CN1309183A - Process for preparing bio-degradable plastics by construction of genetically engineered cytoalgae - Google Patents
Process for preparing bio-degradable plastics by construction of genetically engineered cytoalgae Download PDFInfo
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Abstract
A process for construction of genetically engineered cytoalgae to prepare biodegradable plastics includes such steps as polymerase chain reaction to obtain the agp gene of wild cytoalgae, molecular cloning to remove part of it for obtaining recombined plasmid, and transforming the wild cytoalgae to obtain said genetically engineered cytoalgae, which has no power to synthesize glucogen and can be used to prepare biodegradable plastics PHB.
Description
The present invention relates to a kind of method of producing bio-degradable plastics, belong to biotechnology (particularly biochemical industry) field with the genetically engineered cytoalgae that makes up.
Along with the continuous development of human society, plastics have become our requisite industry and daily necessities, and the waste of the conventional plastic of being made by petroleum products is difficult to degraded.Because the usage quantity of a large amount of waste or used plastics mulch films, packing plastics bag and disposable non-degradable plastic tableware is increased sharply, and abandon arbitrarily in recent years, each big or middle city has generally formed serious white pollution.Its same vehicle exhaust, phosphorus-containing detergent are listed in the emphasis that Environmental Protection in China is administered together.Therefore, people invest development, the exploitation of environmentally friendly plastics-biodegradable plastic to sight and produce.
Poly-beta-hydroxy-butyrate (Poly-β-Hydroxybutyrate is hereinafter to be referred as PHB) is a kind of Biodegradable polymer material of finding the earliest.(as limit nitrogen or limit phosphorus) will accumulate PHB in a lot of microbies when the environmental nutrient condition is uneven, and its physicochemical property are similar to acrylic plastering, and can environment do not polluted by the enzyme liberating of microorganism secretion under field conditions (factors).PHB not only has the mechanical property and the processing characteristics of common macromolecular material, also have the more unexistent properties of chemosynthesis plastics, as biodegradable, biocompatibility, optical activity, piezoelectricity and anticoagulant property etc., in addition, its ratio is great, oxygen-permeable is low, anti-ultraviolet radiation, thereby has uniqueness in fields such as medicine, agricultural, electronics, wrapping material and application prospects is subjected to the attention of international biotechnology circle deeply.
Nineteen ninety, Britain's ICI Imperial Chemical Industries adopts heterotroph bacterium alcaligenes eutrophus, is raw material with glucose and propionic acid, at limit phosphorus condition bottom fermentation, has released beta-hydroxy-butanoic acid and the beta-hydroxy valeric acid copolymer plastics of commodity Biopol by name.Afterwards, people study the colibacillary PHB production of recombinating again, and up to the present, its expression amount generally can reach about 70% of dry cell weight.But will continue the reasons such as separation and purification difficulty of logical oxygen, fermented liquid owing to ferment in raw materials used (glucose, sucrose, propyl alcohol etc.) price height, the fermenting process, make production cost too high, competition capability is low than conventional plastic.For this reason, reduce the production cost of PHB significantly, become the key issue that PHB produced and introduced to the market the essential solution of institute.
Consider CO from raw materials cost
2It is the most cheap carbon source.The used organic carbon source of heterotrophic microorganism fermentative production PHB all is with the CO in the atmosphere basically by photosynthesis
2Be fixed in the green plants, synthetic and come through a series of biochemical reaction.If the CO that green plants photosynthesis is fixed up
2Being directly used in the synthetic of PHB, will be a kind of desirable approach that fundamentally solves the too high problem of raw materials cost.Utilize transgenic technology that bacterium PHB synthase gene is transferred to Arabidopis thaliana (biotechnology (Biotechnology) 13:142-150; American National scientific research progress (Proceedingsof the National Academic Science of USA) 91:12760-12764; International biomacromolecule magazine (International Journal of Biological Macromolecules) 17:7-12), rape, potato (Botany Gazette 37:581-588; Botany Gazette 40:615-621) and cotton (American National scientific research progress (Proceedingsof the National Academic Science of USA) 93:12768-12773; Thermochemistry journal (ThermochimActa) 313:43-53) etc. in the plant materials, just can in plant, set up the PHB route of synthesis.Though from reducing raw materials cost, transgenic plant produce PHB and have very big advantage, but since the plant-growth cycle long, need take arable land, PHB expression amount low (at present the highest level of report only reach dry cell weight 14%), product separation extracts difficulty, has hindered the conversion of this technology to actual production greatly.
Cyanobacteria claims blue-green algae again, is a kind of photosynthetic oxygen evolution autotrophic microorganism.Cyanobacteria is the same with bacterium, all is prokaryotic cell prokaryocyte, and its karyomit(e) is exposed to be present in the tenuigenin.Identical with eucaryon algae, higher plant, cyanobacteria can pass through photosynthesis, utilizes energy of the sun from CO2, water, synthetic its whole cellular materials of inorganic salt.Cell growth and divide other required all molecules, all be by these photosynthetic head product synthetic (cyanobacteria molecular biology (The Biology ofCyanobacteria), 1994, pp217-257).Cytoalgae 6803 (Synechocystis sp.PCC6803) is a kind of unicellular spherical cyanobacteria, it has the following advantages: simple in structure, can under multiple condition, grow, but both can carry out also heterotrophic growth (using phycology magazine (Journal of Applied Physiology) 8:263-273) of photoautotrophy; Whole examining orders of chromosomal DNA are finished, and have predicted possible protein coding zone.S.6803 specific gene can be deleted, modified to study its function, effect, (DNA studies (DNA Research) 3:109-136 also can to change its pathways metabolism at a certain specific site introducing foreign gene; DNA studies (DNA Research) 3:185-209); Easily transform, be suitable for genetic manipulation (Enzymology method (Methods in Enzymology) 297:18-26).Under normal culture condition, do not need pair cell to do any processing, external source wire or near-ring shape plasmid DNA all are incorporated on the chromosomal DNA of cytoalgae 6803 by homologous recombination easily.And, mark with various antibiotic resistant genes, cytoalgae 6803 is transformed and screen all very convenient.
Be accompanied by the physiological research of cyanobacteria, people are some cyanobacterias, as the green glue cyanobacteria of Fo Shi (biological chemistry Acta Biophysica Sinica (Biochima Biophysics Acta) 120:308-310), spirulina (bacteriology magazine (Journalof Bacteriology) 149:361-363; Bacteriology magazine (Journal of Bacteriology) 172:2791-2792), synechococcus (fermentation and biotechnology magazine (Journal of Fermentation and Bioengineering) 82:512-514; Bacteriology magazine (Journal of Bacteriology) 179:5009-5013), in cytoalgae (microbiology collection (Archives of Microbiology) 170:162-170) etc., found with bacterial body in similar PHB particle, make cyanobacteria become possibility as the expression body of PHB.Although these cyanobacterias have the ability of synthetic PHB, PHB is not high at its intravital accumulating level.Up to now, the high expression level amount of bibliographical information is in heat-resisting synechococcus, and PHB can build up to 20% of dry cell weight, and is more much lower than the accumulation volume of bacterium, but is higher than the expression amount in the transgenic plant.1996, the Japan scholar adopts gene engineering method for the first time, transform synechococcus 7942 with the plasmid that contains alcaligenes eutrophus PHB synthase gene, realized the expression of PHB in the cyanobacteria of no PHB synthesis capability, but the content of PHB is very low in the transformant, only reaches 1% (biotechnology communication (Biotechnology Letters) 18:1047-1050) of dry weight.China yet there are no report about the research of this respect.
Cyanobacteria accumulation PHB be studies show that PHB function as storage material in the cyanobacteria body is degenerated greatly.In the cyanobacteria body, carbon source and energy storage material mainly are glycogen (microbiology annual report (Annual Reviews ofMicrobiology) 38:1-25).Under the photoautotrophy condition, the cell of exponential phase of growth can accumulate the 10-20% that glycogen reaches dry weight, and under the nitrogen hunger condition, glycogen can build up to the 40-60% of dry cell weight, the also promptly superfluous carbon source overwhelming majority at first changes glycogen into and stores, and only having seldom, a part changes PHB into.After the balanced growth condition was recovered, glycogen decomposed immediately, discharged carbon source and energy.This shows that in the cyanobacteria body, glycogen has replaced the physiological function of PHB, synthesizing on substrate of glycogen competed with the synthetic formation of PHB.Want to improve the expression amount of PHB in the cyanobacteria body, the synthetic of material glycogen competed in essential control.
In cyanobacteria, glycogen synthetic relates to three enzymes: adenosine diphosphate (ADP) (being called for short ADP)-glucose tetra-sodium carboxylase, glycogen synthetase and q enzyme, ADP-glucose tetra-sodium carboxylase agp genes encoding wherein, catalysis ADP and free glucose molecule are combined into the reaction of ADP-glucose, and ADP-glucose is specificity substrate (biological chemistry biophysics collection (the Archives of Biochemistry and Biophysics) 372:179-188 of cyanobacteria glycogen synthetase; Cyanobacteria biology (The Biology of Cyanobacteria), 1982, pp47-85).Therefore, cut off the supply of ADP-glucose, it is synthetic to suppress glycogen, thereby guides the carbon source bottoms stream into the PHB route of synthesis, improves the expression amount of cyanobacteria PHB.
The objective of the invention is to propose a kind of method that the genetically engineered cytoalgae is produced poly-beta-hydroxy-butyrate that makes up, utilize engineered method to transform the cyanobacteria cytoalgae, to form the glycogen route of synthesis removal of competing reaction with the PHB route of synthesis, and be expression vector with this mutant strain, utilize cheap carbon source CO
2Directly synthetic PHB in its body realizes the high level accumulation of cyanobacteria PHB, thereby reduces the production cost of PHB greatly.
The structure genetically engineered cytoalgae that the present invention proposes is produced the method for bio-degradable plastics, is made up of following steps:
1, at first extract chromosomal DNA from the wild-type cytoalgae: used wild-type cytoalgae 6803 is buied by the upright university of State of Arizona, US.Its process is: picking 5-10 single algae falls from the solid plate, be inoculated in the culturing bottle that 200-400 milliliters of liquid substratum is housed, this substratum is designated as the BG-11 substratum, consisting of of every liter of substratum: yellow soda ash: 20g, dipotassium hydrogen phosphate: 30.5g, ferric ammonium citrate: 6g, SODIUMNITRATE: 1.5g, sal epsom: 75mg, contain 0.25mol/L ethylenediamine-N,N'-diacetic acid(EDDA) disodium, the pH value is 8.0 the aqueous solution 11.2 μ L, calcium chloride: 36mg, citric acid: 6mg, contain 2.86g/L boric acid, 0.222g/L Zinc Sulphate Heptahydrate, 0.079g/L cupric sulfate pentahydrate, 1.81g/L four aqueous magnesium chlorides, 0.39g/L Sodium Molybdate Dihydrate, 0.049g/L the liquid microelement 1mL of cobalt nitrate hexahydrate is 24-32 ℃ in temperature, illumination density is that aerated culture to the light absorption value of nutrient solution under 730 nanometers is 0.3-1.0 in the illumination box of 1000-3000lx; Nutrient solution is transferred in the 200-300 milliliter Centrifuge Cup, under the condition of 15-25 ℃, 3000-5000 rev/min centrifugal 5-15 minute, according to conventional cyanobacteria DNA extraction method the gained frustule is handled, obtain the chromosomal DNA precipitation of wild-type cytoalgae, add in the damping fluid that contains 1.21g/L Tutofusin tris and 0.372g/L two ethylenediamine hydrate oxalic acid disodiums, pH8.0 of 0.2-0.6mL, dissolution precipitation, standby.
2, being template with wild-type cytoalgae chromosomal DNA, is primer by the upstream and the downstream nucleotide order synthetic oligonucleotide of cytoalgae agp gene.Contain deoxyadenosine triphosphoric acid (being designated as A), deoxyguanosine triphosphoric acid (being designated as G), deoxy cytidine triphosphate (being designated as C), deoxythymidine triphosphoric acid (being designated as T) in the primer, institute's synthetic primer one is: 5 '-phosphoric acid-C-A-G-C-T-C-C-G-G-A-T-C-C-C-A-A-C-G-G-C-G-phosphoric acid-3 '; Primer two is: 5 '-phosphoric acid-G-G-C-C-G-A-G-C-T-C-A-G-G-G-A-T-T-T-T-A-C-T-G-C-T-T-T-phosphoric acid-3 ', two kinds of primers are synthetic by Beijing ancient cooking vessel state biotech development center (hereinafter to be referred as ancient cooking vessel state company).After primer is synthetic, be 50-100 μ mol/L with sterilized water dissolution precipitation to concentration, standby.
3, utilization polymerase chain reaction technology amplifying target genes agp fragment from the chromosomal DNA, used polysaccharase and corresponding amplification buffer, four kinds of deoxynucleoside acid solutions (hereinafter to be referred as dNTPs) are buied by ancient cooking vessel state company.The chromosomal DNA solution of getting the preparation of 3-10 μ L step 1 is in a sterilized centrifuge tube, add the amplification buffer of 20-40 μ L sterilized water, 5-15 μ L, four kinds of deoxynucleotides of 5-15 μ L, the Taq archaeal dna polymerase of 0.5-1.5 μ L successively by step 2 synthetic primer two, 0.5-1 μ L by step 2 synthetic primer one, 0.5-1.5 μ L, complementing to cumulative volume with sterilized water is 100-150 μ L, adds the paraffin oil of 60-120 μ L again; Centrifuge tube is placed temperature varying system, be warming up to 92-96 ℃, kept 10-15 minute, be cooled to 50-60 ℃ then, kept 1-5 minute, be warming up to 70-76 ℃ again, kept 1-5 minute, then by be warming up to 92-96 ℃ and keep 1-2 minute, be cooled to 50-60 ℃ and keep 1-5 minute, be warming up to 70-76 ℃ and keep 1-5 minute variation sequential loop 20-30 time, be warming up to 92-96 ℃ and kept 1-2 minute at last, be cooled to 50-60 ℃ and kept 1-5 minute, be warming up to 70-76 ℃ and kept 10-20 minute, finish amplified reaction.
4, adopt restriction endonuclease that agp dna fragmentation and the plasmid pUC118 that the amplification of the 3rd step obtains cut, used restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company.Get 5-10 μ l by the agp dna fragmentation that the amplification of the 3rd step obtains, add 0.5-1.5 μ lBamH I restriction endonuclease, 0.5-1.5 μ lSac I restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 3-5 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃; Get 2-4 μ l plasmid pUC118 simultaneously, add 0.5-1.5 μ l BamH I restriction endonuclease, 0.5-1.5 μ l Sac I restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 6-11 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃.
5, the agpDNA fragment is connected into the multiple clone site of plasmid pUC118 carrier under the ligase enzyme effect, used T4 dna ligase and corresponding damping fluid are buied by ancient cooking vessel state company.Get 2-8 μ l by the agp dna fragmentation of step 4 gained, 5-9 μ l plasmid pUC118 fragment, mix, add 1-2 μ l ligation damping fluid by step 4 gained, 1 μ l T4 dna ligase, 2-6 ℃ of reaction 8-12 hour down, standby.
6, adopt molecular cloning operation preparation colibacillary competent cell (Calcium Chloride Method), used intestinal bacteria DH10B is protected by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber and plants.Picking list bacterium colony from the solid plate of thalline, be inoculated in the 3-10mL liquid nutrient medium, this substratum is designated as the LB substratum, contain 5g yeast extract powder in every liter of substratum, the 10g peptone, 10g sodium-chlor, the pH value is 7.0, at under 25-38 ℃ on 150-300 rev/min shaking table culturing bacterium 6-15 hour, then getting the 0.1-1.0mL nutrient solution is inoculated in the 50-100mL liquid nutrient medium, is 0.3-1.0 be cultured to nutrient solution under 25-38 ℃ on 150-300 rev/min shaking table at the light absorption value of 600 nanometers, and the Lime Chloride according to routine is equipped with competent cell then:
7, the gene clone of agp dna fragmentation, agents useful for same isopropyl-(being called for short IPTG) and 5-bromo-4-chloro-3-indoles-β-D-galactoside (being called for short X-gal) are buied by ancient cooking vessel state company.Get 150-250 μ L competence intestinal bacteria DH10B cell, add the ligation thing of 5-10 μ L by step 5 gained, carry out transformation experiment according to ordinary method, on the LB solid medium that contains 40 μ g/mL IPTG, 40 μ g/mL X-gal and 15g/L agar powder in 37 ℃ of following culturing cells, the picking white colony prepares plasmid DNA according to ordinary method after 16-24 hour;
8, adopt restriction endonuclease respectively plasmid DNA and the plasmid pRL425 that step 7 obtains to be cut, plasmid pRL425 is provided by U.S. Rick professor Debus, and restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company.Get 2-4 μ l by the 7th step gained plasmid DNA, add 0.5-1.5 μ lKpn I restriction endonuclease, 0.5-1.5 μ lSma I restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 3-5 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃, adopt conventional low melting-point agarose gel electrophoresis to reclaim the long dna fragmentation of 4341bp that is; Get 2-4 μ l plasmid pRL425 simultaneously, add 0.5-1.5 μ lKpn I restriction endonuclease, 0.5-1.5 μ lEcoRV restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 6-11 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃, adopt conventional low melting-point agarose gel electrophoresis to reclaim the long dna fragmentation of 1539bp that is;
9, get 4-9 μ l two kinds of dna fragmentations in above-mentioned the 8th step respectively, add 1-2 μ l ligation damping fluid, 1 μ l T4 dna ligase, reacted 8-12 hour down at 2-6 ℃, get 5-10 μ L, add 150-250 μ L by step 6 gained competence intestinal bacteria DH10B cell, carry out transformation experiment according to ordinary method, coat on the LB solid medium that contains 750 μ g/mL erythromycin and 15g/L agar powder, cultivated 16-24 hour down at 37 ℃, picking list bacterium colony prepares plasmid DNA according to ordinary method, and is standby;
10, the screening of cyanobacteria transformation and sudden change engineering strain: contain in the BG-11 liquid nutrient medium of 0.5-1.5g/L glucose at 200-400mL, inoculating 5-10 cytoalgae list algae and fall, is that 24-32 ℃, illumination density are that aerated culture to nutrient solution is 0.3-1.0 at the light absorption value of 730 nanometers in the illumination box of 1000-3000lx in temperature; Get the 10-20mL nutrient solution in 50mL has sterilized centrifuge tube, 15-25 ℃, 3000-5000 rev/min centrifugal 10-20 minute are abandoned supernatant; In precipitation, add in the 2-8mL BG-11 liquid nutrient medium, make solution equal 2.5 at the light absorption value of 730 nanometers; Get the above-mentioned solution of 0.2-0.6mL in the glass culture tube of sterilizing, add 2-8 μ L by step 9 gained plasmid DNA, vibration; It is that 24-32 ℃, illumination density are that the illumination box of 1000-3000lx was cultivated 5-10 hour that test tube is placed temperature, and per vibration half an hour once; Getting 100-300 μ L liquid coats on the BG-11 solid plate that contains glucose; After 20-30 hour filter paper is transferred on the BG-11 solid plate that contains 10-100 μ g/mL erythromycin, continues cultivation and dropped out existing to single algae in 10-15 days;
11, single algae of picking 5-10 step 10 acquisition falls to being inoculated in respectively 200-400mL BG-11 liquid nutrient medium, the BG-11 liquid nutrient medium that contains 0.5-1.0g/L glucose, contain the BG-11 liquid nutrient medium of 5-20mM sodium acetate or contain 0.5-1.0g/L glucose simultaneously and the BG-11 liquid nutrient medium of 5-20mM sodium acetate in, in temperature is 25-32 ℃, illumination density is to cultivate in the illumination box of 1500-2500lx, sampling in per 24 hours, measure cell density, cultivate after 6-8 days, with the rotating speed of whizzer with 4000-8000 rev/min, centrifugal 10-20 minute, abandon supernatant liquor, results gained cell, in 60 ℃ of oven drying precipitations, can obtain product bio-degradable plastics poly-beta-hydroxy-butyrate of the present invention.
Utilize the constructed cytoalgae mutant strain of the present invention not have the ability of glycogen biosynthesis, under the condition that different additional carbons exist, the PHB level of cylinder accumulation can reach wild-type cell 1.3-10 times in the following PHB level that accumulates of same culture conditions, mutant strain can also efficiently utilize and add glucose, under the same terms, the biomass of being gathered in the crops can reach 1.4-1.6 times of wild-type cell biomass.The present invention utilizes engineered method, replace the synthetic glycogen synthetase gene that forms competing reaction by part with purpose product P HB, thereby deletion glycogen route of synthesis has produced really and has suppressed the unusual effect that glycogen synthesizes, the carbon source bottoms stream guided into PHB route of synthesis, raising PHB expression amount and the growth of promotion cytoalgae.The PHB level that the constructed cytoalgae mutant strain of the present invention accumulates under certain culture condition, be the maximum in the similar research, will provide the reference of reality for reducing the PHB production cost, also for utilizing the friendly type material of cleaning raw material and clean energy production environment that possibility is provided.
Introduce embodiments of the invention below:
Embodiment 1:
(1) at first from the wild-type cytoalgae, extracts chromosomal DNA.5 single algaes of picking fall from the solid plate, are inoculated in the culturing bottle that 400 milliliters of liquid substratum are housed.This substratum is designated as the BG-11 substratum, the consisting of of every liter of substratum: yellow soda ash: 20g; Dipotassium hydrogen phosphate: 30.5g; Ferric ammonium citrate: 6g; SODIUMNITRATE: 1.5g; Sal epsom: 75mg; Contain 0.25mol/L ethylenediamine-N,N'-diacetic acid(EDDA) disodium, pH and be 8.0 the aqueous solution: 11.2 μ L; Calcium chloride: 36mg; Citric acid: 6mg; The liquid microelement that contains 2.86g/L boric acid, 0.222g/L Zinc Sulphate Heptahydrate, 0.079g/L cupric sulfate pentahydrate, 1.81g/L four aqueous magnesium chlorides, 0.39g/L Sodium Molybdate Dihydrate, 0.049g/L cobalt nitrate hexahydrate: 1mL.In temperature is that 30 ℃, illumination density are that aerated culture to the light absorption value of nutrient solution under 730 nanometers is 1.0 in the illumination box of 2000lx.Nutrient solution is transferred in 250 milliliters of Centrifuge Cups, under 25 ℃, 4000 rev/mins the condition of son centrifugal 10 minutes, obtains the wet algae precipitation of 1 gram.Adding 40mL in gained precipitation, to contain 10% sucrose, 6.05g/L Tutofusin tris, 3.72g/L two ethylenediamine hydrate oxalic acid, pH value be 8.0 damping fluid, 25 ℃ of placements 1 hour down; Under 25 ℃, 6000 rev/mins condition centrifugal 10 minutes, abandon supernatant; Adding 8mL in precipitation, to contain 6.05g/L Tutofusin tris, 2.93g/L sodium-chlor, 18.6g/L two ethylenediamine hydrate oxalic acid, pH value be 8.0 damping fluid, be transferred in 50 milliliters of centrifuge tubes, adding 1.5mL concentration is the N,O-Diacetylmuramidase aqueous solution of 50mg/mL, mixing, placed 20 minutes for 37 ℃, add 1mL concentration again and be 10% the N-sarcosyl aqueous solution, mixing was placed 20 minutes for 37 ℃; Add the phenol solution that 10.5mL crosses with the damping fluid balance that contains 1.21g/L Tutofusin tris and 0.372g/L two ethylenediamine hydrate oxalic acid disodiums, pH8.0, mixing 1 hour; 4 ℃, 6000 rev/mins centrifugal 10 minutes, draw the 10mL supernatant liquor to another centrifuge tube; In this supernatant, add 10mL chloroform (primary isoamyl alcohol that contains 1/25th volume), mixing 0.5 hour; 4 ℃, 6000 rev/mins centrifugal 10 minutes, draw the 10mL supernatant liquor to another centrifuge tube, the concentration that adds the 1mL volume again is that 3.0mol/L, pH value are 5.0 sodium acetate solution and 20mL dehydrated alcohol ,-20 ℃ of placements 8-12 hour down; 4 ℃, 12000 rev/mins centrifugal 10 minutes, abandon supernatant, in precipitation, add 1mL 70% ethanol, 4 ℃, 12000 rev/mins centrifugal 10 minutes, remove supernatant; The DNA precipitation is put the Bechtop drying.In drying precipitated, add 0.4mL and contain the damping fluid of 1.21g/L Tutofusin tris and 0.372g/L two ethylenediamine hydrate oxalic acid disodiums, pH8.0, dissolution precipitation.Standby.
(2) being template with wild-type cytoalgae chromosomal DNA, is primer by the upstream and the downstream nucleotide order synthetic oligonucleotide of cytoalgae agp gene.Four kinds of involved deoxynucleoside triphosphates are respectively: deoxyadenosine triphosphoric acid (being designated as A), deoxyguanosine triphosphoric acid (being designated as G), deoxy cytidine triphosphate (being designated as C), deoxythymidine triphosphoric acid (being designated as T), and institute's synthetic primer one is: 5 '-phosphoric acid-C-A-G-C-T-C-C-G-G-A-T-C-C-C-A-A-C-G-G-C-G-phosphoric acid-3 '; Primer two is: 5 '-phosphoric acid-G-G-C-C-G-A-G-C-T-C-A-G-G-G-A-T-T-T-T-A-C-T-G-C-T-T-T-phosphoric acid-3 '.Two kinds of primers are synthetic by Beijing ancient cooking vessel state biotech development center (hereinafter to be referred as ancient cooking vessel state company).After primer is synthetic, be 100 μ mol/L with sterilized water dissolution precipitation to concentration, standby.
(3) utilization polymerase chain reaction technology amplifying target genes agp fragment from the chromosomal DNA, used polysaccharase and corresponding amplification buffer, four kinds of deoxynucleoside acid solutions (hereinafter to be referred as dNTPs) are buied by ancient cooking vessel state company.Get 5 μ L by the chromosomal DNA solution of step 1 preparation in a sterilized centrifuge tube, add 30 μ L sterilized waters, 10 μ L amplification buffers, 10 μ LdNTPs, 1 μ L successively by step 2 synthetic primer one, 1 μ L Taq archaeal dna polymerase by step 2 synthetic primer two, 0.5 μ L, add 42.5 μ L sterilized waters again, add the paraffin oil of 60 μ L at last; Centrifuge tube is placed temperature varying system, be warming up to 92 ℃, kept 10 minutes, be cooled to 52 ℃ then, kept 2 minutes, be warming up to 71 ℃ again, kept 3 minutes, then by be warming up to 92 ℃ and keep 1 minute, be cooled to 52 ℃ and keep 2 minutes, be warming up to 71 ℃ and keep 3 minutes variation sequential loop 25 times, be warming up to 92 ℃ and kept 1 minute at last, be cooled to 52 ℃ and kept 2 minutes, be warming up to 71 ℃ and kept 10 minutes, finish amplified reaction.
(4) adopt restriction endonuclease that agp dna fragmentation and the plasmid pUC118 that the amplification of the 3rd step obtains cut, used restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company, and plasmid pUC118 is provided by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber.The consisting of of reaction solution when enzyme is cut the agp dna fragmentation:
Agp dna fragmentation 10 μ l
BamHⅠ(10U/μl) 1μl
SacⅠ(10U/μl) 1μl
Beffer?E(10×) 2μl
Sterilized water 6 μ l
Cumulative volume 20 μ l
Reacted 3 hours down at 37 ℃; Get 2 μ l plasmid pUC118 simultaneously, carry out enzyme and cut, reaction solution consists of:
Puc 118 2 μ l
BamHⅠ(10U/μl) 1μl
SacⅠ(10U/μl) 1μl
Buffer?E(10×) 2μl
Sterilized water 14 μ l
Cumulative volume 20 μ l
Reacted 3 hours down at 37 ℃.
(5) the agp dna fragmentation is connected into the multiple clone site of plasmid pUC118 carrier under the ligase enzyme effect, used T4 phage DNA ligase enzyme and corresponding damping fluid are buied by ancient cooking vessel state company.Ligation application of sample ratio is:
Plasmid pUC118 enzyme is cut liquid 9 μ l
Agp dna fragmentation enzyme is cut liquid 8.5 μ l
10 * connection damping fluid, 2 μ l
T4 phage DNA ligase enzyme 0.5 μ l
Cumulative volume 20 μ l
Reacted 12 hours down at 2 ℃.Standby.
(6) adopt molecular cloning operation preparation colibacillary competent cell (Calcium Chloride Method), used intestinal bacteria DH10B is protected by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber and plants.Picking list bacterium colony from the solid plate of thalline is inoculated in and is equipped with in the 5mL liquid nutrient medium.This substratum is designated as the LB substratum, is made up of 5g/L yeast extract powder, 10g/L peptone, 10g/L sodium-chlor, and the pH value is 7.0.Under 37 ℃ on 200 rev/mins shaking table culturing bacterium 10 hours, then get the 0.5mL nutrient solution and be inoculated in the 50mL liquid nutrient medium, be 0.375 on 200 rev/mins shaking table, being cultured to nutrient solution under 37 ℃ at the light absorption value of 600 nanometers; Nutrient solution is changed in the centrifuge tube of sterilization of precooling, the ice bath cooling is centrifugal 15 minutes with 5000 rev/mins on 4 ℃ whizzer after 10 minutes; Pour out supernatant liquor, under aseptic condition, will manage and be inverted 1 minute so that residual supernatant liquor flows to end; In precipitation, add 10 milliliters of ice-cold 0.1mmol/L CaCl
2Solution, ice bath was placed 15 minutes; On 4 ℃ whizzer with 5000 rev/mins centrifugal 15 minutes, abandon supernatant; Add 2 milliliters of 0.1mol/LCaCl with the ice precooling
2The resuspended once more precipitation of solution is placed on ice and can be obtained competent cell a moment.
(7) gene clone of agp dna fragmentation, agents useful for same isopropyl-(being called for short IPTG) and 5-bromo-4-chloro-3-indoles-β-D-galactoside (being called for short X-gal) are buied by ancient cooking vessel state company.Get the competence intestinal bacteria DH10B cell of 200 μ L steps (6) preparation, add 5 μ L step (5) gained ligation things, placed 30 minutes on ice, immediately pipe is transferred in 42 ℃ the circulator bath, placed 90 seconds, immediately pipe is transferred in the ice bath, placed 2 minutes, every pipe adds the LB liquid nutrient medium of 0.8mL, places 45 minutes under the room temperature with 5000 rev/mins speed centrifugal 2 minutes down at 37 ℃, abandon supernatant, stay about 50 μ L, coat after shaking up and contain 40 μ g/mL IPTG, on the LB solid medium of 40 μ g/mL X-gal and 15g/L agar powder, cultivated 16 hours in 37 ℃, the picking white colony, prepare plasmid DNA according to ordinary method, this plasmid is long to be 4647bp, called after pUCA.
(8) adopt restriction endonuclease respectively the plasmid pUCA that plasmid pRL425 and step 7 obtain to be cut, plasmid pRL425 is provided by U.S. Rick professor Debus, and restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company.The consisting of of reaction solution when enzyme is cut pUCA:
Plasmid pUCA 4 μ l
SmaⅠ(10U/μl) 1μl
KpnⅠ(10U/μl) 1μl
Many with enzyme cutting buffering liquid (10 *) 2 μ l
Sterilized water 14 μ l
Cumulative volume 20 μ l
Reacted 3 hours down at 37 ℃, adopt conventional low melting-point agarose gel electrophoresis to reclaim the long DNA of 4341bp that is
Fragment, standby.Get 2 μ l plasmid pRL425 simultaneously and carry out enzyme and cut, reaction solution application of sample ratio is:
Plasmid pRL425 2 μ l
EcoRV(10U/μl) 1μl
KpnⅠ(10U/μl) 1μl
Many with enzyme cutting buffering liquid (10 *) 2 μ l
Sterilized water 14 μ l
Cumulative volume 20 μ l
Reacted 3 hours down at 37 ℃, adopt conventional low melting-point agarose gel electrophoresis to reclaim the long DNA of 1539bp that is
Fragment, standby.
(9) respectively get two kinds of dna fragmentations of the above gained of 8.5 μ l, add 2 μ l ligation damping fluids, 1 μ l T4 phage DNA ligase enzyme is 2 ℃ of reactions 12 hours down.Get 5 μ L ligation liquid, add 200 μ L step (6) gained competence intestinal bacteria DH10B cells, carry out transformation experiment according to ordinary method, coat on the LB solid medium that contains 750 μ g/mL erythromycin and 15g/L agar powder, cultivated 16 hours down at 37 ℃, picking list bacterium colony prepares plasmid DNA according to ordinary method.This plasmid is long to be 5881bp, called after pUCAE.
(10) screening of cyanobacteria transformation and sudden change engineering strain: contain in the BG-11 liquid nutrient medium of 1.5g/L glucose at 200mL, inoculating 5 cytoalgae list algaes and fall, is that 30 ℃, illumination density are that aerated culture to nutrient solution is 0.5 at the light absorption value of 730 nanometers in the illumination box of 2000lx in temperature; Get the 20mL nutrient solution in 50mL has sterilized centrifuge tube, 25 ℃, 4000 rev/mins centrifugal 10 minutes, abandon supernatant; In precipitation, add in the 4.0mL BG-11 liquid nutrient medium, make solution equal 2.5 at the light absorption value of 730 nanometers; Get the above-mentioned solution of 0.4mL in the glass culture tube of sterilizing, add 6 μ L step 6 gained plasmid DNA, vibration; It is that 30 ℃, illumination density are that the illumination box of 2000lx was cultivated 6 hours that test tube is placed temperature, and per vibration half an hour once; Getting 100 μ L liquid coats on the BG-11 solid plate that contains glucose; After 22 hours filter paper is transferred on the BG-11 solid plate that contains 20 μ g/mL erythromycin, continues cultivation and dropped out existing to single algae in 11 days.
(11) single algae of picking step (10) acquisition falls to being inoculated in the 300mLBG-11 liquid nutrient medium, in temperature is that 30 ℃, illumination density are aerated culture (photoautotrophy) in the illumination box of 2000lx, the cell speed of growth is identical with wild-type cell, cultivate after 7 days, born of the same parents' inner chlorophyll content is 1.25 times of wild-type cell; PHB content is about 15% of dry cell weight, is 4.4 times of wild-type cell PHB content; Do not detect the existence of glycogen in the born of the same parents.
Embodiment 2:
(1) with (1) among the embodiment 1, at first from the wild-type cytoalgae, extracts chromosomal DNA.
(2) with (2) among the embodiment 1, be template with wild-type cytoalgae chromosomal DNA, press the upstream and the downstream nucleotide order synthetic oligonucleotide primer thing of cytoalgae agp gene.Be 100 μ mol/L with sterilized water dissolution precipitation to concentration, standby.
(3) get the chromosomal DNA solution of 10 μ L steps (1) preparation in a sterilized centrifuge tube, add successively 45 μ L sterilized waters, 15 μ L amplification buffers, 15 μ LdNTPs, 1.5 μ L by step (2) synthetic primer one, 1.5 μ L by step (2) synthetic primer two, 1 μ LTaq archaeal dna polymerase, add 61 μ L sterilized waters again, add the paraffin oil of 80 μ L at last; Centrifuge tube is placed temperature varying system, be warming up to 94 ℃, kept 10 minutes, be cooled to 54 ℃ then, kept 2 minutes, be warming up to 72 ℃ again, kept 3 minutes, then by be warming up to 94 ℃ and keep 1 minute, be cooled to 54 ℃ and keep 2 minutes, be warming up to 72 ℃ and keep 3 minutes variation sequential loop 25 times, be warming up to 94 ℃ and kept 1 minute at last, be cooled to 54 ℃ and kept 2 minutes, be warming up to 72 ℃ and kept 10 minutes, finish amplified reaction.
(4) adopt restriction endonuclease that agp dna fragmentation and the plasmid pUC118 that the amplification of (3) step obtains cut, used restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company, and plasmid pUC118 is provided by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber.Application of sample volume, temperature of reaction and time have just made enzyme cutting buffering liquid into many with enzyme cutting buffering liquid (10 *) with embodiment 1 step (4).
(5) the agp dna fragmentation is connected into the multiple clone site of plasmid pUC118 carrier under the ligase enzyme effect, used T4 phage DNA ligase enzyme and corresponding damping fluid are buied by ancient cooking vessel state company.Ligation application of sample ratio is:
Plasmid pUC118 enzyme is cut liquid 4 μ l
Agp dna fragmentation enzyme is cut liquid 3.5 μ l
10 * connection damping fluid, 2 μ l
T4 phage DNA ligase enzyme 0.5 μ l
Cumulative volume 10 μ l
Reacted 8 hours down at 4 ℃.Standby.
(6) with step (6) among the embodiment 1, adopt molecular cloning operation preparation colibacillary competent cell (Calcium Chloride Method), used intestinal bacteria DH10B is protected by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber and plants.
(7), obtain plasmid pUCA with step (7) among the embodiment 1.
(8), just make enzyme cutting buffering liquid into Buffer J with step (8) among the embodiment 1.
(9) respectively get two kinds of dna fragmentations of 3.5 μ l step (8) gained, add 2 μ l ligation damping fluids, 1 μ l T4DNA ligase enzyme is 4 ℃ of reactions 8 hours down.Get 5 μ L ligation liquid, add 200 μ L step (6) gained competence intestinal bacteria DH10B cells, carry out transformation experiment according to ordinary method, coat on the LB solid medium that contains 750 μ g/mL erythromycin and 15g/L agar powder, cultivated 20 hours down at 37 ℃, picking list bacterium colony prepares plasmid DNA according to ordinary method, called after pUCAE.
(10) screening of cyanobacteria transformation and sudden change engineering strain: contain in the BG-11 liquid nutrient medium of 1.0g/L glucose at 400mL, inoculating 10 cytoalgae list algaes and fall, is that 28 ℃, illumination density are that aerated culture to nutrient solution is 0.45 at the light absorption value of 730 nanometers in the illumination box of 3000lx in temperature; Get the 10mL nutrient solution in 50mL has sterilized centrifuge tube, 25 ℃, 4000 rev/mins centrifugal 10 minutes, abandon supernatant; In precipitation, add in the 1.8mL BG-11 liquid nutrient medium, make solution equal 2.5 at the light absorption value of 730 nanometers; Get the above-mentioned solution of 0.4mL in the glass culture tube of sterilizing, add 8 μ L step 6 gained plasmid DNA, vibration; It is that 28 ℃, illumination density are that the illumination box of 3000lx was cultivated 8 hours that test tube is placed temperature, and per vibration half an hour once; Getting 150 μ L liquid coats on the BG-11 solid plate that contains glucose; After 24 hours filter paper is transferred on the BG-11 solid plate that contains 30 μ g/mL erythromycin, continues cultivation and dropped out existing to single algae in 12 days.
(11) single algae of obtaining of picking step (10) falls to inoculating in the BG-11 liquid nutrient medium that sub-400mL contains 1.0g/L glucose, in temperature is that 28 ℃, illumination density are aerated culture (photosynthetic raising together with) in the illumination box of 3000lx, and the cell maximum growth rate is 1.7 times of wild-type cell; The cell concentration of final results is 1.5 times of wild-type cells; Cultivate after 7 days, born of the same parents' inner chlorophyll content and wild-type cell are close; PHB content is about 1.6% of dry cell weight, is 1.3 times of wild-type cell PHB content; Do not detect the existence of glycogen in the born of the same parents.
Embodiment 3:
(1) with (1) among the embodiment 1, at first from the wild-type cytoalgae, extracts chromosomal DNA.
(2) with (2) among the embodiment 1, be template with wild-type cytoalgae chromosomal DNA, press the upstream and the downstream nucleotide order synthetic oligonucleotide primer thing of cytoalgae agp gene.With sterilized water dissolution precipitation to 50 μ mol/L, standby.
(3) get the chromosomal DNA solution of 5 μ L steps (1) preparation in a sterilized centrifuge tube, add 60 μ L sterilized waters, 10 μ L amplification buffers, 10 μ LdNTPs, 2 μ L successively by step (2) synthetic primer one, 2 μ L Taq archaeal dna polymerase by step (2) synthetic primer two, 0.5 μ L, add 9.5 μ L sterilized waters again, add the paraffin oil of 80 μ L at last; Centrifuge tube is placed temperature varying system, be warming up to 96 ℃, kept 10 minutes, be cooled to 56 ℃ then, kept 2 minutes, be warming up to 74 ℃ again, kept 3 minutes, then by be warming up to 96 ℃ and keep 1 minute, be cooled to 56 ℃ and keep 2 minutes, be warming up to 74 ℃ and keep 3 minutes variation sequential loop 25 times, be warming up to 96 ℃ and kept 1 minute at last, be cooled to 56 ℃ and kept 2 minutes, be warming up to 74 ℃ and kept 10 minutes, finish amplified reaction.
(4) adopt restriction endonuclease that agp dna fragmentation and the plasmid pUC118 that the amplification of (3) step obtains cut, used restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company, and plasmid pUC118 is provided by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber.Application of sample volume, temperature of reaction and time are with embodiment 1 step (4).
(5) the agp dna fragmentation is connected into the multiple clone site of plasmid pUC118 carrier under the ligase enzyme effect, used T4 dna ligase and corresponding damping fluid are buied by ancient cooking vessel state company.Ligation application of sample ratio is:
Plasmid pUC118 enzyme is cut liquid 9 μ l
Agp dna fragmentation enzyme is cut liquid 8 μ l
10 * connection damping fluid, 2 μ l
T4 phage DNA ligase enzyme 1 μ l
Cumulative volume 20 μ l
Reacted 10 hours down at 6 ℃.Standby.
(6) with step (6) among the embodiment 1, adopt molecular cloning operation preparation colibacillary competent cell (Calcium Chloride Method), used intestinal bacteria DH10B is protected by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber and plants.
(7), obtain plasmid pUCA with step (7) among the embodiment 1.
(8), just make enzyme cutting buffering liquid into Buffer J with step (8) among the embodiment 1.
(9) respectively get two kinds of dna fragmentations of 3.5 μ l step (8) gained, add 2 μ l ligation damping fluids, 1 μ l T4DNA ligase enzyme is 6 ℃ of reactions 10 hours down.Get 8 μ L ligation liquid, add 200 μ L step (6) gained competence intestinal bacteria DH10B cells, carry out transformation experiment according to ordinary method, coat on the LB solid medium that contains 750 μ g/mL erythromycin and 15g/L agar powder, cultivated 20 hours down in 37 ℃, picking list bacterium colony prepares plasmid DNA according to ordinary method, called after pUCAE.
(10) screening of cyanobacteria transformation and sudden change engineering strain: contain in the BG-11 liquid nutrient medium of 1g/L glucose at 400mL, inoculating 5 cytoalgae list algaes and fall, is that 26 ℃, illumination density are that aerated culture to nutrient solution is 0.475 at the light absorption value of 730 nanometers in the illumination box of 35001x in temperature; Get the 10mL nutrient solution in 50mL has sterilized centrifuge tube, 25 ℃, 4000 rev/mins centrifugal 10 minutes, abandon supernatant; In precipitation, add in the 1.9mL BG-11 liquid nutrient medium, make solution equal 2.5 at the light absorption value of 730 nanometers; Get the above-mentioned solution of 0.4mL in the glass culture tube of sterilizing, add 8 μ L step 6 gained plasmid DNA, vibration; It is that 26 ℃, illumination density are that the illumination box of 3500lx was cultivated 8 hours that test tube is placed temperature, and per vibration half an hour once; Getting 200 μ L liquid coats on the BG-11 solid plate that contains glucose; After 26 hours filter paper is transferred on the BG-11 solid plate that contains 40 μ g/mL erythromycin, continues cultivation and dropped out existing to single algae in 13 days.
(11) single algae of obtaining of picking step (10) falls to being inoculated in 400mL and contains in the BG-11 liquid nutrient medium of 10mM sodium acetate, in temperature is that 26 ℃, illumination density are aerated culture (photosynthetic raising together with) in the illumination box of 3500lx, and the cell maximum growth rate is 60% of a wild-type cell; The cell concentration of final results is 74% of a wild-type cell; Cultivate after 7 days, born of the same parents' inner chlorophyll content is 1.13 times of wild-type cell; PHB content is about 30% of dry cell weight, is 10 times of wild-type cell PHB content; Do not detect the existence of glycogen in the born of the same parents.
Embodiment 4:
(1) with (1) among the embodiment 1, at first from the wild-type cytoalgae, extracts chromosomal DNA.
(2) with (2) among the embodiment 1, be template with wild-type cytoalgae chromosomal DNA, press the upstream and the downstream nucleotide order synthetic oligonucleotide primer thing of cytoalgae agp gene.With sterilized water dissolution precipitation to 75 μ mol/L, standby.
(3) get the chromosomal DNA solution of 10 μ L steps (1) preparation in a sterilized centrifuge tube, add 30 μ L sterilized waters, 15 μ L amplification buffers, 15 μ LdNTPs, 2 μ L successively by step (2) synthetic primer one, 2 μ L Taq archaeal dna polymerase by step (2) synthetic primer two, 1 μ L, add 75 μ L sterilized waters again, add 100 μ L paraffin oils at last; Centrifuge tube is placed temperature varying system, be warming up to 94 ℃, kept 10 minutes, be cooled to 56 ℃ then, kept 2 minutes, be warming up to 72 ℃ again, kept 3 minutes, then by be warming up to 94 ℃ and keep 1 minute, be cooled to 56 ℃ and keep 2 minutes, be warming up to 72 ℃ and keep 3 minutes variation sequential loop 25 times, be warming up to 94 ℃ and kept 1 minute at last, be cooled to 56 ℃ and kept 2 minutes, be warming up to 72 ℃ and kept 10 minutes, finish amplified reaction.
(4) adopt restriction endonuclease that agp dna fragmentation and the plasmid pUC118 that the amplification of (3) step obtains cut, used restriction endonuclease and corresponding damping fluid are buied by ancient cooking vessel state company, and plasmid pUC118 is provided by biology department of Tsing-Hua University little algae Biotechnology Experiment chamber.Application of sample volume, temperature of reaction and time are with embodiment 1 step (4).
(5) with step (5) among the embodiment 1.
(6), adopt the colibacillary competent cell of molecular cloning operation preparation with step (6) among the embodiment 1.
(7), obtain plasmid pUCA with step (7) among the embodiment 1.
(8) with step (8) among the embodiment 1.
(9), obtain plasmid pUCAE with step (9) among the embodiment 1.
(10) with step (10) among the embodiment 1, single algae that acquisition has the erythromycin resistance falls.
(11) single algae of obtaining of picking step (10) falls to being inoculated in 400mL and contains in the BG-11 liquid nutrient medium of 1.0g/L glucose and 10mM sodium acetate, in temperature is that 30 ℃, illumination density are aerated culture (photosynthetic raising together with) in the illumination box of 3000lx, and the cell maximum growth rate is 1.1 times of wild-type cell; The cell concentration of final results is 1.2 times of wild-type cell; Cultivate after 7 days, born of the same parents' inner chlorophyll content is 1.16 times of wild-type cell; PHB content is about 5.0% of dry cell weight, is 2.7 times of wild-type cell PHB content; Do not detect the existence of glycogen in the born of the same parents.
Claims (1)
1, a kind of method that makes up genetically engineered cytoalgae production bio-degradable plastics is characterized in that this method is made up of following steps:
(1) at first extract chromosomal DNA from the wild-type cytoalgae: its process is: picking 5-10 single algae falls from the solid plate, be inoculated in the culturing bottle that 200-400 milliliters of liquid substratum is housed, this substratum is designated as the BG-11 substratum, consisting of of every liter of substratum: yellow soda ash: 20g, dipotassium hydrogen phosphate: 30.5g, ferric ammonium citrate: 6g, SODIUMNITRATE: 1.5g, sal epsom: 75mg, contain 0.25mol/L ethylenediamine-N,N'-diacetic acid(EDDA) disodium, the pH value is 8.0 the aqueous solution 11.2 μ L, calcium chloride: 36mg, citric acid: 6mg, contain 2.86g/L boric acid, 0.222g/L Zinc Sulphate Heptahydrate, 0.079g/L cupric sulfate pentahydrate, 1.81g/L four aqueous magnesium chlorides, 0.39g/L Sodium Molybdate Dihydrate, 0.049g/L the liquid microelement 1mL of cobalt nitrate hexahydrate is 24-32 ℃ in temperature, illumination density is that aerated culture to the light absorption value of nutrient solution under 730 nanometers is 0.3-1.0 in the illumination box of 1000-3000lx; Nutrient solution is transferred in the 200-300 milliliter Centrifuge Cup, under the condition of 15-25 ℃, 3000-5000 rev/min centrifugal 5-15 minute, according to conventional cyanobacteria DNA extraction method the gained frustule is handled, obtain the chromosomal DNA precipitation of wild-type cytoalgae, add in the damping fluid that contains 1.21g/L Tutofusin tris and 0.372g/L two ethylenediamine hydrate oxalic acid disodiums, pH8.0 of 0.2-0.6mL, dissolution precipitation, standby;
(2) being template with wild-type cytoalgae chromosomal DNA, is primer by the upstream and the downstream nucleotide order synthetic oligonucleotide of cytoalgae agp gene.Contain deoxyadenosine triphosphoric acid (being designated as A), deoxyguanosine triphosphoric acid (being designated as G), deoxy cytidine triphosphate (being designated as C), deoxythymidine triphosphoric acid (being designated as T) in the primer, institute's synthetic primer one is: 5-phosphoric acid-C-A-G-C-T-C-C-G-G-A-T-C-C-C-A-A-C-G-G-C-G-phosphoric acid-3 '; Primer two is: 5 '-phosphoric acid-G-G-C-C-G-A-G-C-T-C-A-G-G-G-A-T-T-T-T-A-C-T-G-C-T-T-T-phosphoric acid-3 ', after primer is synthetic, be 50-100 μ mol/L with sterilized water dissolution precipitation to concentration, and standby;
(3) get the chromosomal DNA solution of 3-10 μ L step 1 preparation in a sterilized centrifuge tube, add the amplification buffer of 20-40 μ L sterilized water, 5-15 μ L, four kinds of deoxynucleotides of 5-15 μ L, the Taq archaeal dna polymerase of 0.5-1.5 μ L successively by step 2 synthetic primer two, 0.5-1 μ L by step 2 synthetic primer one, 0.5-1.5 μ L, complementing to cumulative volume with sterilized water is 100-150 μ L, adds the paraffin oil of 60-120 μ L again; Centrifuge tube is placed temperature varying system, be warming up to 92-96 ℃, kept 10-15 minute, be cooled to 50-60 ℃ then, kept 1-5 minute, be warming up to 70-76 ℃ again, kept 1-5 minute, then by be warming up to 92-96 ℃ and keep 1-2 minute, be cooled to 50-60 ℃ and keep 1-5 minute, be warming up to 70-76 ℃ and keep 1-5 minute variation sequential loop 20-30 time, be warming up to 92-96 ℃ and kept 1-2 minute at last, be cooled to 50-60 ℃ and kept 1-5 minute, be warming up to 70-76 ℃ and kept 10-20 minute, finish amplified reaction;
(4) get 5-10 μ l by the agp dna fragmentation that the amplification of the 3rd step obtains, add 0.5-1.5 μ LBamH I restriction endonuclease, 0.5-1.5 μ lSac I restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 3-5 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃; Get 2-4 μ l plasmid pUC118 simultaneously, add 0.5-1.5 μ lBamH I restriction endonuclease, 0.5-1.5 μ lSac I restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 6-11 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃;
(5) get 2-8 μ l by the agp dna fragmentation of step 4 gained, 5-9 μ l plasmid pUC118 fragment, mix, add 1-2 μ l ligation damping fluid by step 4 gained, 1 μ l T4 dna ligase, 2-6 ℃ of reaction 8-12 hour down, standby:
(6) picking list bacterium colony from the solid plate of thalline, be inoculated in the 3-10mL liquid nutrient medium, this substratum is designated as the LB substratum, contain 5g yeast extract powder in every liter of substratum, the 10g peptone, 10g sodium-chlor, the pH value is 7.0, at under 25-38 ℃ on 150-300 rev/min shaking table culturing bacterium 6-15 hour, then getting the 0.1-1.0mL nutrient solution is inoculated in the 50-100mL liquid nutrient medium, is 0.3-1.0 be cultured to nutrient solution under 25-38 ℃ on 150-300 rev/min shaking table at the light absorption value of 600 nanometers, and the Lime Chloride according to routine is equipped with competent cell then;
(7) get 150-250 μ L competence intestinal bacteria DH10B cell, add the ligation thing of 5-10 μ L by step 5 gained, carry out transformation experiment according to ordinary method, on the LB solid medium that contains 40 μ g/mL IPTG, 40 μ g/mL X-gal and 15g/L agar powder in 37 ℃ of following culturing cells, the picking white colony prepares plasmid DNA according to ordinary method after 16-24 hour;
(8) get 2-4 μ l by the 7th step gained plasmid DNA, add 0.5-1.5 μ lKpn I restriction endonuclease, 0.5-1.5 μ lSma I restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 3-5 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃, adopt conventional low melting-point agarose gel electrophoresis to reclaim the long dna fragmentation of 4341bp that is; Get 2-4 μ l plasmid pRL425 simultaneously, add 0.5-1.5 μ lKpn I restriction endonuclease, 0.5-1.5 μ lEcoRV restriction endonuclease, the corresponding enzyme cutting buffering liquid of 1-2 μ l, 6-11 μ l sterilized water successively, reacted 1-4 hour down at 25-38 ℃, adopt conventional low melting-point agarose gel electrophoresis to reclaim the long dna fragmentation of 1539bp that is;
(9) get 4-9 μ l two kinds of dna fragmentations in above-mentioned the 8th step respectively, add 1-2 μ l ligation damping fluid, 1 μ l T4DNA ligase enzyme, reacted 8-12 hour down at 2-6 ℃, get 5-10 μ L, add 150-250 μ L by step 6 gained competence intestinal bacteria DH10B cell, carry out transformation experiment according to ordinary method, coat on the LB solid medium that contains 750 μ g/mL erythromycin and 15g/L agar powder, cultivated 16-24 hour down at 37 ℃, picking list bacterium colony prepares plasmid DNA according to ordinary method, and is standby;
(10) contain in the BG-11 liquid nutrient medium of 0.5-1.5g/L glucose at 200-400mL, inoculating 5-10 cytoalgae list algae and fall, is that 24-32 ℃, illumination density are that aerated culture to nutrient solution is 0.3-1.0 at the light absorption value of 730 nanometers in the illumination box of 1000-3000lx in temperature; Get the 10-20mL nutrient solution in 50mL has sterilized centrifuge tube, 15-25 ℃, 3000-5000 rev/min centrifugal 10-20 minute are abandoned supernatant; In precipitation, add in the 2-8mL BG-11 liquid nutrient medium, make solution equal 2.5 at the light absorption value of 730 nanometers; Get the above-mentioned solution of 0.2-0.6mL in the glass culture tube of sterilizing, add 2-8 μ L by step 9 gained plasmid DNA, vibration; It is that 24-32 ℃, illumination density are that the illumination box of 1000-3000lx was cultivated 5-10 hour that test tube is placed temperature, and per vibration half an hour once; Getting 100-300 μ L liquid coats on the BG-11 solid plate that contains glucose; After 20-30 hour filter paper is transferred on the BG-11 solid plate that contains 10-100 μ g/mL erythromycin, continues cultivation and dropped out existing to single algae in 10-15 days;
(11) single algae of picking 5-10 step 10 acquisition falls to inoculating sub-200-400mL BG-11 liquid nutrient medium respectively, the BG-11 liquid nutrient medium that contains 0.5-1.0g/L glucose, contain the BG-11 liquid nutrient medium of 5-20mM sodium acetate or contain 0.5-1.0g/L glucose simultaneously and the BG-11 liquid nutrient medium of 5-20mM sodium acetate in, in temperature is 25-32 ℃, illumination density is to cultivate in the illumination box of 1500-2500lx, sampling in per 24 hours, measure cell density, cultivate after 6-8 days, with the rotating speed of whizzer with 4000-8000 rev/min, centrifugal 10-20 minute, abandon supernatant liquor, results gained cell, in 60 ℃ of oven drying precipitations, can obtain product bio-degradable plastics poly-beta-hydroxy-butyrate of the present invention.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104293803A (en) * | 2014-09-29 | 2015-01-21 | 天津大学 | Ethanol-tolerant related gene slr0982 of synechocystis 6803 and applications of gene |
| US9765205B2 (en) | 2011-08-24 | 2017-09-19 | Algix, Llc | Macrophyte-based bioplastic |
| CN114951254A (en) * | 2022-05-11 | 2022-08-30 | 福建工程学院 | Method for preventing and controlling co-migration of micro-plastic-drug-resistant gene in soil |
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2000
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9765205B2 (en) | 2011-08-24 | 2017-09-19 | Algix, Llc | Macrophyte-based bioplastic |
| CN104293803A (en) * | 2014-09-29 | 2015-01-21 | 天津大学 | Ethanol-tolerant related gene slr0982 of synechocystis 6803 and applications of gene |
| CN114951254A (en) * | 2022-05-11 | 2022-08-30 | 福建工程学院 | Method for preventing and controlling co-migration of micro-plastic-drug-resistant gene in soil |
| CN114951254B (en) * | 2022-05-11 | 2023-11-14 | 福建工程学院 | Method for preventing and controlling microplastic-drug-resistant gene co-migration in soil |
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