CN106191029B - A method of the high flux screening aureomycin superior strain based on flow cytometry - Google Patents
A method of the high flux screening aureomycin superior strain based on flow cytometry Download PDFInfo
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Abstract
The method of the invention discloses a kind of high flux screening aureomycin superior strain based on flow cytometry, belongs to High Throughput Screening Assay field.The present invention is by carrying out PI streptomyces aureus spore staining, using flow cytometry to the Activity determination of streptomyces aureus spore, realizes to the activity differentiation of streptomyces aureus spore and the absolute counting of spore.Further sorting culture is carried out to the work spore population analyzed, provides a kind of mode for the high flux screening of streptomyces aureus.The high-throughput culture scheme that solid-liquid combination is used to the work spore after sorting, effectively increases streptomyces aureus orifice plate growth rate.Chromogenic reaction and porous board detecting method can detect simultaneously great amount of samples as a kind of detection means easily and fast.The present invention is based on the active streptomyces aureus of selected by flow cytometry apoptosis, provide a kind of novel effective mode for high flux screening aureomycin superior strain.
Description
Technical field
The method of the present invention relates to a kind of high flux screening aureomycin superior strain based on flow cytometry, belongs to high pass
Measure screening technique field.
Background technique
Aureomycin (Chorotetracycline, i.e. CTC) belongs to a kind of broad-spectrum antibiotic of Tetracyclines.Aureomycin energy
Enough antibacterial, growth promotions, efficiency of feed utilization is high, residual quantity is low in human body.Its production technology have been relatively mature, production cost
It is lower, become the maximum antimicrobial growth promoter of dosage in current feed industry.The yield of country facilities aureomycin is also lower at present
(potency is in 18000 μ g/mL or so), therefore the yield for improving aureomycin is necessary.Aureomycin is by streptomyces aureus
The secondary metabolite that (Streptomyces aureofaciens) fermentation obtains, yield synthesize character and are determined by polygenes,
Metabolism network is complicated.The factor for influencing Ferment of DM process is also very much, such as the quality of seed, the composition of culture medium, technique item
Part and the Metabolism control of fermentation process etc..Microbial Breeding technology develops to generation from mutagenesis, reasoning, recombinant technique at present
Thank the methods of engineering, systems biology and heterologous organisms synthesis.But classic mutagenesis breeding still has many advantages.It does not need such as to know
Road biological heredity background, metabolic model, it is only necessary to obtain effective mutation library and directed screening.High flux screening and routine mutagenesis knot
It closes, suitable for improve industrial producing strain breeding of the secondary metabolite yield as target, reducing leakage sieve phenomenon as far as possible.
Current classical mutagenesis combination high-throughput screening method, may lead since classical mutagenesis can have a large amount of dead cells
Cause subsequent cultivate cannot achieve, at the same the bacterial strain after classical mutagenesis further to isolate and purify, expands cultivate after could be into
The high flux screenings such as the subsequent ELISA Plate of row and exist the screening period it is long the problems such as.
Summary of the invention
To solve the above-mentioned problems, the present invention combines classical mutagenesis, flow cytometry, the screening of high pass orifice, solid-liquid connection
Culture is closed, a kind of method of high flux screening aureomycin superior strain based on flow cytometry is provided.The present invention is based on streams
Formula cell art can analyze mass efficient bacterial strain aureomycin high flux screening in a short time, so that it is living to sort purpose
Spore, and then carry out high-throughput culture detection.
The method of high flux screening aureomycin superior strain based on flow cytometry of the invention, mainly such as including step
Under:
(1) spore to be screened including the dead spore of multiple contents of streptomyces aureus is obtained；
(2) it is dyed using to be screened spore of the PI dyestuff to step (1)；
(3) using flow cytomery spore activity；
(4) the active spore of selected by flow cytometry apoptosis is utilized, directly single active spore is sorted into equipped with solid
In the high pass orifice of culture medium；
(5) to cultivate 2~3 days on the solid medium of high pass orifice, direct adding liquid seed culture medium continues to train
It supports and obtains seed liquor；Cultured seed liquor is transferred in the new high pass orifice containing fermentation medium, fermented and cultured；
(6) the aureomycin standard items for configuring a certain concentration gradient, draw Tris-HCl buffer solution, Sm (NO)3It is solution, each
Concentration standards solution, concussion are mixed to developing the color completely, are detected with microplate reader, obtain the light absorption value OD under aureomycin and 405nm
Relationship；After fermented and cultured, take fermented liquid supernatant, be added under suitable concentration containing Tris-HCl buffer solution and
In the orifice plate of samarium solution, concussion, which is mixed to color, becomes faint yellow, then measures the light absorption value OD under 405nm with microplate reader, into
And learn the opposite height of bacterial strain pan mycin content to be screened；
(7) relatively high more plants of bacterial strain of aureomycin content obtained in the previous step are chosen, secondary screening is carried out.
In one embodiment of the invention, the spore to be screened of the step (1) can be the spore of different activities,
Including dead spore.
In one embodiment of the invention, the spore to be screened of the step (1) is to lure streptomyces aureus
It is obtained after change.
In one embodiment of the invention, in the spore to be screened of the step (1), dead spore content is up to 90%
More than, preferably up to 95% or more more preferably up to 99% or more.
In one embodiment of the invention, the mutagenesis can be physical mutagenesis or chemical mutagenesis, such as normal pressure
Room-temperature plasma (ARTP) mutagenesis, ultraviolet mutagenesis, ethylmethane sulfonate (EMS) mutagenesis, nitrosoguanidine (NTG) mutagenesis, sulfuric acid
Diethylester (DES) mutagenesis etc..
In one embodiment of the invention, the dyeing of the step (2), specifically: spore to be screened is adjusted
To concentration 105~106The suspension of a spore/mL mixes isometric suspension with 10 μ g/mL PI coloring agents, avoid light place 4
DEG C refrigerator is incubated for 20min.
In one embodiment of the invention, the step (3) and step (4) are specifically: any glimmering with no label
The sample of light element is negative control, sets the fully inactive sample in conjunction with PI coloring agent to positive control, determines yin
Property with positive spore group；Streptomyces aureus spore suspension after taking dyeing, is filtered again, dilutes certain multiple, up flow type
Cytometric Analysis is detected with the channel FL3 ((610 ± 15) nm), and unstressed configuration region most concentrated part gating is sorted to equipped with solid
In the high pass orifice of body culture medium, every hole sorting is set as single spore.
In one embodiment of the invention, solid in the high pass orifice equipped with solid medium in the step (4)
The volume of body culture medium is 50 holes μ L/.
It in one embodiment of the invention, is to cultivate to growing adding liquid after visible spore in the step (5)
Seed culture medium.
In one embodiment of the invention, the additive amount of liquid seed culture medium is 150 μ L/ in the step (5)
Hole.
In one embodiment of the invention, the additive amount of fermentation culture is 900 holes μ L/ in the step (5).
In one embodiment of the invention, the step (6), in linear concentration range, target concentration is bigger,
OD value is bigger.
In one embodiment of the invention, a certain concentration gradient of the step (6) is 0~360 μ g/mL.
In one embodiment of the invention, the step (6) is the light absorption value OD drawn under aureomycin and 405nm
Standard curve；After fermented and cultured, fermented supernatant fluid is diluted to suitable concentration, is added to that be added to Tris-HCl in advance slow
It rushes in the orifice plate of solution and samarium solution, concussion, which is mixed to color, becomes faint yellow, and the extinction under 405nm is then measured with microplate reader
Light absorption value OD is substituted into standard curve, obtains corresponding aureomycin content by value OD.
In one embodiment of the invention, the step (6) is by 20 μ L Tris-HCl buffer solutions, 60 μ L
Sm(NO)3Fermentation liquid after solution, each concentration standards aureomycin solution of 120 μ L or dilution, which shakes, to be mixed.
In one embodiment of the invention, the pH of the Tris-HCl buffer solution is 6.7, Sm (NO)3Solution
Concentration is 5 × 10-4mol/L。
In one embodiment of the invention, the high pass orifice can be 96 deep-well plates, 96 shallow bore hole plates, 48 deep holes
Any one culture vessel with high flux screening effect such as plate, 24 deep-well plates, 12 deep-well plates.Preferably, it is trained equipped with solid
The high pass orifice for supporting base is 96 shallow bore hole plates, and the high pass orifice equipped with fermentation medium is 48 deep-well plates.
Beneficial effects of the present invention:
The method of the present invention combine atmospheric pressure at room plasma (ARTP), PI decoration method, flow cytometry separating method,
Microplate reader detection, solid-liquid combination cultural method and High Throughput Screening Assay, to sorting active streptomyces aureus Spore cultivation
Provide a kind of accurate quick method.The method of the present invention, which is based on flow cytometer, in a short time to be detected monospore,
To establish huge analysis target group, the sorting culture of flow cytometry high throughput reduces the mistake grown on plate
Journey does not leak each spore of sieve, and separation velocity and screening efficiency are improved.In addition, the cultural method using solid-liquid combination is direct
The growth rate for improving single streptomyces aureus spore solves the problems, such as that single spore grows difficulty in liquid medium.
Specific embodiment
Culture medium:
(1) solid medium (g/L): sucrose 20g, import yeast powder 0.5g, KH2PO40.8g, agar 18g.
(2) seed culture medium (g/L): sucrose 30g, import yeast powder 5g, MgSO40.5g, (NH4)2SO43g, NaCl4g,
KH2PO41g, CaCO30.2g。
(3) orifice plate fermentation medium (g/L): sucrose 30g, MgSO40.5g, CaCO30.33g, citric acid 2.55g, (NH4)2SO43g, 1mL mixed liquor (0.3%CuSO4·5H2O, 0.5%ZnSO4·7H2O, 0.25%MnSO4`4H2O)。
Fluorescent dye is prepared: being weighed 0.01g PI, is settled to 10mL with PBS (pH 7.4) buffer solution.Gradient dilution is extremely
10μg/mL.4 DEG C are kept in dark place.
PI dyestuff is to streptomyces aureus spore staining:
Isometric streptomyces aureus spore suspension and 10 μ g/mLPI dyestuffs is taken to mix, 4 DEG C of refrigerator dyeing of avoid light place
20min。
Flow cytomery:
Streptomyces aureus spore suspension after taking dyeing, is filtered again, dilutes certain multiple, flow cytometer
(MoFlo XDP) analysis.Emit fluorescence selection appropriate channel detection according to coloring agent.The data obtained 5.4 software of Summit point
Analysis.
1 ARTP mutagenesis of embodiment or other mutagenic obtained different activities spores
Starting strain streptomyces aureus slant strains are cultivated 4 days under the conditions of 34 DEG C, until spore is mature, inclined-plane bacterium colony
In Slate grey.For the appropriate mature slant pore of scraping in the test tube equipped with 2mL PBS buffer solution, oscillator shake is even to be made bacteria suspension
Afterwards, aseptic filter paper filters.The bacteria suspension for drawing 10 μ L suitable concentrations is spread evenly across sterile slide surface, and slide glass is placed in load
On platform, mutagenesis is carried out with ARTP mutagenesis instrument.Condition: incident power 100W, helium gas flow 10SLM, respectively one timing of irradiation
Between obtain different activities spore.Irradiation certain time obtains the spore of different activities respectively.Other physics, chemical mutagenesis can also
Obtain the spore of different activities.
2 PI dyestuff of embodiment is to streptomyces aureus spore staining:
It takes sample to be disposed, slide glass is put into the EP pipe equipped with PBS (pH 7.4) buffer solution with aseptic nipper, is filled
Divide oscillation 1min, the thallus being attached on slide glass is thoroughly eluted in liquid PBS.Filter paper filtering, 5000 × g are centrifuged 5min,
It discards supernatant, PBS is added and is resuspended.It repeats this operating procedure 2~3 times, obtains pure streptomyces aureus spore suspension.Use PBS
Pure streptomyces aureus spore suspension is diluted to concentration 105~106A spore/mL.Isometric streptomyces aureus spore suspension
It is mixed with 10 μ g/mLPI dyestuffs, 4 DEG C of refrigerator stain incubation 20min of avoid light place.
3 flow cytomery of embodiment:
Detection will establish check experiment and remove non-specific binding i.e. background.Do not mark the sample of any fluorescein for yin
Property control, represent cellular context fluorescence signal.Fundamental voltage, setting feminine gender and positive population boundary are adjusted by negative control.
Will be fully inactive, the sample in conjunction with PI coloring agent is set as positive control, determines negative and positive spore group.Fixed strip
Sample detection is carried out after part.Streptomyces aureus spore suspension after taking dyeing, is filtered again, dilutes certain multiple, up flow type
Cell instrument (MoFlo XDP) analysis.Emit red fluorescence wavelength according to PI, is detected with the channel FL3 ((610 ± 15) nm).Pass through
FSC, SSC, fluorescence channel coordinate parameters, voltage and threshold parameter are adjusted, the group position for obtaining analysis object preliminary first.
To group position substantially gating, FSC, SSC threshold value are adjusted again, and voltage parameter removes most background noise.Institute's total
It is analyzed according to 5.4 software of Summit.From software diagram, it can be seen that after the inactive sporocyst PI dyeing of streptomyces aureus,
Issue red fluorescent.Since PI cannot penetrate living cell membrane, active streptomyces aureus spore fails to be colored, nothing
Fluorescence signal is shown.So as to further sub-elect active streptomyces aureus spore.
4 aureomycin chromogenic reaction of embodiment
According to the chromogenic reaction of aureomycin and samarium ion as prescreening method.Aureomycin and samarium ion are under near-neutral sulfite deinking
Light yellow complex is formed, there is maximum light absorption at 405nm.Consider after fermentation, containing with coloured original in culture medium
Material generates certain influence to measurement result.With production fermentation liquor formulation, while in the orifice plate culture with do not cultivate thallus.As a result table
Bright, not cultivating thallus fermentation liquid also has certain OD value, illustrates that the color of fermentation liquid itself is worth measurement to be implicitly present in shadow OD
The fermentation liquid OD value rung, but cultivate thallus is greater than the fermentation liquid for not cultivating thallus, therefore fermentation liquid intrinsic colour interferes not shadow
Ring screening aureomycin superior strain.Configuration concentration successively draws 20 μ L in the aureomycin standard solution of 0.5~20mg/L
Tris-HCl buffer solution, 60 μ L Sm (NO)3Solution, each concentration standards solution concussion of 120 μ L are mixed, have been developed the color after 10min
Entirely, microplate reader measures the OD405 value of each concentration.Determine OD405With the regression equation of chlortetracycline concentration C (mg/L): y=
0.0107x-0.006, R2=0.99305.X is standard concentration (μ g/mL), y OD405nm, standard concentration range be 0~
360μg/mL。
The culture and screening of 5 superior strain of embodiment
Single streptomyces aureus spore is directly sorted into 96 shallow bore hole plates (180 μ of culture medium loading amount by flow cytometer
L).Since streptomyces aureus spore volume is too small, directly growth rate only has 15% or so in liquid medium.It is asked to solve this
Topic, improves culture scheme.To after growing visible spore on the 96 orifice plate solid mediums equipped with 50 μ L (about 2~
3d), 150 μ L of seed culture fluid is drawn on this orifice plate solid medium.It is trained on 750r/min, 30~32 DEG C of plate shaker
Support 23~25h.Through this improved culture scheme, streptomyces aureus is in orifice plate cultured on solid medium rate up to 95% left side
It is right.With the inoculum concentration of 5% (v/v), the seed liquor in 96 orifice plates is forwarded to 48 orifice plates (equipped with 1000 μ of fermentation culture in parallel
L in), 750r/min, 30~31 DEG C of 96~110h of fermented and cultured.Seed liquor in remaining 96 orifice plates is stored in 4 with 40% glycerol
DEG C refrigerator.After the thallus centrifugation that 48 orifice plates are obtained, draws supernatant and suitably dilute, draw 120 μ L dilutions to 96 orifice plates (in advance
Tris-HCl buffer solution and samarium solution is added), shaking table concussion mixes 20min, and color is become faint yellow, measured using microplate reader
OD405Value picks out the higher bacterial strain of aureomycin relative amount and carries out the experiment of shaking flask secondary screening.
Shaking flask secondary screening can be screened from numerous spores and obtain aureomycin the results show that the method for the present invention accuracy is very high
The relatively high bacterial strain of yield.
In addition, inventor also provides a comparison of the method for the invention based on flow cytometry Yu orifice plate high flux screening, and pass
The method that system mutagenesis and orifice plate high flux screening are bound directly, it is found that method screening effect of the invention is fabulous, will not leak sifter device
Active spore, separation velocity and screening efficiency significantly improve.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (8)
1. a kind of method of the high flux screening aureomycin superior strain based on flow cytometry, which is characterized in that mainly include
Steps are as follows:
(1) spore to be screened including the dead spore of multiple contents of streptomyces aureus is obtained；
(2) it is dyed using to be screened spore of the PI dyestuff to step (1)；
(3) using flow cytomery spore activity: using the sample of any fluorescein of no label as negative control, will be complete
The inactive sample in conjunction with PI coloring agent is set as positive control, determines negative with positive spore group: not mark
The sample of any fluorescein is set as positive control for negative control, by the fully inactive sample in conjunction with PI coloring agent,
Determine negative and positive spore group；
(4) the active spore of selected by flow cytometry apoptosis is utilized, directly single active spore is sorted into equipped with solid culture
In the high pass orifice of base: the streptomyces aureus spore suspension after taking dyeing filters again, dilutes certain multiple, up flow type
Cytometric Analysis is detected with the channel FL3 (610 ± 15) nm, and unstressed configuration region most concentrated part gating is sorted to equipped with solid
In the high pass orifice of culture medium, every hole sorting is set as single spore；
(5) to cultivate 2 ~ 3 days on the solid medium of high pass orifice, direct adding liquid seed culture medium continues culture and obtains
Seed liquor；Cultured seed liquor is transferred in the new high pass orifice containing fermentation medium, fermented and cultured；
(6) the aureomycin standard items for configuring a certain concentration gradient draw Tris-HCl buffer solution, concentration is 5 × 10-4 mol/L
Sm (NO)3Solution, each concentration standards solution, by 20 μ L Tris-HCl buffer solutions, 60 μ L Sm (NO)3Solution, 120
Fermentation liquid concussion after each concentration standards aureomycin solution of μ L or dilution is mixed to developing the color completely, is detected, is obtained with microplate reader
The relationship of light absorption value OD under to aureomycin and 405 nm；After fermented and cultured, fermented liquid supernatant is taken, under suitable concentration
It is added in the orifice plate containing Tris-HCl buffer solution and samarium solution, concussion, which is mixed to color, becomes faint yellow, then uses enzyme
The light absorption value OD under 405 nm of instrument measurement is marked, and then learns the opposite height of bacterial strain pan mycin content to be screened；
(7) relatively high more plants of bacterial strain of aureomycin content obtained in the previous step are chosen, secondary screening is carried out.
2. the method according to claim 1, wherein the spore to be screened of the step (1) is different activities
Spore, including dead spore.
3. the method according to claim 1, wherein the spore to be screened of the step (1) is by golden strepto-
It is obtained after bacterium progress mutagenesis.
4. the method according to claim 1, wherein the mutagenesis, is physical mutagenesis or chemical mutagenesis.
5. the method according to claim 1, wherein the dyeing of the step (2), specifically: will be to be screened
Spore is adjusted to concentration 105~106The suspension of a spore/mL mixes isometric suspension with 10 μ g/mL PI coloring agents,
4 DEG C of refrigerators of avoid light place are incubated for 20 min.
6. the method according to claim 1, wherein in the step (4), the high throughput equipped with solid medium
The volume of solid medium is 50 holes μ L/ in orifice plate.
7. the method according to claim 1, wherein the step (6), is drawn under aureomycin and 405 nm
The standard curve of light absorption value OD；After fermented and cultured, fermented supernatant fluid is diluted to suitable concentration, is added to and is added in advance
In the orifice plate of Tris-HCl buffer solution and samarium solution, concussion, which is mixed to color, becomes faint yellow, then with microplate reader measurement 405
Light absorption value OD is substituted into standard curve, obtains corresponding aureomycin content by the light absorption value OD under nm.
8. the method according to the description of claim 7 is characterized in that the pH of the Tris-HCl buffer solution is 6.7.
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| CN109652314A (en) * | 2019-02-26 | 2019-04-19 | 焦作健康元生物制品有限公司 | A kind of demethylchlortetra cylinum bacterial strain is cultivated and screening technique |
| CN111778135A (en) * | 2020-06-23 | 2020-10-16 | 浙江大学 | FACS-Ichip in-situ microorganism culture device and culture method |
| CN113549579B (en) * | 2021-08-02 | 2022-08-16 | 金河生物科技股份有限公司 | Streptomyces aureofaciens mutant strain with high aureomycin yield and low impurity proportion and application thereof |
| CN117761027A (en) * | 2023-12-22 | 2024-03-26 | 深圳栅极芯致生物科技有限公司 | High-throughput screening method for strains |
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