CN104560725B - Kit and its application based on Flow Cytometry high flux screening producing and ethanol fungi - Google Patents
Kit and its application based on Flow Cytometry high flux screening producing and ethanol fungi Download PDFInfo
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Abstract
The invention discloses a kind of kit based on Flow Cytometry high flux screening producing and ethanol fungi and its application, which includes box body and reagent, further includes Tissue Culture Plate, centrifuge tube and copper sieve, the reagent and is：Reagent A：Citric acid：6.15g；Sodium citrate：17.544g；1000mL；Reagent B：Propidium iodide（PI）：0.3mg；2,7 dihydro dichlorofluorescein sodium Diacetates：0.3mg；Reagent C：Glucose：2g, 50mL；Reagent D：Glucose：12g, 50mL；Reagent E：Dusty yeast：0.5g；Tryptone：0.5g；Diammonium hydrogen phosphate：0.05g；Magnesium sulfate：0.025g, pH 5.5,50mL；Reagent F：Dusty yeast：0.05g；Tryptone：0.05g；Diammonium hydrogen phosphate：0.05g；Magnesium sulfate：0.025g, pH 5.5,50mL.
Description
Technical field
The invention belongs to technical field of microbial fermentation, and in particular to one kind is produced based on Flow Cytometry high flux screening
The kit of ethanol fungi and its application.
Background technology
Biomass energy is the important component of following sustainable energy system, since this century, biological energy source into
Enter the research field of renewable resource, its with pollution-free, it is efficient the advantages that as in the world utilize the energy Main way, because
This develops biological energy source at present has become the common objective of various countries.And among bioenergy, research at present is most extensive, most has
Effect be exactly bio-ethanol utilization and exploitation.For current social scientific and technological level and the degree of development, can
Alcohol fuel is entirely possible to realize industrialization production in the coming few decades of prediction.
Excellent microorganism fungus kind is the basic and crucial of fermentation industry, to make the species of fermentation industry product, yield and
Quality has greatly improved it may first have to which the production bacterial strain of selection and breeding function admirable, the bacterial strain of function admirable can bring for production
Very high economic benefit.Mainly have in the method for high-yield ethanol saccharomyces cerevisiae screening at present, alcohol in high concentration plate screening model,
TTC compound slab screening methods etc..These method workloads are all very big, when carrying out bacterium colony screening, often rely on experimenter's warp
Test, by visually observing, select purpose bacterial strain, there are blindness, greatly reduces screening efficiency.
Active oxygen (reactive oxygen species, ROS) refers to some derivatives such as superoxide anion of oxygen
(O2-), hydroxy radical (OH) and hydrogen peroxide (H2O2) etc., excessive ROS can make large biological molecule such as DNA, protein,
Peroxidating chain reaction occurs for lipid, causes cyto-architectural damage, influences physiological activity.Howlett have detected copper ion institute
The change of caused saccharomyces cerevisiae intracellular ROS, it is found that the horizontal cell cycles of ROS have certain influence.Some researches show that yeast is thin
Born of the same parents' overall activity and production alcohol ability and the level of its internal ROS are in a degree of negative correlativing relation, this is us with logarithm
The content of ROS provides strong support as screening conditions in phase yeast.
Flow Cytometry (Flow cytometry, FCM) rapidly can carry out spectrochemical analysis to individual cells,
Researcher is set to establish multi-dimensional table of the individual cells in colony as having compared with conventional method for cell count many excellent
Gesture, as accuracy is high, it is reproducible the advantages that.It is used to the molecule being suspended in fluid is counted and sorted.It is logical
Cross and sample is dyed using specific dyestuff, so that the fluorescence signal of excitation-detection mark, realizes high speed, cell one by one
Quantitative analysis and sorting.
The content of the invention
The object of the present invention is to provide a kind of kit based on Flow Cytometry high flux screening producing and ethanol fungi and
It is applied, which is based on Flow Cytometry, using fungal cell's overall activity and production alcohol ability with its internal ROS's
Level is in a degree of negative correlativing relation, can be rapidly and efficiently using the kit to judge the production alcohol ability of tested fungi
Screening high-yield ethanol fungi, improve industrial screening efficiency.
In order to achieve the above object, the technical solution adopted by the present invention is：
Based on the kit of Flow Cytometry high flux screening producing and ethanol fungi, including box body and reagent, further include thin
Born of the same parents' culture plate, centrifuge tube and copper sieve, the reagent include reagent A, each one bottle of B, C, D, E, F：
Reagent A：Citric acid：6.15g；Sodium citrate：17.544g；Add distilled water to be settled to 1000mL, sealed after autoclaving
Dress；
Reagent B：Propidium iodide（PI）：0.3mg；2,7- dihydro dichlorofluorescein sodium Diacetates：0.3mg；
Reagent C：Glucose：2g, adds distilled water to be settled to 50mL, autoclaving post package；
Reagent D：Glucose：12g, adds distilled water to be settled to 50mL, autoclaving post package；
Reagent E：Dusty yeast：0.5g；Tryptone：0.5g；Diammonium hydrogen phosphate：0.05g；Magnesium sulfate：0.025g, adds double steamings
Water 40mL dissolves, and adjust pH with NaOH solution is settled to 50mL, autoclaving post package to 5.5, then with distilled water；
Reagent F：Dusty yeast：0.05g；Tryptone：0.05g；Diammonium hydrogen phosphate：0.05g；Magnesium sulfate：0.025g, adds double
Water 40mL dissolvings are steamed, adjust pH with NaOH solution is settled to 50mL, autoclaving post package to 5.5, then with distilled water.
The Tissue Culture Plate is the porocyte culture plates of 48 holes ~ 96.
The centrifuge tube is 0.5 ~ 1.5mL centrifuge tubes.
The copper sieve is 200 ~ 400 mesh.
The Tissue Culture Plate is 1 ~ 2 piece, and centrifuge tube is 48 ~ 96, and copper sieves 1.
The reagent B is divided in brown centrifuge tube.
The NaOH solution concentration is 1 ~ 5mol/L.
The Tissue Culture Plate, centrifuge tube and copper sieve carried out sterilization treatment, can be ultraviolet irradiation sterilizings, can also
It is high-temperature heating sterilizing.The kit is stored under the conditions of 4 DEG C.
The kit based on Flow Cytometry high flux screening producing and ethanol fungi is in producing and ethanol fungi is screened
Using.
The application, includes the following steps：
Reagent B：Propidium iodide（PI）：0.3mg；2,7- dihydro dichlorofluorescein sodium Diacetates（DCFH-DA）：0.3mg；Face
Used time adds aseptic double-distilled water to be settled to 3mL, and dark place filtration disinfection is now with the current；
Reagent E is sufficiently mixed with reagent C, and 4 DEG C save backup；
Reagent F is sufficiently mixed with reagent D, and 4 DEG C save backup；
Kit application method of the present invention is as follows：
1）, reagent B configuration：Propidium iodide（PI）：0.3mg；2,7- dihydro dichlorofluorescein sodium Diacetates（DCFH-
DA）：0.3mg；Facing the used time adds aseptic double-distilled water to be settled to 3mL, and dark place filtration disinfection is now with the current；
2）, bacteria suspension preparation
Tested bacterium solution 1mL is taken to discard supernatant liquor, bacterial sediment reagent A is clear in 4 DEG C, 5500r/min centrifugation 10min
Both sides are washed, then are suspended with 1mL reagent As, obtain bacteria suspension, it is spare；
3）, various mutagenic treatments, such as mutagenesis, ultraviolet irradiation mutagenesis, ion beam are carried out to bacteria suspension as needed
The bacteria suspension after mutagenesis is obtained after injecting the processing such as mutagenesis, it is spare；
4）, reagent C and reagent E be sufficiently mixed, add reagent C+E in the orifice plate, additive amount is 500-1000 μ l/ holes；
To step 2）30 μ l reagent B are added in the bacteria suspension of acquisition, in 37 DEG C of avoid light place 10min, after being sieved through filter with 300 mesh copper, in
Detect on flow cytometer, be collected with orifice plate, each hole collects 1 × 104A thalline；It is put into constant-temperature table, 37 DEG C,
250r/min cultivates 24h；
5）, reagent D and reagent F be sufficiently mixed, dispense into sterile 10mL centrifuge tubes, 5mL/ pipes；
6）, by step 4）In obtained bacterium solution, add to correspondingly in the ready centrifuge tube of step 5 institute, 500-
1000 μ l/ are managed；Constant-temperature table is put into, 37 DEG C, 150r/min cultivates 60h；
7）, detecting step 6）The content of alcohol in obtained zymotic fluid.
Beneficial effect：Kit provided by the invention based on Flow Cytometry high flux screening producing and ethanol fungi and its
Using the kit is based on Flow Cytometry, utilizes fungal cell's overall activity and production alcohol ability and the water of its internal ROS
Flat is in a degree of negative correlativing relation, to judge the production alcohol ability of tested fungi, can be rapidly and efficiently using the kit
The fungi of high-yield ethanol is screened, industrial screening efficiency is improved, saves labour cost, while kit test dose is few, can be same
When screen 48 ~ 96 samples, greatly improve efficiency.
Embodiment
With reference to instantiation, the present invention is described in detail.
Embodiment 1
With influence of the kit measurement ion beam mutation of the present invention to ROS contents in saccharomyces cerevisiae body
First, solution is configured
Reagent B：Propidium iodide（PI）：0.3mg；2,7- dihydro dichlorofluorescein sodium Diacetates（DCFH-DA）：0.3mg, point
In brown centrifuge tube, it is kept in dark place, adds aseptic double-distilled water to be settled to 3mL, dark place filtration disinfection is now with the current；
2nd, influence of the ion beam mutation to ROS contents in saccharomyces cerevisiae body is measured
1. pair saccharomyces cerevisiae carries out ion beam mutation
（a）Cultured bacterium solution centrifuges 10min in 4 DEG C, 5500r/min, discards supernatant liquor, and reagent A is cleaned twice, then
With reagent A suspended yeast cell pellet, N is remained+Injection is handled.
（b）With the protection liquid of sterilizing（0.5% (wt) starch and 0.5% (wt) glucose）Above-mentioned bacteria suspension is diluted, takes dilution
100 μ L of liquid, the sterile petri dish center of a diameter of 90mm is spread evenly across with sterile glass spatula, and bacterium is made in sterile wind drying
Film.The mycoderm of preparation is respectively placed on the sterile target platform of ion implantation apparatus vacuum target chamber, 1 × 10-3Adopted under pa vacuum states
It is 15KeV with energy, dosage is respectively 0,4 × 1015、5×1015、6×1015、8×1015、10×1015、11×1015ions/
cm2, N+ injection yeast mycoderm, respectively with 1.5mL reagent As immersion, elution, the bacterium solution eluted is obtained, by the bacterium of acquisition
Liquid names ck, a, b, c, d, e, f successively.
2. kit measurement
（a）Take tested each 1mL of bacterium solution ck, a, b, c, d, e, f that it is clear to discard upper strata in 4 DEG C, 5500r/min centrifugation 10min
Liquid, bacterial sediment are cleaned one time with reagent A, then are suspended with 1mL reagent As, obtain bacteria suspension, spare；
（b）Reagent C and reagent E are sufficiently mixed, reagent C+E is added in 48 orifice plates, additive amount is 500-1000 μ l/ holes；
（c）To step（a）30 μ l reagent B are added in the bacteria suspension of acquisition, in 37 DEG C of avoid light place 10min, with 300 mesh copper
After being sieved through filter, in being detected on flow cytometer, step is used（b）Middle ready 48 orifice plate of institute is collected, and each hole collection 1 ×
104A thalline, constant-temperature table is put into, 37 DEG C, 250r/min culture 24h, obtain bacterium solution；
（d）Reagent D and reagent F are sufficiently mixed, dispensed into 48 sterile 10mL centrifuge tubes, 5mL/ pipes；
（e）By step（c）In obtained bacterium solution, add to step correspondingly（d）In the ready centrifuge tube of institute,
1000 μ l/ are managed；Constant-temperature table is put into, 37 DEG C, 150r/min cultivates 60h；
3rd, testing result such as table 1.
Table 1：
| N+ ion implantation dosages（×1015ions/cm2） | 0 | 4 | 5 | 6 | 8 | 10 | 11 |
| DCF fluorescence intensities | 65.38 | 142.38 | 234.65 | 139.63 | 198.55 | 248.89 | 265.87 |
Embodiment 2
With influence of the kit measurement ultraviolet radiation mutagenesis of the present invention to ROS contents in saccharomyces cerevisiae body
First, solution is configured
Reagent B：Propidium iodide（PI）：0.3mg；2,7- dihydro dichlorofluorescein sodium Diacetates（DCFH-DA）：0.3mg, point
In brown centrifuge tube, it is kept in dark place, adds aseptic double-distilled water to be settled to 3mL, dark place filtration disinfection is now with the current；
2nd, influence of the ultraviolet irradiation to ROS contents in saccharomyces cerevisiae body is measured
1. pair saccharomyces cerevisiae carries out ultraviolet irradiation
（a）Cultured bacterium solution centrifuges 10min in 4 DEG C, 5500r/min, discards supernatant liquor, and reagent A is cleaned twice, then
With reagent A suspended yeast cell pellet, ultraviolet irradiation injection processing is remained.
（b）With sterile water dilution step（a）In bacteria suspension, take 2 mL in the sterile petri dish of the mm of U=75,
It is placed under 30W ultraviolet lamps at 30cm, respectively treatment with irradiation：30 s, 45 s, 90 s, 120 s, 180 s, 240 s, each processing
Repeat 3 times, 30min is stood in dark culturing case after each ultraviolet irradiation.To be compared without irradiation, ultraviolet shines
Penetrate later operation all to carry out in the dark, prevent light reparation.By the bacterium solution of acquisition name successively ck1, a1, b1, c1, d1,
e1、f1。
2. kit measurement
（a）Take tested bacterium solution ck1, a1, b1, c1, d1, e1, f1.Each 1mL is discarded in 4 DEG C, 5500r/min centrifugation 10min
Supernatant liquor, bacterial sediment are cleaned on one side with reagent A, then are suspended with 1mL reagent As, obtain bacteria suspension, spare；
（b）Reagent C and reagent E are sufficiently mixed, reagent C+E is added in 48 orifice plates, additive amount is 500-1000 μ l/ holes；
（c）To step（a）30 μ l reagent B are added in the bacteria suspension of acquisition, in 37 DEG C of avoid light place 10min, with 300 mesh copper
After being sieved through filter, in being detected on flow cytometer, step is used（b）Middle ready 48 orifice plate of institute is collected, and each hole collection 1 ×
104A thalline,；Constant-temperature table is put into, 37 DEG C, 250r/min cultivates 24h；
（d）Reagent D and reagent F are sufficiently mixed, dispensed into 48 sterile 10mL centrifuge tubes, 5mL/ pipes；
（e）By step（c）In obtained bacterium solution, add to step correspondingly（d）In the ready centrifuge tube of institute,
1000 μ l/ are managed；Constant-temperature table is put into, 37 DEG C, 150r/min cultivates 60h；
3rd, testing result is as shown in table 2.
Table 2：
| The ultraviolet irradiation time（s） | 0 | 30 | 45 | 90 | 120 | 180 | 240 |
| DCF fluorescence intensities | 65.38 | 142.38 | 234.65 | 348.69 | 473.82 | 643.19 | 753.28 |
Embodiment 3
With kit high flux screening producing and ethanol saccharomyces cerevisiae of the present invention
First, mutagenesis is carried out to saccharomyces cerevisiae
By starting strain Saccharomyces Cerevisiae in S 23（Starting strain used obtains for laboratory screening, in patent
It has been reported that mistake in 201410495426.9, and the present invention is only as an example, be not related to the protection of specific bacterial strain）Cell is made
Suspension, adjustment cell concentration are 106A/milliliter, then centrifuges 10min in 4 DEG C, 5500r/min by bacterium solution, it is clear to discard upper strata
Liquid, reagent A are cleaned twice, then with reagent A suspended yeast cell pellet, carry out mutagenic treatment.Mutagenic treatment is：It is different ultraviolet
Exposure intensity treatment with irradiation, different ions beam injection processing, then selects different mutagenesis bacterium solutions and adds up to 48 parts.
2nd, kit screens
（a）Each 1mL of the bacterium solution after mutagenesis is taken to discard supernatant liquor, bacterial sediment in 4 DEG C, 5500r/min centrifugation 10min
Cleaned on one side with reagent A, then suspended with 1mL reagent As, obtain bacteria suspension, it is spare；
（b）Reagent C and reagent E are sufficiently mixed, reagent C+E is added in 48 orifice plates, additive amount is 500-1000 μ l/
Hole；
（c）To step（a）30 μ l reagent B are added in the bacteria suspension of acquisition, in 37 DEG C of avoid light place 10min, with 300 mesh copper
After being sieved through filter, in being detected on flow cytometer, step is used（b）Middle ready 48 orifice plate of institute is collected, and it is strong to collect DCF fluorescence
Thalline of the degree less than 200, each hole collect 1 × 104A thalline, is put into constant-temperature table, 37 DEG C, 250r/min cultivates 24h；
（d）Reagent D and reagent F are sufficiently mixed, dispensed into 48 sterile 10mL centrifuge tubes, 5mL/ pipes；
（e）By step（c）In obtained bacterium solution, add to step correspondingly（d）In the ready centrifuge tube of institute,
1000 μ l/ are managed；Constant-temperature table is put into, 37 DEG C, 150r/min cultivates 60h；
（f）Determination step（e）Alcohol content in obtained zymotic fluid, screens high-proof bacterial strain.
3rd, experimental result
At the same time in 48 plants of bacterium solutions of screening, ethanol content (mL/L) is as shown in table 3 respectively, and the ethanol of wherein starting strain contains
Measure as 60 mL/L.
Table 3：
| [60,70） | [70,80） | [80,90） | [90,100） | [100,110） | [110,120） | [120,+∞） |
| 2 | 2 | 4 | 7 | 18 | 9 | 6 |
[a, b in table）It is scope between a and b, including a, but do not include b.From table 3 it is observed that obtained without mutagenesis
To the yeast of different producing and ethanol abilities, screened using the kit, the fermentability of multi-strain bacteria strain can be investigated at the same time, reagent is used
Amount is few, and screening efficiency is high.
Above example is only that the present invention is further explained, and is not intended as the limit to the scope of the present invention
System, kit of the present invention can select the hole count of Tissue Culture Plate and the number of centrifuge tube according to the number of strain to be tested, together
When producing and ethanol ability screening is carried out to the bacterial strains of different mutagenic conditions, easy to operate, reagent dosage is few, and screening efficiency is high, significantly
Reduce labour payment.
Claims (6)
1. based on the kit of Flow Cytometry high flux screening producing and ethanol fungi, including box body and reagent, it is characterised in that：
Further include Tissue Culture Plate, centrifuge tube and copper sieve, the Tissue Culture Plate is the porocyte culture plates of 48 holes ~ 96, the reagent
Including each one bottle of reagent A, B, C, D, E, F：
Reagent A：Citric acid：6.15g；Sodium citrate：17.544g；Distilled water is added to be settled to 1000mL, autoclaving post package；
Reagent B：Propidium iodide：0.3mg；2,7- dihydro dichlorofluorescein sodium Diacetates：0.3mg；Reagent B is divided in brown centrifugation
Guan Zhong；
Reagent C：Glucose：2g, adds distilled water to be settled to 50mL, autoclaving post package；
Reagent D：Glucose：12g, adds distilled water to be settled to 50mL, autoclaving post package；
Reagent E：Dusty yeast：0.5g；Tryptone：0.5g；Diammonium hydrogen phosphate：0.05g；Magnesium sulfate：0.025g, adds distilled water
40mL dissolves, and adjust pH with NaOH solution is settled to 50mL, autoclaving post package to 5.5, then with distilled water；
Reagent F：Dusty yeast：0.05g；Tryptone：0.05g；Diammonium hydrogen phosphate：0.05g；Magnesium sulfate：0.025g, adds distilled water
40mL dissolves, and adjust pH with NaOH solution is settled to 50mL, autoclaving post package to 5.5, then with distilled water.
2. the kit according to claim 1 based on Flow Cytometry high flux screening producing and ethanol fungi, its feature exist
In：The centrifuge tube is 0.5 ~ 1.5mL centrifuge tubes.
3. the kit according to claim 1 based on Flow Cytometry high flux screening producing and ethanol fungi, its feature exist
In：The copper sieve is 200 ~ 400 mesh.
4. the kit according to claim 1 based on Flow Cytometry high flux screening producing and ethanol fungi, its feature exist
In：The Tissue Culture Plate is 1 ~ 2 piece, and centrifuge tube is 48 ~ 96, and copper sieves 1.
5. the kit according to claim 1 based on Flow Cytometry high flux screening producing and ethanol fungi, its feature exist
In：The NaOH solution concentration is 1 ~ 5mol/L.
6. the kit in claim 1 ~ 5 based on Flow Cytometry high flux screening producing and ethanol fungi described in any one exists
Screen the application in producing and ethanol fungi.
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| CN106191028A (en) * | 2016-07-26 | 2016-12-07 | 江南大学 | A kind of method of high flux screening high yield tylosin bacterial strain based on flow cytometry |
| CN106191029B (en) * | 2016-07-26 | 2019-05-31 | 江南大学 | A method of the high flux screening aureomycin superior strain based on flow cytometry |
| CN106222162A (en) * | 2016-07-26 | 2016-12-14 | 江南大学 | A kind of method of high flux screening avilamycin superior strain based on flow cytometry |
| CN111693499A (en) * | 2020-06-11 | 2020-09-22 | 北京市农林科学院 | Active oxygen free radical detection method for edible fungus hyphae and application of active oxygen free radical detection method in strain viability detection |
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| CA2832308C (en) * | 2010-10-25 | 2023-05-09 | Synthetic Genomics, Inc. | A method for identification of microbial antagonists against plant pathogens |
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| 活性氧在酿酒酵母乙醇胁迫中的作用;赵素娟;《中国优秀硕士学位论文全文数据库.工程科技I辑》;20130215(第2期);摘要、第14页倒数第1段-第15页第3段 * |
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