CN104560725A - Flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof - Google Patents
Flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof Download PDFInfo
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 63
- 238000012216 screening Methods 0.000 title claims abstract description 33
- 241000233866 Fungi Species 0.000 title claims abstract description 22
- 238000000684 flow cytometry Methods 0.000 title claims abstract description 20
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 77
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 20
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 claims abstract description 13
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 12
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 12
- 229910052802 copper Inorganic materials 0.000 claims abstract description 12
- 239000010949 copper Substances 0.000 claims abstract description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims abstract description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 6
- 239000000843 powder Substances 0.000 claims abstract description 6
- 239000001509 sodium citrate Substances 0.000 claims abstract description 3
- 238000012360 testing method Methods 0.000 claims description 26
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 230000004907 flux Effects 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 14
- VFNKZQNIXUFLBC-UHFFFAOYSA-N 2',7'-dichlorofluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(Cl)=C(O)C=C1OC1=C2C=C(Cl)C(O)=C1 VFNKZQNIXUFLBC-UHFFFAOYSA-N 0.000 claims description 6
- OPGYRRGJRBEUFK-UHFFFAOYSA-L disodium;diacetate Chemical compound [Na+].[Na+].CC([O-])=O.CC([O-])=O OPGYRRGJRBEUFK-UHFFFAOYSA-L 0.000 claims description 6
- 239000001632 sodium acetate Substances 0.000 claims description 6
- 235000017454 sodium diacetate Nutrition 0.000 claims description 6
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 4
- 108010046845 tryptones Proteins 0.000 claims description 4
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 2
- 229940038773 trisodium citrate Drugs 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 abstract 2
- 229910019142 PO4 Inorganic materials 0.000 abstract 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract 2
- 239000010452 phosphate Substances 0.000 abstract 2
- 239000012137 tryptone Substances 0.000 abstract 2
- 238000004113 cell culture Methods 0.000 abstract 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 abstract 1
- 241000894006 Bacteria Species 0.000 description 32
- 239000007788 liquid Substances 0.000 description 27
- 239000000725 suspension Substances 0.000 description 13
- 238000000034 method Methods 0.000 description 11
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- 230000001954 sterilising effect Effects 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 231100000350 mutagenesis Toxicity 0.000 description 7
- 238000002703 mutagenesis Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 230000001678 irradiating effect Effects 0.000 description 5
- 231100000219 mutagenic Toxicity 0.000 description 5
- 230000003505 mutagenic effect Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 238000010884 ion-beam technique Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000013049 sediment Substances 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical compound CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 210000002287 horizontal cell Anatomy 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000005468 ion implantation Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 150000002926 oxygen Chemical class 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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Abstract
The invention discloses a flow-cytometry-based kit for high-flux screening of ethanol-producing fungi and application thereof. The kit comprises a box body, reagents, a cell culture plate, a centrifuge tube and a copper screen. The reagents are as follows: 1000 mL of reagent A composed of 6.15g of citric acid and 17.544g of sodium citrate, reagent B composed of 0.3mg of propidium iodide (PI) and 0.3mg of DCFH-DA, 50 mL of reagent C composed of 2g of glucose, 50 mL of reagent D composed of 10g of glucose, 50 mL of reagent E (pH value is 5.5) composed of 0.5g of yeast powder, 0.5g of tryptone, 0.05g of ammonium monoacid phosphate and 0.025g of magnesium sulfate, and 50 mL of reagent F (pH value is 5.5) composed of 0.05g of yeast powder, 0.05g of tryptone, 0.05g of ammonium monoacid phosphate and 0.025g of magnesium sulfate.
Description
Technical field
The invention belongs to technical field of microbial fermentation, be specifically related to a kind of test kit based on Flow Cytometry high flux screening producing and ethanol fungi and application thereof.
Background technology
Biomass energy is the important component part of following sustainable energy system, from this century, bioenergy enters the research field of renewable resources, it is with pollution-free, efficiency advantages of higher becomes the Main way utilizing the energy in the world, therefore develops the common objective that bioenergy has become various countries at present.And in the middle of bioenergy, at present research is the most extensive, utilization and the exploitation of the most effective is exactly bio-ethanol.By the scientific and technological level of current society and the degree of development, in the coming few decades that can predict, alcohol fuel completely likely realizes industrialization to produce.
Excellent microbial strains is basis and the key of fermentation industry, make the kind of fermentation industry product, seed output and quality has greatly improved, the production bacterial strain of first necessary seed selection excellent property, the bacterial strain of excellent property can bring very high economic benefit for producing.Mainly contain in the method for high-yield ethanol yeast saccharomyces cerevisiae screening at present, alcohol in high concentration plate screening model, TTC compound slab sieve method etc.These method workloads are all very large, when carrying out bacterium colony screening, often rely on experimenter's experience, by means of visual inspection, select object bacterial strain, there is blindness, greatly reduce screening efficiency.
Active oxygen (reactive oxygen species, ROS) refers to that some derivative of oxygen is as superoxide anion (O
2-), hydroxy radical qiao (OH) and hydrogen peroxide (H
2o
2) etc., excessive ROS can make biomacromolecule as DNA, protein, lipid generation peroxidation chain reaction, causes cyto-architectural damage, affects physiologically active.Howlett have detected the change of ROS in the yeast saccharomyces cerevisiae born of the same parents caused by cupric ion, finds that the horizontal cell cycle of ROS has certain influence.There are some researches show, yeast cell overall activity and the level of producing ROS in alcohol ability and its body are negative correlativing relation to a certain degree, and this is for we provide strong support using the content of ROS in logarithmic phase yeast body as screening conditions.
Flow Cytometry (Flow cytometry, FCM) spectrochemical analysis can be carried out to individual cells rapidly, researchist is made to set up the multidimensional presentation of individual cells in colony, there is a lot of advantage compared with the method for cell count of routine, as advantages such as tolerance range are high, reproducible.It is for counting the molecule be suspended in fluid and sorting.By using specific dyestuff to dye to sample, thus the fluorescent signal of excitation-detection mark, realize cell quantitative at a high speed, one by one and sorting.
Summary of the invention
The object of this invention is to provide a kind of test kit based on Flow Cytometry high flux screening producing and ethanol fungi and application thereof, this test kit is based on Flow Cytometry, the level utilizing fungal cell's overall activity and produce ROS in alcohol ability and its body is negative correlativing relation to a certain degree, judge the product alcohol ability of tested fungi, applying this test kit can the fungi of screening high-yield ethanol rapidly and efficiently, improves industrial screening efficiency.
In order to achieve the above object, the technical solution used in the present invention is:
Based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, comprise box body and reagent, also comprise Tissue Culture Plate, centrifuge tube and copper sieve, described reagent comprises each one bottle of reagent A, B, C, D, E, F:
Reagent A: citric acid: 6.15g; Trisodium Citrate: 17.544g; Add distilled water and be settled to 1000mL, encapsulate after autoclaving;
Reagent B: propidium iodide (PI): 0.3mg; 2,7-dihydro dichlorofluorescein sodium Diacetate: 0.3mg;
Reagent C: glucose: 2g, adds distilled water and is settled to 50mL, encapsulate after autoclaving;
Reagent D: glucose: 12g, adds distilled water and is settled to 50mL, encapsulate after autoclaving;
Reagent E: yeast powder: 0.5g; Tryptones: 0.5g; Secondary ammonium phosphate: 0.05g; Magnesium sulfate: 0.025g, adds distilled water 40mL and dissolves, and regulates pH to 5.5, then is settled to 50mL with distilled water, encapsulate after autoclaving by NaOH solution;
Reagent F: yeast powder: 0.05g; Tryptones: 0.05g; Secondary ammonium phosphate: 0.05g; Magnesium sulfate: 0.025g, adds distilled water 40mL and dissolves, and regulates pH to 5.5, then is settled to 50mL with distilled water, encapsulate after autoclaving by NaOH solution.
Described Tissue Culture Plate is 48 hole ~ 96 porocyte culture plates.
Described centrifuge tube is 0.5 ~ 1.5mL centrifuge tube.
Described copper sieve is 200 ~ 400 orders.
Described Tissue Culture Plate is 1 ~ 2 piece, and centrifuge tube is 48 ~ 96, and copper sieves 1.
Described reagent B is divided in brown centrifuge tube.
Described NaOH solution concentration is 1 ~ 5mol/L.
Described Tissue Culture Plate, centrifuge tube and copper sieve all carried out sterilising treatment, and can be uv irradiating sterilizing, also can be heat sterilizing.Under described test kit is kept at 4 DEG C of conditions.
The described application of test kit in screening producing and ethanol fungi based on Flow Cytometry high flux screening producing and ethanol fungi.
Described application, comprises the steps:
Reagent B: propidium iodide (PI): 0.3mg; 2,7-dihydro dichlorofluorescein sodium Diacetate (DCFH-DA): 0.3mg; Facing the used time adds aseptic double-distilled water and is settled to 3mL, and dark place filters sterilization, now with the current;
Reagent E fully mixes with reagent C, and 4 DEG C save backup;
Reagent F fully mixes with reagent D, and 4 DEG C save backup;
Test kit using method of the present invention is as follows:
1), the configuration of reagent B: propidium iodide (PI): 0.3mg; 2,7-dihydro dichlorofluorescein sodium Diacetate (DCFH-DA): 0.3mg; Facing the used time adds aseptic double-distilled water and is settled to 3mL, and dark place filters sterilization, now with the current;
2), the preparation of bacteria suspension
Get tested bacterium liquid 1mL in 4 DEG C, the centrifugal 10min of 5500r/min, discard supernatant liquid, bacterial sediment reagent A cleans both sides, then suspends by 1mL reagent A, obtains bacteria suspension, for subsequent use;
3), as required various mutagenic treatment is carried out to bacteria suspension, such as, after the process such as chemomorphosis, uv irradiating mutagenesis, ion beam mutation mutagenesis, obtain the bacteria suspension after mutagenesis, for subsequent use;
4), by reagent C fully mix with reagent E, add reagent C+E in the orifice plate, addition is 500-1000 μ l/ hole; To step 2) add 30 μ l reagent B in the bacteria suspension that obtains, place 10min in 37 DEG C of lucifuges, with the sieved filter of 300 order copper after, detect on flow cytometer, collect with orifice plate, each hole collection 1 × 10
4individual thalline; Put into constant-temperature table, 37 DEG C, 250r/min cultivates 24h;
5), by reagent D fully mix with reagent F, divide and be filled in aseptic 10mL centrifuge tube, 5mL/ manages;
6), the bacterium liquid that will obtain in step 4), add to one to one in the ready centrifuge tube of step 5 institute, 500-1000 μ l/ manages; Put into constant-temperature table, 37 DEG C, 150r/min cultivates 60h;
7), detecting step 6) content of alcohol in the fermented liquid that obtains.
Beneficial effect: the test kit based on Flow Cytometry high flux screening producing and ethanol fungi provided by the invention and application thereof, this test kit is based on Flow Cytometry, the level utilizing fungal cell's overall activity and produce ROS in alcohol ability and its body is negative correlativing relation to a certain degree, judge the product alcohol ability of tested fungi, applying this test kit can the fungi of screening high-yield ethanol rapidly and efficiently, improve industrial screening efficiency, save labour cost, test kit agents useful for same amount is few simultaneously, 48 ~ 96 samples can be screened simultaneously, greatly raise the efficiency.
Embodiment
Below in conjunction with specific examples, the present invention is described in detail.
embodiment 1
With the impact of kit measurement ion beam mutation of the present invention on ROS content in Saccharomyces cerevisiae
One, solution is configured
Reagent B: propidium iodide (PI): 0.3mg; 2,7-dihydro dichlorofluorescein sodium Diacetate (DCFH-DA): 0.3mg, is divided in brown centrifuge tube, keeps in Dark Place, and adds aseptic double-distilled water and is settled to 3mL, and dark place filters sterilization, now with the current;
Two, ion beam mutation is measured on the impact of ROS content in Saccharomyces cerevisiae
1. pair yeast saccharomyces cerevisiae carries out ion beam mutation
A () cultured bacterium liquid, in 4 DEG C, the centrifugal 10min of 5500r/min, discards supernatant liquid, reagent A cleaning twice, then uses reagent A suspended yeast cell precipitation thing, waits until N
+inject process.
B () dilutes above-mentioned bacteria suspension with the protection liquid (0.5% (wt) starch and 0.5% (wt) glucose) of sterilizing; get diluent 100 μ L; be spread evenly across with sterile glass spatula the sterile petri dish central authorities that diameter is 90mm, sterile wind dries up makes mycoderm.The mycoderm of preparation is placed in respectively on the aseptic target platform of ion implanter vacuum target chamber, 1 × 10
-3adopt energy to be 15KeV under pa vacuum state, dosage is respectively 0,4 × 10
15, 5 × 10
15, 6 × 10
15, 8 × 10
15, 10 × 10
15, 11 × 10
15ions/cm
2, N+ inject yeast mycoderm, to soak with 1.5mL reagent A respectively, wash-out, obtain the bacterium liquid eluted, the bacterium liquid of acquisition is named ck, a, b, c, d, e, f successively.
2. kit measurement
A () gets each 1mL of tested bacterium liquid ck, a, b, c, d, e, f in 4 DEG C, the centrifugal 10min of 5500r/min, discard supernatant liquid, bacterial sediment reagent A cleans one time, then suspends by 1mL reagent A, obtains bacteria suspension, for subsequent use;
B reagent C fully mixes with reagent E by (), add reagent C+E in 48 orifice plates, and addition is 500-1000 μ l/ hole;
Add 30 μ l reagent B in c bacteria suspension that () obtains to step (a), place 10min in 37 DEG C of lucifuges, with the sieved filter of 300 order copper after, detect on flow cytometer, collect with ready 48 orifice plates in step (b), each hole collection 1 × 10
4individual thalline, put into constant-temperature table, 37 DEG C, 250r/min cultivates 24h, obtains bacterium liquid;
D reagent D fully mixes with reagent F by (), point to be filled in 48 aseptic 10mL centrifuge tubes, 5mL/ manages;
E bacterium liquid that () will obtain in step (c), add to one to one in the ready centrifuge tube of step (d) institute, 1000 μ l/ manage; Put into constant-temperature table, 37 DEG C, 150r/min cultivates 60h;
Three, detected result is as table 1.
Table 1:
| N+ ion implantation dosage (× 10 15ions/cm 2） | 0 | 4 | 5 | 6 | 8 | 10 | 11 |
| DCF fluorescence intensity | 65.38 | 142.38 | 234.65 | 139.63 | 198.55 | 248.89 | 265.87 |
embodiment 2
With the impact of kit measurement ultraviolet radiation mutagenesis of the present invention on ROS content in Saccharomyces cerevisiae
One, solution is configured
Reagent B: propidium iodide (PI): 0.3mg; 2,7-dihydro dichlorofluorescein sodium Diacetate (DCFH-DA): 0.3mg, is divided in brown centrifuge tube, keeps in Dark Place, and adds aseptic double-distilled water and is settled to 3mL, and dark place filters sterilization, now with the current;
Two, uviolizing is measured on the impact of ROS content in Saccharomyces cerevisiae
1. pair yeast saccharomyces cerevisiae carries out uv irradiating
A () cultured bacterium liquid, in 4 DEG C, the centrifugal 10min of 5500r/min, discards supernatant liquid, reagent A cleaning twice, then uses reagent A suspended yeast cell precipitation thing, waits until uv irradiation and injects process.
B () uses the bacteria suspension in sterilized water dilution step (a), get 2 mL in the sterile petri dish of U=75 mm, be placed in 30cm place under 30W ultraviolet lamp, radiation treatment respectively: 30 s, 45 s, 90 s, 120 s, 180 s, 240 s, each process repetition 3 times, leaves standstill 30min after each uv irradiating in dark culturing case.Compare not carry out irradiation, the later operation of uviolizing is carried out all in the dark, prevents light reparation.The bacterium liquid of acquisition is named ck1, a1, b1, c1, d1, e1, f1 successively.
2. kit measurement
A () gets tested bacterium liquid ck1, a1, b1, c1, d1, e1, f1.Each 1mL, in 4 DEG C, the centrifugal 10min of 5500r/min, discards supernatant liquid, bacterial sediment reagent A cleaning, then suspends by 1mL reagent A, obtains bacteria suspension, for subsequent use;
B reagent C fully mixes with reagent E by (), add reagent C+E in 48 orifice plates, and addition is 500-1000 μ l/ hole;
Add 30 μ l reagent B in c bacteria suspension that () obtains to step (a), place 10min in 37 DEG C of lucifuges, with the sieved filter of 300 order copper after, detect on flow cytometer, collect with ready 48 orifice plates in step (b), each hole collection 1 × 10
4individual thalline; Put into constant-temperature table, 37 DEG C, 250r/min cultivates 24h;
D reagent D fully mixes with reagent F by (), point to be filled in 48 aseptic 10mL centrifuge tubes, 5mL/ manages;
E bacterium liquid that () will obtain in step (c), add to one to one in the ready centrifuge tube of step (d) institute, 1000 μ l/ manage; Put into constant-temperature table, 37 DEG C, 150r/min cultivates 60h;
Three, detected result is as shown in table 2.
Table 2:
| Uv irradiation time (s) | 0 | 30 | 45 | 90 | 120 | 180 | 240 |
| DCF fluorescence intensity | 65.38 | 142.38 | 234.65 | 348.69 | 473.82 | 643.19 | 753.28 |
Embodiment 3
With test kit high flux screening producing and ethanol yeast saccharomyces cerevisiae of the present invention
One, mutagenesis is carried out to yeast saccharomyces cerevisiae
Be that laboratory screening obtains by starting strain used for starting strain Saccharomyces Cerevisiae in S 23(, reported in patent 201410495426.9, and the present invention be only exemplarily, does not relate to the protection of concrete bacterial strain) make cell suspension, adjustment cell concn is 10
6individual/milliliter, then by bacterium liquid in 4 DEG C, the centrifugal 10min of 5500r/min, discard supernatant liquid, reagent A cleaning twice, then uses reagent A suspended yeast cell precipitation thing, carries out mutagenic treatment.Mutagenic treatment is: the process of different uv irradiating intensity illumination, and different ions bundle injects process, then selects different mutagenic bacteria liquid and adds up to 48 parts.
Two, test kit screening
A () gets each 1mL of bacterium liquid after mutagenesis in 4 DEG C, the centrifugal 10min of 5500r/min, discard supernatant liquid, bacterial sediment reagent A cleaning, then suspends by 1mL reagent A, obtains bacteria suspension, for subsequent use;
B reagent C fully mixes with reagent E by (), add reagent C+E in 48 orifice plates, and addition is 500-1000 μ l/ hole;
30 μ l reagent B are added in c bacteria suspension that () obtains to step (a), 10min is placed in 37 DEG C of lucifuges, after the sieved filter of 300 order copper, detect on flow cytometer, collect with ready 48 orifice plates in step (b), collect the thalline that DCF fluorescence intensity is less than 200, each hole collects 1 × 10
4individual thalline, puts into constant-temperature table, 37 DEG C, and 250r/min cultivates 24h;
D reagent D fully mixes with reagent F by (), point to be filled in 48 aseptic 10mL centrifuge tubes, 5mL/ manages;
E bacterium liquid that () will obtain in step (c), add to one to one in the ready centrifuge tube of step (d) institute, 1000 μ l/ manage; Put into constant-temperature table, 37 DEG C, 150r/min cultivates 60h;
Ethanol content in f fermented liquid that () determination step (e) obtains, screens high-proof bacterial strain.
Three, experimental result
In the 48 strain bacterium liquid simultaneously screened, ethanol content (mL/L) is as shown in table 3 respectively, and wherein the ethanol content of starting strain is 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 |
In table [a, b) for scope is between a and b, comprise a, but do not comprise b.As can be seen from Table 3, obtain the yeast of different producing and ethanol ability without mutagenesis, use this test kit to screen, can investigate the fermentation capacity of multi-strain bacteria strain, reagent dosage is few, and screening efficiency is high simultaneously.
Above embodiment is only to further explanation explanation of the present invention; not as limiting the scope of the invention; test 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 test strains; the screening of producing and ethanol ability is carried out to the bacterial strain of different mutagenic condition simultaneously; simple to operate; reagent dosage is few, and screening efficiency is high, greatly reduces labour payment.
Claims (8)
1. based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, comprise box body and reagent, it is characterized in that: also comprise Tissue Culture Plate, centrifuge tube and copper sieve, described reagent comprises each one bottle of reagent A, B, C, D, E, F:
Reagent A: citric acid: 6.15g; Trisodium Citrate: 17.544g; Add distilled water and be settled to 1000mL, encapsulate after autoclaving;
Reagent B: propidium iodide: 0.3mg; 2,7-dihydro dichlorofluorescein sodium Diacetate: 0.3mg;
Reagent C: glucose: 2g, adds distilled water and is settled to 50mL, encapsulate after autoclaving;
Reagent D: glucose: 12g, adds distilled water and is settled to 50mL, encapsulate after autoclaving;
Reagent E: yeast powder: 0.5g; Tryptones: 0.5g; Secondary ammonium phosphate: 0.05g; Magnesium sulfate: 0.025g, adds distilled water 40mL and dissolves, and regulates pH to 5.5, then is settled to 50mL with distilled water, encapsulate after autoclaving by NaOH solution;
Reagent F: yeast powder: 0.05g; Tryptones: 0.05g; Secondary ammonium phosphate: 0.05g; Magnesium sulfate: 0.025g, adds distilled water 40mL and dissolves, and regulates pH to 5.5, then is settled to 50mL with distilled water, encapsulate after autoclaving by NaOH solution.
2. according to claim 1 based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, it is characterized in that: described Tissue Culture Plate is 48 hole ~ 96 porocyte culture plates.
3. according to claim 1 based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, it is characterized in that: described centrifuge tube is 0.5 ~ 1.5mL centrifuge tube.
4. according to claim 1 based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, it is characterized in that: described copper sieve is 200 ~ 400 orders.
5. according to claim 1 based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, it is characterized in that: described Tissue Culture Plate is 1 ~ 2 piece, centrifuge tube is 48 ~ 96, and copper sieves 1.
6. according to claim 1 based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, it is characterized in that: described reagent B is divided in brown centrifuge tube.
7. according to claim 1 based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi, it is characterized in that: described NaOH solution concentration is 1 ~ 5mol/L.
8. screening the application in producing and ethanol fungi based on the test kit of Flow Cytometry high flux screening producing and ethanol fungi described in any one in claim 1 ~ 7.
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