CN106191085B - Utilize cellulose restriction endonuclease and the safe grade carrier of β-glucosyl enzym structuring food prods and screening and culturing medium - Google Patents
Utilize cellulose restriction endonuclease and the safe grade carrier of β-glucosyl enzym structuring food prods and screening and culturing medium Download PDFInfo
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- CN106191085B CN106191085B CN201610597141.5A CN201610597141A CN106191085B CN 106191085 B CN106191085 B CN 106191085B CN 201610597141 A CN201610597141 A CN 201610597141A CN 106191085 B CN106191085 B CN 106191085B
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- glucosyl enzym
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- restriction endonuclease
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- 125000000188 beta-D-glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 title claims abstract description 25
- 229920002678 cellulose Polymers 0.000 title claims abstract description 23
- 239000001913 cellulose Substances 0.000 title claims abstract description 23
- 108091008146 restriction endonucleases Proteins 0.000 title claims abstract description 22
- 238000012216 screening Methods 0.000 title claims abstract description 18
- 238000012258 culturing Methods 0.000 title claims abstract description 15
- 235000013305 food Nutrition 0.000 title claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims abstract description 8
- 240000001972 Gardenia jasminoides Species 0.000 claims abstract 3
- 239000002609 medium Substances 0.000 claims description 13
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 11
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 4
- 108010061982 DNA Ligases Proteins 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
- 210000005253 yeast cell Anatomy 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 3
- 230000003115 biocidal effect Effects 0.000 abstract description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 3
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 244000005700 microbiome Species 0.000 abstract description 3
- 239000013612 plasmid Substances 0.000 description 26
- 239000007788 liquid Substances 0.000 description 19
- 239000000243 solution Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000029087 digestion Effects 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 238000001962 electrophoresis Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 238000004321 preservation Methods 0.000 description 10
- 238000001179 sorption measurement Methods 0.000 description 10
- 239000000499 gel Substances 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- 241000157835 Gardenia Species 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 229920000297 Rayon Polymers 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000012160 loading buffer Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 241000228245 Aspergillus niger Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 229910009891 LiAc Inorganic materials 0.000 description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 3
- 229960005542 ethidium bromide Drugs 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 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 description 2
- 241000235342 Saccharomycetes Species 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 238000011095 buffer preparation Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- Genetics & Genomics (AREA)
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- Microbiology (AREA)
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Abstract
The invention discloses using cellulose restriction endonuclease and the safe grade carrier of β-glucosyl enzym structuring food prods and screening and culturing medium, belong to genetic engineering field.A kind of food safety grade carrier, containing cellulose inscribe enzyme coding gene, β-glucosyl enzym encoding gene, can express cellulose restriction endonuclease and β-glucosyl enzym using cellulose restriction endonuclease, β-glucosyl enzym as selection markers.The carbon source that the screening and culturing medium of the carrier does not have microorganism that can directly utilize includes sodium cellulose glycolate and gardenia blue.Cellulose inscribe enzyme coding gene, β-glucosyl enzym encoding gene are cloned on carrier and obtain food safety grade carrier.The present invention utilizes special screening and culturing medium using cellulose restriction endonuclease and β-glucosyl enzym as selection markers enzyme, can fast and accurately filter out transformant；It since selection markers are not antibiotic, thus is the carrier of aliment security level.
Description
Technical field
The invention belongs to genetic engineering fields, relate to the use of cellulose restriction endonuclease and β-glucosyl enzym structuring food prods safety level
Carrier and screening and culturing medium.
Background technique
Most of carrier is all using the resistance of antibiotic as the selection markers of carrier now, and antibiotic eats the mankind
The harm of product safety is very much, therefore its application just has many limitations using antibiotic as the carrier of selection markers.
Must have the following characteristics that as selection markers can distinguish transformant and non-transformed son easily；It can allow purpose
Genetic fragment or purpose unit are incorporated into genome, or stabilization exists in host strain.In building genetic engineering bacterium, for
Integrative plasmid has more preferences, and integrative plasmid ring will not go out easily, in contrast has better stability.
Summary of the invention
It is an object of the invention to be to provide a kind of food safety grade carrier and its construction method and screening and culturing medium.
The purpose of the invention is achieved by the following technical solution:
A kind of food safety grade carrier contains cellulose inscribe using cellulose restriction endonuclease, β-glucosyl enzym as selection markers
Enzyme coding gene, β-glucosyl enzym encoding gene, can express cellulose restriction endonuclease and β-glucosyl enzym.Preferably, the fibre
Tie up plain inscribe enzyme coding gene, β-glucosyl enzym encoding gene derives from aspergillus niger.
The sequence of the cellulose inscribe enzyme coding gene is preferably as shown in SEQ ID NO.1.
The sequence of the β-glucosyl enzym encoding gene is preferably as shown in SEQ ID NO.2.
The construction method of the food safety grade carrier includes the following steps: cellulose inscribe enzyme coding gene, β-
Alpha-glucosidase encoding gene is cloned on carrier, and carrier is enable to express cellulose restriction endonuclease and β-glucosyl enzym.
Preferably, the construction method of the food safety grade carrier includes the following steps: sequence shown in SEQ ID NO.3
Sequence shown in column, SEQ ID NO.4 is building up on pPIC9K carrier by restriction enzyme and DNA ligase；Wherein limit
Property restriction endonuclease be SnaBI, EcoR I and AvrII, NotI, SnaBI, EcoR I correspond to sequence shown in digestion SEQ ID NO.3,
AvrII, NotI correspond to sequence shown in digestion SEQ ID NO.4.
The screening and culturing medium of the food safety grade carrier includes sodium cellulose glycolate and gardenia blue.The culture medium
The carbon source for not having microorganism that can directly utilize, after cellulose restriction endonuclease and β-glucosyl enzym only on carrier are expressed simultaneously,
Sodium cellulose glycolate can be converted to glucose, make microorganism growth increment.β-glucosyl enzym is converted to gardenia blue simultaneously
Gardenia red, to make periphery of bacterial colonies that light red be presented.Based on this, cellulose inscribe enzyme coding gene and β-glucosyl enzym are encoded
The assortment of genes can be used for the safe grade carrier of structuring food prods.
Preferably, the formula of the screening and culturing medium are as follows: sodium cellulose glycolate 10g/L, gardenia blue 1.5g/L,
NH4NO3 1.0g/L, NaCl 2g/L, YGM003A yeast cells full-synthetic culture medium YGM003A-1 1.5g/L, agar 20g/L.
The present invention utilizes the sieve of special designing using cellulose restriction endonuclease and β-glucosyl enzym as selection markers enzyme
Culture medium flat plate is selected, transformant can be fast and accurately filtered out.It since selection markers are not antibiotic, thus is food safety
The carrier of grade.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto.
Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art.
1, material
Restriction enzyme is purchased from Takara；Plasmid extraction kit (B518188), DNA ligase, kanamycins are purchased from
Shanghai bioengineering limited liability company；Bacillus coli DH 5 alpha containing plasmid pPIC9K is preserved in laboratory.Artificial synthesized
Composition sequence 1, composition sequence 2 as shown in SEQ ID NO.3,4 respectively.
2, the extraction of plasmid pPIC9K
Bacillus coli DH 5 alpha containing expression plasmid pPIC9K, (10 μ g/mL kanamycins of the addition) training in LB culture medium
12h is supported, cultivation temperature is 30 DEG C, extracts plasmid with plasmid extraction kit (B518188).Extraction step is as follows:
(1) 0.5mL bacterium solution is taken, 10000rpm is centrifuged 3min and collects thallus, most or Aspirate medium.
(2) 700 μ L Lysis Buffer UF are added in bacterial sediment, suction is beaten or vibrated to thorough suspension thalline, room
Temperature places 3min.
(3) lysate is all carefully moved into adsorption column, is placed at room temperature for 1min, 8000rpm is centrifuged 2min.Outwell collecting pipe
In liquid, adsorption column is put into the same collecting pipe.
(4) 500 μ L Prewash Solution, 8000rpm are added into adsorption column and are centrifuged 2min.It outwells in collecting pipe
Liquid, adsorption column is put into the same collecting pipe.
(5) 500 μ L Wash Solution, 8000rpm are added into adsorption column and are centrifuged 1min.Outwell the liquid in collecting pipe
Adsorption column is put into the same collecting pipe by body.
(6) it repeats that 500 μ L Wash Solution, 8000rpm centrifugation 1min are added into adsorption column.Outwell collecting pipe
In liquid, adsorption column is put into the same collecting pipe.
(7) adsorption column and collecting pipe are put into centrifuge, 8000rpm is centrifuged 2min.
(8) adsorption column is put into clean 1.5mL centrifuge tube, 50 μ L Elution is added in adsorbed film center
Buffer, is stored at room temperature 2min, and 8000 rpm are centrifuged 2min.Obtained plasmid DNA solution is placed in -20 DEG C of preservations.
3, the digestion of composition sequence 1 shown in plasmid pPIC9K and SEQ ID NO.3
Each 0.2 μ L of SnaBI and EcoR I enzyme is added into the plasmid pPIC9K that 25 μ L are extracted, enzyme cutting buffering liquid and double is added
24.6 μ L of water is steamed, in 30 DEG C of heat preservation 60min, 5 μ L Loading Buffer, 60 DEG C of heat preservation 10min is added and obtain plasmid
PPIC9K digested liquid.
Composition sequence 1 dilutes 10 times with distilled water, takes 25 μ L, and each 0.2 μ L of SnaBI and EcoR I enzyme is added, and digestion is added
5 μ L Loading Buffer are added in 30 DEG C of heat preservation 60min in 24.6 μ L of buffer and distilled water, and 60 DEG C of heat preservation 10min are obtained
To 1 digested liquid of sequence.
4, digested liquid electrophoresis
(1) electrophoresis reagents are prepared
1) 5 × TBE(tris- boric acid and EDTA) buffer preparation (1000mL): Tris 54g, boric acid 27.5g,
0.5mol/L EDTA 20mL, is transferred to 8.0 for pH, is settled to 1000mL.4 DEG C of refrigerators save, and the used time dilutes 5 times.
2) preparation of sample loading buffer: 0.25% bromophenol blue, 40%(W/V) aqueous sucrose solution, 4 DEG C of refrigerators preservations.
3) preparation of ethidium bromide: weighing 0.1g ethidium bromide, is dissolved in 10mL water, is made into the mother of final concentration of 10mg/mL
Liquid, 4 DEG C of refrigerators save.When dyeing, the mother liquor of 12.5 μ L is drawn, is added in the water of 250mL, makes its final concentration of 0.5 μ g/mL,
It is uniformly mixed.
4) preparation of 100 × TE buffer: 1mol/L Tris-HCl(pH8.0), 100mmol/L EDTA.Weigh Tris
121.1g, EDTA-Na 237.23g first use 800mL distilled water, after heating stirring dissolution, then about with hydrochloric acid tune pH to 8.0(
Hydrochloric acid 20mL is added), then it is settled to 1000mL.
5) preparation of 100 × electrophoretic buffer: 4mol/L Tris-HCl(pH8.0), 2mol/L sodium acetate, 200mmol/L
EDTA.Tris 242.2g, anhydrous sodium acetate 82.03g, EDTA-Na 237.23g are weighed, 400mL distilled water heating stirring is first used
After dissolution, then with glacial acetic acid tune pH to 8.0(glacial acetic acid 50mL is about added), then it is settled to 500mL.Used time dilutes 100 times.
(2) electrophoresis method
1) electrophoresis tank is installed: the running gel bed of organic glass being cleaned, is dried, is sealed the opening at both ends with adhesive tape,
It is placed on horizontal workbench, plugs sample comb.
2) preparation of 1% Ago-Gel: weighing agarose and be dissolved in electrophoretic buffer, sets in micro-wave oven or boiling water bath
It is heated to dissolving completely, taking-up shakes up.
3) encapsulating: the agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board.
4) after agarose gel solidification, electrophoretic buffer is added in electrophoresis tank, then extracts comb.
5) it is loaded: after the 4:1 mixing by volume of DNA sample and sample loading buffer, being added mixed liquor with micropipettor
Into sample cell, every slot adds 20 μ L, records the point sample order and sample-adding amount of sample.
6) electrophoresis: installing electrode cable, and loading wells one terminates cathode, and another termination anode opens power supply, adjusts voltage extremely
3-5V/cm, electrophoresis 1-3hr stop electrophoresis when bromophenol blue is moved on to away from gel front 1-2cm.
7) it dyes and observes: taking out gel, be placed in the dyeing liquor containing ethidium bromide and dye 30min, it can be in 254nm
Ultraviolet lamp under observe, have the position of fluorescent red-orange band, as DNA band.
5, the recycling and purifying of endonuclease bamhi
Use the DNA fragmentation of the plastic recovery kit recovery purifying digestion of Takara.It is cut out in the UV lamp containing purposeful
The Ago-Gel of DNA exhausts the liquid of gel surface with paper handkerchief.
Blob of viscose weight is weighed, blob of viscose volume is calculated.It is added blob of viscose lysate Buffer GM, Buffer GM's into blob of viscose
3 gel volume of dosage.25 DEG C of dissolution blob of viscose 10min of room temperature after evenly mixing.After gel is completely dissolved, it is molten that 3M sodium acetate is added
10 μ L of liquid (pH5.2) is uniformly mixed to solution and is restored yellow.Spin Column in kit is placed in Collection
On Tube.0.2mL glue lysate is transferred in Spin Column, and 12000rpm is centrifuged 1 minute, abandons filtrate.By 700 μ L's
Buffer WB is added in Spin Column, and room temperature 12000rpm is centrifuged 30 seconds, abandons filtrate.Again by the Buffer of 700 μ L
WB is added in Spin Column, and room temperature 12000rpm is centrifuged 30 seconds, abandons filtrate.Spin Column is placed in
On Collection Tube, room temperature 12000rpm is centrifuged 1min.Spin Column is placed in the centrifuge tube of new 1.5mL
On, 30 μ L sterile purified waters or Elution Buffer is added in the centre of Spin Column film, is stored at room temperature 1 minute.Room
Warm 12000rpm is centrifuged 1min eluted dna.
6, the connection and conversion of the pPIC9K plasmid Yu composition sequence 1 of digestion
Linked system are as follows: the 17 μ L of composition sequence of digestion recycling, 1 μ L, the T4 DNA of pPIC9K plasmid of digestion recycling
11 μ L of μ L, Buffer of ligase.4 DEG C connect 24 hours.
The 50 μ L of bacillus coli DH 5 alpha (without plasmid) competent cell for taking -80 DEG C of pipe preservations, sets and melts on ice, be added
The 5 above-mentioned connection products of μ L, gently rotating centrifugal pipe is to mix content, ice bath 30min；Centrifuge tube is placed in 42 DEG C of thermal shock 60-
It 90 seconds, then sets rapidly ice bath 3 minutes.500 μ L LB liquid mediums are added into centrifuge tube, mixing is placed on 37 DEG C of shaking table vibrations
Swing 45 minutes (150 revs/min) of culture；Purpose is to make relevant resistant maker gene expression on plasmid, and thallus is made to recover.It draws
100 μ L culture solutions are added on the LB solid medium of the 5mg/L containing kanamycins, with sterile elbow glass rod with gentle that cell is uniform
It is spreadable.By plate be placed in room temperature until liquid be absorbed, be inverted plate, 37 DEG C culture 12-16 hours.Picking individual colonies, preservation
Transformant is obtained, the bacillus coli DH 5 alpha of the carrier containing pPIC9K-1 is obtained.
7, plasmid extraction and digestion
Recombinant plasmid pPIC9K-1, composition sequence 2 are subjected to digestion, connection, conversion, identification according to above-mentioned steps, wherein
Recombinant plasmid pPIC9K-1, composition sequence 2 digestion used in restriction endonuclease be AvrII and NotI, finally obtain recombinant plasmid
pPIC9K-1-2。
8, plasmid pPIC9K -1-2 transformed yeast bacterium
(1) prepared by yeast bacterium competence
1) from a little yeast SMD1168 on plate or in conservation pipe, is chosen, in YPDA plate (YPDA culture medium: tryptone
20g/L, yeast extract 10g/L, agar 20g/L(solid), 0.2% adenine of adenine 15mL/L, adjust pH=7.5,121 DEG C
Sterilize 15min) on draw monoclonal, 30 DEG C cultivate 3 days or so.
2) (3 are done in parallel from picking them separately monoclonal (diameter 2-3mm) on plate into the teat glass containing 3mL YPDA
Pipe).
3) 30 DEG C, 50rpm culture 8-12h.200 μ L bacterium solutions are taken, OD is surveyed600.Choose OD600It is worth a maximum test tube (i.e.
Highest one of vigor), it draws 5 μ L and is transferred in the fresh YPDA culture medium of 50mL (culture medium is in 250mL triangular flask).
4) 30 DEG C, 230rpm culture 16-20h, until OD600=0.15-0.3。
5) cultured bacterium solution is transferred to 2 50mL centrifuge tubes, room temperature 3000g is centrifuged 3min, abandons supernatant, new with 100mL
Fresh YPDA has hanged the thallus of tube bottom, and pours into clean triangular flask.30 DEG C, 230rpm culture until OD600=0.4-0.5
(3-5 hours).
6) bacterium solution is poured into 2 50mL centrifuge tubes, room temperature 3000g is centrifuged 3min, abandons supernatant, each effective 30mL of centrifugation
Milipore water is resuspended.
7) 3000g is centrifuged 3min again, abandons supernatant, is resuspended per effective 1.1 × TE/LiAc of 1.4mL.
8) bacterium solution of resuspension is transferred in 2 EP pipes, 12000rpm is centrifuged 15s.
9) supernatant is abandoned, is resuspended per 1.1 × TE/LiAc of effective 600 μ L, two pipe resuspended bacterium solutions is incorporated to a pipe, obtain yeast
Competent cell.
(2) it converts
Plasmid is linearized with SacI, and 0.2 μ L of restriction enzyme SacI enzyme is added into 25 μ L pPIC9K-1-2, is added
5 μ L Loading Buffer, 60 DEG C of heat preservations are added in 30 DEG C of heat preservation 60min in 24.8 μ L of enzyme cutting buffering liquid and distilled water
10min obtains digested liquid.The pPIC9K-1-2 plasmid that pPIC9K-1-2 digested liquid is linearized by electrophoresis recovery purifying.
The 10 μ L of pPIC9K-1-2 plasmid of linearisation, salmon sperm dna (denaturation, 10mg/mL) 5 μ are added into centrifuge tube
L is added 50 μ L of competent yeast cells, flicks mixing；500 μ L PEG/LiAc are added and flick mixing.30 DEG C of water-bath 30min, often
10-15min flicks mixing；20 μ L DMSO are added, flick mixing；42 DEG C of water-baths 15min, every 5-10min flick mixing；
12000rpm is centrifuged 0.5min, abandons supernatant；It is resuspended with 1mL liquid PDA culture medium；30 DEG C, 200rpm culture 90min；12000r/
M is centrifuged 0.5min, abandons supernatant；Distilled water 1mL is added, 12000r/m is centrifuged 10min after resuspension, abandons supernatant, and distilled water is added
1mL obtains final re-suspension liquid.
9, screening and culturing medium design and screening
Screening and culturing medium: sodium cellulose glycolate 10g/L, gardenia blue 1.5g/L, NH4NO3 1.0g/L, NaCl 2g/L,
The general Jino Science and Technology Ltd. in the Beijing YGM003A yeast cells full-synthetic culture medium YGM003A-1() 1.5g/L, agar 20g/
L, 115 DEG C of 20 min of sterilizing, inverted plate.Final re-suspension liquid is coated on screening flat board, and 28 DEG C of culture 48h, periphery of bacterial colonies has light
Red is transformant.
Because in screening and culturing medium, without the carbon source that saccharomycete can be utilized directly, only the cellulose inscribe on plasmid
After enzyme and β-glucosyl enzym are expressed simultaneously, sodium cellulose glycolate could be converted to glucose, Yeast Growth is made to rise in value.Together
When β-glucosyl enzym gardenia blue is converted to gardenia red, to make periphery of bacterial colonies that light red be presented.
Control experiment: the saccharomycete dilution spread screening and culturing medium plate containing plasmid pPIC9K or without plasmid is carried out
Culture is grown without bacterium colony.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Hubei University Of Technology
<120>cellulose restriction endonuclease and the safe grade carrier of β-glucosyl enzym structuring food prods and screening and culturing medium are utilized
<130> 1
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 720
<212> DNA
<213> Aspergillus niger
<400> 1
atgaagttgc cagtttcttt ggctatgttg gctgctaccg ctatgggtca aaccatgtgt 60
tctcaatacg actctgcttc ttctccacca tactctgtta accaaaactt gtggggtgaa 120
taccaaggta ccggttctca atgtgtttac gttgacaagt tgtcttcttc tggtgcttct 180
tggcacaccg aatggacctg gtctggtggt gaaggtaccg ttaagtctta ctctaactct 240
ggtgttacct tcaacaagaa gttggtttct gacgtttctt ctatcccaac ctctgttgaa 300
tggaagcaag acaacaccaa cgttaacgct gacgttgctt acgacttgtt caccgctgct 360
aacgttgacc acgctacctc ttctggtgac tacgaattga tgatctggtt ggctagatac 420
ggtaacatcc aaccaatcgg taagcaaatc gctaccgcta ccgttggtgg taagtcttgg 480
gaagtttggt acggttctac cacccaagct ggtgctgaac aaagaaccta ctctttcgtt 540
tctgaatctc caatcaactc ttactctggt gacatcaacg ctttcttctc ttacttgacc 600
caaaaccaag gtttcccagc ttcttctcaa tacttgatca acttgcaatt cggtaccgaa 660
gctttcaccg gtggtccagc taccttcacc gttgacaact ggaccgcttc tgttaactag 720
<210> 2
<211> 2583
<212> DNA
<213> Aspergillus niger
<400> 2
atgagattca ccttgatcga agctgttgct ttgaccgctg tttctttggc ttctgctgac 60
gaattggctt actctccacc atactaccca tctccatggg ctaacggtca aggtgactgg 120
gctgaagctt accaaagagc tgttgacatc gtttctcaaa tgaccttggc tgaaaaggtt 180
aacttgacca ccggtaccgg ttgggaattg gaattgtgtg ttggtcaaac cggtggtgtt 240
ccaagattgg gtgttccagg tatgtgtgct caagactctc cattgggtgt tagagactct 300
gactacaact ctgctttccc agctggtgtt aacgttgctg ctacctggga caagaacttg 360
gcttacttga gaggtcaagc tatgggtcaa gacttctctg acaagggtgc tgacatccaa 420
ttgggtccag ctgctggtcc attgggtaga tctccagacg gtggtagaaa ctgggaaggt 480
ttctctccag acccagcttt gtctggtgtt ttgttcgctg aaaccatcaa gggtatccaa 540
gacgctggtg ttgttgctac cgctaagcac tacatcgctt acgaacaaga acacttcaga 600
caagctccag aagctcaagg ttacggtttc aacatcaccg aatctggttc tgctaacttg 660
gacgacaaga ccatgcacga attgtacttg tggccattcg ctgacgctat cagagctggt 720
gctggtgctg ttatgtgttc ttacaaccaa atcaacaact cttacggttg tcaaaactct 780
tacaccttga acaagttgtt gaaggctgaa ttgggtttcc aaggtttcgt tatgtctgac 840
tgggctgctc accacgctgg tgtttctggt gctttggctg gtttggacat gtctatgcca 900
ggtgacgttg actacgactc tggtacctct tactggggta ccaacttgac catctctgtt 960
ttgaacggta ccgttccaca atggagagtt gacgacatgg ctgttagaat catggctgct 1020
tactacaagg ttggtagaga cagattgtgg accccaccaa acttctcttc ttggaccaga 1080
gacgaatacg gtttcaagta ctactacgtt tctggtggtc catacgaaaa ggttaaccaa 1140
ttcgttaacg ttcaaagaaa ccactctgaa ttgatcagaa gaatcggtgc tgactctacc 1200
gttttgttga agaacgacgg tgctttgcca ttgaccggta aggaaagatt ggttgctttg 1260
atcggtgaag acgctggttc taacccatac ggtgctaacg gttgttctga cagaggttgt 1320
gacaacggta ccttggctat gggttggggt tctggtaccg ctaacttccc atacttggtt 1380
accccagaac aagctatctc taacgaagtt ttgaagaaca agaacggtgt tttcaccgct 1440
accgacaact gggctatcga ccaaatcgaa gctttggcta agaccgcttc tgtttctttg 1500
gttttcgtta acgctgactc tggtgaaggt tacatcaacg ttgacggtaa cttgggtgac 1560
agaagaaact tgaccttgtg gagaaacggt gacaacgtta tcaaggctgc tgcttctaac 1620
tgtaacaaca ccatcgttat catccactct gttggtccag ttttggttaa cgaatggtac 1680
gacaacccaa acgttaccgc tatcttgtgg ggtggtttgc caggtcaaga atctggtaac 1740
tctttggctg acgttttgta cggtagagtt aacccaggtg ctaagtctcc attcacctgg 1800
ggtaagacca gagaagctta ccaagactac ttgtacaccg aaccaaacaa cggtaacggt 1860
gctccacaag aagacttcgt tgaaggtgtt ttcatcgact acagaggttt cgacaagaga 1920
aacgaaaccc caatctacga cttcggttac ggtttgtctt acaccacctt caactactct 1980
aacttgcaag ttgaagtttt gtctgctcca gcttacgaac cagcttctgg tgaaaccgaa 2040
gctgctccaa ccttcggtga agttggtaac gcttctgact acttgtaccc agacggtttg 2100
caaagaatca ccaagttcat ctacccatgg ttgaactcta ccgacttgga agcttcttct 2160
ggtgacgctt cttacggtca agacgcttct gactacttgc cagaaggtgc taccgacggt 2220
tctgctcaac caatcttgcc agctggtggt ggtgctggtg gtaacccaag attgtacgac 2280
gaattgatca gagttaccgt taccatcaag aacaccggta aggttgctgg tgacaaggtt 2340
ccacaattgt acgtttcttt gggtggtcca aacgaaccaa agatcgtttt gagacaattc 2400
gaaagaatca ccttgcaacc atctgaagaa acccaatggt ctaccacctt gaccagaaga 2460
gacttggcta actggaacgt tgaaacccaa gactgggaaa tcacctctta cccaaagatg 2520
gttttcgttg gttcttcttc tagaaagttg ccattgagag cttctttgcc aaccgttcac 2580
taa 2583
<210> 3
<211> 1292
<212> DNA
<213> Artificial Sequence
<220>
<223>composition sequence 1
<400> 3
cctacgtagt tcgatcaaca acccgaatcc tatcgtaatg atgttttgcc cgatcagcct 60
caatcgacaa ttttacgcgt ttcgatcgaa gcagggatga caattggctg ggaacggtat 120
actggaataa atggtcttcg ttatggtatt gatgtttttg gtgcatcggc cccggcgaat 180
gatctatatg ctcatttcgg cttgaccgca gtcggcatca cgaacaaggt gttggccgcg 240
atcgccggta agtcggcacg ttaaaaaata gctatggaat atagtagcta ctaataagtt 300
aggagaataa actggatcca aacgatgaga tttccttcaa tttttactgc agttttattc 360
gcagcatcct ccgcattagc tgctccagtc aacactacaa cagaagatga aacggcacaa 420
attccggctg aagctgtcat cggttactca gatttagaag gggatttcga tgttgctgtt 480
ttgccatttt ccaacagcac aaataacggg ttattgttta taaatactac tattgccagc 540
attgctgcta aagaagaagg ggtaatgaag ttgccagttt ctttggctat gttggctgct 600
accgctatgg gtcaaaccat gtgttctcaa tacgactctg cttcttctcc accatactct 660
gttaaccaaa acttgtgggg tgaataccaa ggtaccggtt ctcaatgtgt ttacgttgac 720
aagttgtctt cttctggtgc ttcttggcac accgaatgga cctggtctgg tggtgaaggt 780
accgttaagt cttactctaa ctctggtgtt accttcaaca agaagttggt ttctgacgtt 840
tcttctatcc caacctctgt tgaatggaag caagacaaca ccaacgttaa cgctgacgtt 900
gcttacgact tgttcaccgc tgctaacgtt gaccacgcta cctcttctgg tgactacgaa 960
ttgatgatct ggttggctag atacggtaac atccaaccaa tcggtaagca aatcgctacc 1020
gctaccgttg gtggtaagtc ttgggaagtt tggtacggtt ctaccaccca agctggtgct 1080
gaacaaagaa cctactcttt cgtttctgaa tctccaatca actcttactc tggtgacatc 1140
aacgctttct tctcttactt gacccaaaac caaggtttcc cagcttcttc tcaatacttg 1200
atcaacttgc aattcggtac cgaagctttc accggtggtc cagctacctt caccgttgac 1260
aactggaccg cttctgttaa ctaggaattc gg 1292
<210> 4
<211> 3624
<212> DNA
<213> Artificial Sequence
<220>
<223>composition sequence 2
<400> 4
ggcctagggt tcgatcaaca acccgaatcc tatcgtaatg atgttttgcc cgatcagcct 60
caatcgacaa ttttacgcgt ttcgatcgaa gcagggatga caattggctg ggaacggtat 120
actggaataa atggtcttcg ttatggtatt gatgtttttg gtgcatcggc cccggcgaat 180
gatctatatg ctcatttcgg cttgaccgca gtcggcatca cgaacaaggt gttggccgcg 240
atcgccggta agtcggcacg ttaaaaaata gctatggaat atagtagcta ctaataagtt 300
aggagaataa actggatcca aacgatgaga tttccttcaa tttttactgc agttttattc 360
gcagcatcct ccgcattagc tgctccagtc aacactacaa cagaagatga aacggcacaa 420
attccggctg aagctgtcat cggttactca gatttagaag gggatttcga tgttgctgtt 480
ttgccatttt ccaacagcac aaataacggg ttattgttta taaatactac tattgccagc 540
attgctgcta aagaagaagg ggtaatgaga ttcaccttga tcgaagctgt tgctttgacc 600
gctgtttctt tggcttctgc tgacgaattg gcttactctc caccatacta cccatctcca 660
tgggctaacg gtcaaggtga ctgggctgaa gcttaccaaa gagctgttga catcgtttct 720
caaatgacct tggctgaaaa ggttaacttg accaccggta ccggttggga attggaattg 780
tgtgttggtc aaaccggtgg tgttccaaga ttgggtgttc caggtatgtg tgctcaagac 840
tctccattgg gtgttagaga ctctgactac aactctgctt tcccagctgg tgttaacgtt 900
gctgctacct gggacaagaa cttggcttac ttgagaggtc aagctatggg tcaagacttc 960
tctgacaagg gtgctgacat ccaattgggt ccagctgctg gtccattggg tagatctcca 1020
gacggtggta gaaactggga aggtttctct ccagacccag ctttgtctgg tgttttgttc 1080
gctgaaacca tcaagggtat ccaagacgct ggtgttgttg ctaccgctaa gcactacatc 1140
gcttacgaac aagaacactt cagacaagct ccagaagctc aaggttacgg tttcaacatc 1200
accgaatctg gttctgctaa cttggacgac aagaccatgc acgaattgta cttgtggcca 1260
ttcgctgacg ctatcagagc tggtgctggt gctgttatgt gttcttacaa ccaaatcaac 1320
aactcttacg gttgtcaaaa ctcttacacc ttgaacaagt tgttgaaggc tgaattgggt 1380
ttccaaggtt tcgttatgtc tgactgggct gctcaccacg ctggtgtttc tggtgctttg 1440
gctggtttgg acatgtctat gccaggtgac gttgactacg actctggtac ctcttactgg 1500
ggtaccaact tgaccatctc tgttttgaac ggtaccgttc cacaatggag agttgacgac 1560
atggctgtta gaatcatggc tgcttactac aaggttggta gagacagatt gtggacccca 1620
ccaaacttct cttcttggac cagagacgaa tacggtttca agtactacta cgtttctggt 1680
ggtccatacg aaaaggttaa ccaattcgtt aacgttcaaa gaaaccactc tgaattgatc 1740
agaagaatcg gtgctgactc taccgttttg ttgaagaacg acggtgcttt gccattgacc 1800
ggtaaggaaa gattggttgc tttgatcggt gaagacgctg gttctaaccc atacggtgct 1860
aacggttgtt ctgacagagg ttgtgacaac ggtaccttgg ctatgggttg gggttctggt 1920
accgctaact tcccatactt ggttacccca gaacaagcta tctctaacga agttttgaag 1980
aacaagaacg gtgttttcac cgctaccgac aactgggcta tcgaccaaat cgaagctttg 2040
gctaagaccg cttctgtttc tttggttttc gttaacgctg actctggtga aggttacatc 2100
aacgttgacg gtaacttggg tgacagaaga aacttgacct tgtggagaaa cggtgacaac 2160
gttatcaagg ctgctgcttc taactgtaac aacaccatcg ttatcatcca ctctgttggt 2220
ccagttttgg ttaacgaatg gtacgacaac ccaaacgtta ccgctatctt gtggggtggt 2280
ttgccaggtc aagaatctgg taactctttg gctgacgttt tgtacggtag agttaaccca 2340
ggtgctaagt ctccattcac ctggggtaag accagagaag cttaccaaga ctacttgtac 2400
accgaaccaa acaacggtaa cggtgctcca caagaagact tcgttgaagg tgttttcatc 2460
gactacagag gtttcgacaa gagaaacgaa accccaatct acgacttcgg ttacggtttg 2520
tcttacacca ccttcaacta ctctaacttg caagttgaag ttttgtctgc tccagcttac 2580
gaaccagctt ctggtgaaac cgaagctgct ccaaccttcg gtgaagttgg taacgcttct 2640
gactacttgt acccagacgg tttgcaaaga atcaccaagt tcatctaccc atggttgaac 2700
tctaccgact tggaagcttc ttctggtgac gcttcttacg gtcaagacgc ttctgactac 2760
ttgccagaag gtgctaccga cggttctgct caaccaatct tgccagctgg tggtggtgct 2820
ggtggtaacc caagattgta cgacgaattg atcagagtta ccgttaccat caagaacacc 2880
ggtaaggttg ctggtgacaa ggttccacaa ttgtacgttt ctttgggtgg tccaaacgaa 2940
ccaaagatcg ttttgagaca attcgaaaga atcaccttgc aaccatctga agaaacccaa 3000
tggtctacca ccttgaccag aagagacttg gctaactgga acgttgaaac ccaagactgg 3060
gaaatcacct cttacccaaa gatggttttc gttggttctt cttctagaaa gttgccattg 3120
agagcttctt tgccaaccgt tcactaaagc tttggacttc ttcgccagag gtttggtcaa 3180
gtctccaatc aaggttgtcg gcttgtctac cttgccagaa atttacgaaa agatggaaaa 3240
gggtcaaatc gttggtagat acgttgttga cacttctaaa taagcgaatt tcttatgatt 3300
tatgattttt attattaaat aagttataaa aaaaataagt gtatacaaat tttaaagtga 3360
ctcttaggtt ttaaaacgaa aattcttatt cttgagtaac tctttcctgt aggtcaggtt 3420
gctttctcag gtatagcatg aggtcgctct tattgaccac acctctaccg gcatgccgag 3480
caaatgcctg caaatcgctc cccatttcac ccaattgtag atatgctaac tccagcaatg 3540
agttgatgaa tctcggtgtg tattttatgt cctcagagga caacacctgt tgtaatcgtt 3600
cttccacacg gatcgcggcc gcgg 3624
Claims (4)
1. cellulose inscribe enzyme coding gene and the combination of β-glucosyl enzym encoding gene are in structuring food prods safety level yeast vector
Application, it is characterised in that: the construction method of the aliment security level yeast vector includes the following steps: SEQ ID
Sequence shown in NO.3, sequence shown in SEQ ID NO.4 are building up to pPIC9K carrier by restriction enzyme and DNA ligase
On；Wherein restriction enzyme is SnaBI, EcoR I and AvrII, NotI.
2. the construction method of aliment security level yeast vector described in claim 1, characterized by the following steps:
Sequence shown in sequence shown in SEQ ID NO.3, SEQ ID NO.4 is building up to by restriction enzyme and DNA ligase
On pPIC9K carrier；Wherein restriction enzyme is SnaBI, EcoR I and AvrII, NotI.
3. cellulose inscribe enzyme coding gene according to claim 1 and the combination of β-glucosyl enzym encoding gene are eaten in building
Application in product safety level yeast vector, it is characterised in that: the screening and culturing medium of the aliment security level yeast vector includes
Sodium cellulose glycolate and gardenia blue.
4. cellulose inscribe enzyme coding gene according to claim 3 and the combination of β-glucosyl enzym encoding gene are eaten in building
Application in product safety level yeast vector, it is characterised in that: the formula of the screening and culturing medium are as follows: sodium cellulose glycolate
10g/L, gardenia blue 1.5g/L, NH4NO3 1.0g/L, NaCl 2g/L, YGM003A yeast cells full-synthetic culture medium YGM003A-
1 1.5g/L, agar 20g/L.
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|---|---|---|---|
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| Title |
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| 以淀粉酶基因作为筛选标记的E.coli克隆载体的构建;刘国奇等;《南开大学学报（自然科学）》;19980320;64-71 * |
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