with FL3 passage ((610 ± 15) nm), with FCS-Log-Height as abscissa, and SSC-Log-Height
For vertical coordinate, SSC threshold value is 0.03, and fluorescence channel voltage is 513, unstressed configuration region concentrated part is set sorting to equipped with
In the high flux orifice plate of solid medium, the sorting of every hole is set as single spore.
In one embodiment of the invention, in described step (4), equipped with solid in the high flux orifice plate of solid medium
The volume of body culture medium is 50 μ L/ holes.
In one embodiment of the invention, in described step (5), it is to cultivate to growing adding liquid after visible spore
Seed culture medium.
In one embodiment of the invention, in described step (5), the addition of liquid seed culture medium is 150 μ L/
Hole.
In one embodiment of the invention, in described step (5), the addition of fermentation culture is 900 μ L/ holes.
In one embodiment of the invention, described step (6), in linear concentration range, target concentration is the biggest,
OD value is the biggest.
In one embodiment of the invention, the finite concentration gradient of described step (6) refers to 0～210 μ g/mL.
In one embodiment of the invention, described step (6), is to draw avilamycin and the light absorption value under 240nm
The standard curve of OD；After fermentation culture terminates, ethanol is centrifugal after suitably diluting ultrasonic extraction, measures supernatant under 240nm
Light absorption value OD, substitutes into standard curve by light absorption value OD, obtains corresponding avilamycin content.
In one embodiment of the invention, described high flux orifice plate can be 96 deep-well plates, 96 shallow bore hole plates, 48 deep holes
Plate, 24 deep-well plates, 12 deep-well plates etc. any one there is the culture vessel of high flux screening effect.Preferably, train equipped with solid
The high flux orifice plate supporting base is 96 shallow bore hole plates, and the high flux orifice plate equipped with fermentation medium is 48 deep-well plates.
Beneficial effects of the present invention:
The inventive method combine atmospheric pressure at room plasma (ARTP), PI staining, flow cytometry separating method,
Microplate reader detection, the cultural method of solid-liquid combination and High Throughput Screening Assay, to sorting active Avid kyowamycin Spore cultivation
Provide a kind of the most quickly method.Monospore can be detected at short notice by the inventive method based on flow cytometer,
Thus set up huge analysis target group, the high-throughout sorting of flow cytometry is cultivated and is decreased the mistake of growth on flat board
Journey, does not leak each spore of sieve, and separation velocity and screening efficiency are improved.Additionally, utilize the cultural method of solid-liquid combination direct
Improve the rate of growth of single Avid kyowamycin spore, solve the problem growing difficulty in single spore liquid medium within.
Detailed description of the invention
Culture medium:
(1) solid medium (g/L): soluble starch 4g, yeast powder 3g, import Fructus Hordei Germinatus powder 10g, CoCl2·6H2O5mg,
Agar powder 20g.
(2) orifice plate seed culture medium (g/L): corn starch 28.5g, soybean cake powder 7.5g, yeast powder 4.5g, yeast extract 4g,
CoCl230mg, amylase 0.67g.
(3) orifice plate fermentation medium (g/L): corn starch 140g, soybean cake powder 25g, yeast powder 10g, (NH4)2SO4
0.25g, CoCl2·6H2O 20mg, NaMoO422mg, MnSO42.3mg, amylase 0.67g.
Fluorescent dye is prepared:
Weigh 0.01g PI, be settled to 10mL with PBS (pH 7.4) buffer solution.Gradient dilution is to 10 μ g/mL.Keep away for 4 DEG C
Light preserves.
PI stain is to Avid kyowamycin spore staining:
Taking equal-volume Avid kyowamycin spore suspension and the mixing of 10 μ g/mL PI stains, lucifuge places 4 DEG C of refrigerator dyeing
Certain time.
Sample detection:
Take the Avid kyowamycin spore suspension after dyeing, again filter, dilute certain multiple, flow cytometer
(MoFlo XDP) analyzes.Launch fluorescence according to stain and select appropriate channel detection.Set suitable Photomultiplier tube voltage, threshold
The parameters such as value.The data obtained Summit 5.4 software analysis.
Sorting is cultivated:
By the sample flow cytometer detection after enrichment, make separation condition by oneself, Avid kyowamycin spore is sorted into conjunction
Suitable culture medium is cultivated.
The mutation of embodiment 1ARTP or other method of mutagenesis obtain different activities spore
Starting strain Avid kyowamycin slant strains, under the conditions of 28 DEG C, is cultivated 5 days, until spore is ripe, and inclined-plane bacterium colony
In canescence.The appropriate ripe slant pore of scraping is in the test tube equipped with 2mLPBS buffer, and oscillator shake is even makes bacteria suspension
After, aseptic filter paper filters.The bacteria suspension drawing 10 μ L suitable concns is spread evenly across aseptic slide surface, and slide glass is placed in load
On platform, carry out mutation with ARTP mutation instrument.Condition: incident power is 100W, helium gas flow is 10SLM, irradiate a timing respectively
Between obtain the spore of different activities.The spore that other method of mutagenesis obtain different activities is equally applicable.
Embodiment 2PI stain is to Avid kyowamycin spore staining
Sample treatment is complete, is put to the EP pipe equipped with PBS (pH 7.4) buffer solution by slide glass with aseptic nipper, fully
Vibration 1min, the thalline that will be attached on slide glass is thoroughly eluted in liquid PBS.Filter paper filtering, 5000 × g is centrifuged 5min, abandons
Remove supernatant, add PBS resuspended.Repeat this operating procedure 2～3 times, obtain pure Avid kyowamycin spore suspension.Will with PBS
Pure Avid kyowamycin spore suspension is diluted to concentration 105～106Individual spore/mL.Take equal-volume Avid kyowamycin spore suspension
With 10 μ g/mLPI stain mixing, lucifuge 4 DEG C of refrigerators of placement hatch 15min.
Embodiment 3 flow cytomery sorts
Detection controlled trial to be set up removes non-specific binding i.e. background.The sample not having any fluorescein of labelling is the moon
Property comparison, represent cellular context fluorescence signal.Regulate fundamental voltage by negative control, set negative and positive population boundary.
By fully inactive, the sample being combined with PI stain is set to positive control, determines negative and positive spore colony.Fixed strip
Sample detection is carried out after part.Take the Avid kyowamycin spore suspension after dyeing, again filter, dilute certain multiple, up flow type
Cell instrument (MoFlo XDP) is analyzed.The data obtained Summit 5.4 software analysis.Launch red fluorescence wavelength according to PI, use
FL3 passage ((610 ± 15) nm) detects.By regulation FSC, SSC, fluorescence channel coordinate parameters, voltage and threshold parameter, head
The most preliminary group position obtaining analysis object.Group position is substantially set door, again regulates FSC, SSC threshold value, voltage parameter,
Most background noise is removed.Finally determining with FCS-Log-Height as abscissa, SSC-Log-Height sits for vertical
Mark, SSC threshold value is 0.03, and fluorescence channel voltage is 513 for optimized analysis parameter.From software illustrates, it can be seen that A Weilian
After mycete inactive sporocyst PI dyeing, send red fluorescent.Owing to PI can not pass through living cell membrane, active Ah
Dimension streptomycete spore fails to be colored, and unstressed configuration signal shows.Unstressed configuration region concentrated part is set a sorting to 96 orifice plates
In solid medium (every hole 50 μ L).The sorting of every hole is set as single spore.
Embodiment 4 microplate reader detects
At 240nm, have a maximum light absorption according to avilamycin, and blank cultures at 240nm without this of absorption value
Feature, selects microplate reader detection avilamycin as prescreening method.The standard substance of configuration finite concentration gradient, examine by microplate reader
Survey, prepare standard curve equation y=0.01782x+0.06191, R2=0.99715, x are standard concentration (μ g/mL), and y is
OD240nm, standard concentration scope is 0～210 μ g/mL.Pre fermentation experiment in 48 orifice plates shows that the titer of avilamycin exists
1000 μ about g/mL.Draw culture fluid 50 μ L in 48 deep-well plates, ethanol avilamycin is diluted to the standard curve range of linearity it
In, after ultrasonic extraction, 4000r/min is centrifuged 15min, takes 200 μ L of supernatant liquid in 96 ELISA Plate, measures 240nm by microplate reader visible
Light absorption value OD under light.Extract with EtOH Sonicate, within avilamycin is diluted to the standard curve range of linearity, survey at 240nm
OD value.
The cultivation of embodiment 5 superior strain and screening
Directly single Avid kyowamycin spore is sorted to (fluid medium loading amount in 96 shallow bore hole plates through flow cytometer
180μL).Owing to Avid kyowamycin spore volume is too small, directly in 180 μ L fluid mediums, rate of growth only has about 15%.
For solving this problem, culture scheme is improved.Treat equipped with grow on the 96 orifice plate solid mediums of 50 μ L visible spore it
Afterwards (about 3～4d), seed culture fluid 150 μ L is drawn on this orifice plate solid medium.Seed culture fluid in 750r/min, 28 DEG C
Plate shaker on cultivate about 47h.Culture scheme after this improves, Avid kyowamycin is at orifice plate cultured on solid medium
Rate is up to about 93%.In seed culture fluid, Avid kyowamycin upgrowth situation is normal.With the inoculum concentration of 5% (v/v), by 96 orifice plates
In seed liquor parallel be forwarded in 48 orifice plates (equipped with fermentation culture 900 μ L), 28 DEG C, 750r/min fermentation culture 10d.Surplus
The seed liquor Yued in 96 orifice plates is stored in 4 DEG C of refrigerators with 40% glycerol.Microplate reader high flux primary dcreening operation is utilized to detect.Draw 48 deep holes
Culture fluid 50 μ L in plate, after ethanol suitably dilutes ultrasonic extraction, 4000r/min is centrifuged 15min, takes 200 μ L of supernatant liquid in 96 enzyme marks
Plate, measures the light absorption value OD under 245nm visible ray by microplate reader.Using starting strain as comparison during whole, according to right
Comparison according to bacterial strain OD value selects the obvious mutagenic strain of Mutagenic Effect to carry out shaking flask and sieve experiment again；Or directly choose
Bacterial strain bigger for light absorption value OD under 245nm visible ray carries out shaking flask and sieves experiment again.
Shaking flask is sieved result again and is shown, the inventive method accuracy is the highest, it is possible to from numerous spores, screening obtains Avermectin
The bacterial strain that element yield is of a relatively high.
Additionally, inventor also provide a comparison the method based on flow cytometry Yu orifice plate high flux screening of the present invention, and pass
System mutation and orifice plate high flux screening directly in conjunction with method, find that the method screening effect of the present invention is fabulous, sifter device will not be leaked
Activated spore, separation velocity and screening efficiency significantly improve.
Although the present invention is open the most as above with preferred embodiment, but it is not limited to the present invention, any is familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be with being as the criterion that claims are defined.
Claims (10)
1. the method for a high flux screening avilamycin superior strain based on flow cytometry, it is characterised in that described side
Method mainly comprises the following steps that
(1) the dead spore of multiple content of Avid kyowamycin is obtained at interior spore to be screened；
(2) utilize PI dyestuff that the spore to be screened of step (1) is dyeed；
(3) flow cytomery spore activity is used；
(4) utilize the active spore of selected by flow cytometry apoptosis, directly single activated spore is sorted into equipped with solid culture
In the high flux orifice plate of base；
(5) treating to cultivate 3～4 days on the solid medium of high flux orifice plate, direct adding liquid seed culture medium continues to cultivate
To seed liquor；Cultured seed liquor is transferred in the new high flux orifice plate containing fermentation medium, fermentation culture；
(6) configure the avilamycin standard substance of finite concentration gradient, detect by microplate reader, obtain Avermectin in the range of linear concentration
The relation of the light absorption value OD under element and 240nm；After fermentation culture terminates, drawing fermentation liquid, centrifuging and taking supernatant after extraction, properly
Concentration under measure the light absorption value OD under 240nm by microplate reader, and then learn that bacterial strain to be screened produces the relative of avilamycin content
Just；
(7) choose the many strains of bacterial strain that avilamycin content obtained in the previous step is of a relatively high, carry out multiple sieve.
Method the most according to claim 1, it is characterised in that the spore to be screened of described step (1) is different activities
Spore, including dead spore.
Method the most according to claim 1, it is characterised in that the spore to be screened of described step (1) is by A Wei strepto-
Bacterium obtains after carrying out mutation.
Method the most according to claim 3, it is characterised in that described mutation is physical mutagenesis or chemomorphosis.
Method the most according to claim 1, it is characterised in that described step (3) and step (4) specifically: with not mark
The sample remembering any fluorescein is negative control, it is positive right to be set to by the fully inactive sample being combined with PI stain
According to, determine negative and positive spore colony；Take the Avid kyowamycin spore suspension after dyeing, again filter, certain times of dilution
Number, flow cytometer analysis, use FL3 Air conduct measurement, with FCS-Log-Height as abscissa, SSC-Log-Height is vertical
Coordinate, SSC threshold value is 0.03, and fluorescence channel voltage is 513, unstressed configuration region concentrated part sets a sorting to equipped with solid
In the high flux orifice plate of culture medium, the sorting of every hole is set as single spore.
Method the most according to claim 1, it is characterised in that the dyeing of described step (2), specifically: by be screened
Spore adjusts to concentration 105～106The suspension of individual spore/mL, mixes equal-volume suspension and 10 μ g/mL PI stains,
Lucifuge 4 DEG C of refrigerators of placement hatch 15min.
Method the most according to claim 1, it is characterised in that in described step (4), equipped with the high flux of solid medium
In orifice plate, the volume of solid medium is 50 μ L/ holes.
Method the most according to claim 1, it is characterised in that in described step (5), is to cultivate to after growing visible spore
Adding liquid seed culture medium.
Method the most according to claim 1, it is characterised in that the addition of liquid seed culture medium in described step (5)
It is 150 μ L/ holes.
Method the most according to claim 1, it is characterised in that described step (6), is to draw under avilamycin and 240nm
The standard curve of light absorption value OD；After fermentation culture terminates, ethanol is centrifugal after suitably diluting ultrasonic extraction, measures supernatant and exists
Light absorption value OD under 240nm, substitutes into standard curve by light absorption value OD, obtains corresponding avilamycin content.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825495A (en) * | 2019-03-01 | 2019-05-31 | 江南大学 | A kind of method of high flux screening High-productive Monascus Pigment Strain |
| CN110591917A (en) * | 2019-09-20 | 2019-12-20 | 江南大学 | High-throughput screening method for plasmid-lost strains |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268378A (en) * | 2011-07-14 | 2011-12-07 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
| CN104198357A (en) * | 2014-09-24 | 2014-12-10 | 广东省农业科学院动物卫生研究所 | Application of flow cytometry method to vaccine production real-time monitoring |
| CN104560725A (en) * | 2014-12-11 | 2015-04-29 | 南京工业大学 | Flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof |
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2016
- 2016-07-26 CN CN201610597287.XA patent/CN106222162A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268378A (en) * | 2011-07-14 | 2011-12-07 | 华东理工大学 | Method for screening high yield strains from aerobic bacteria at high flux |
| CN104198357A (en) * | 2014-09-24 | 2014-12-10 | 广东省农业科学院动物卫生研究所 | Application of flow cytometry method to vaccine production real-time monitoring |
| CN104560725A (en) * | 2014-12-11 | 2015-04-29 | 南京工业大学 | Flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825495A (en) * | 2019-03-01 | 2019-05-31 | 江南大学 | A kind of method of high flux screening High-productive Monascus Pigment Strain |
| CN109825495B (en) * | 2019-03-01 | 2021-05-28 | 江南大学 | Method for high-throughput screening of monascus pigment high-yield strains |
| CN110591917A (en) * | 2019-09-20 | 2019-12-20 | 江南大学 | High-throughput screening method for plasmid-lost strains |
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Application publication date: 20161214 |