CN100374570C - Electric conversion method of making shuttle plasmid guide into yellow brevibacterium - Google Patents
Electric conversion method of making shuttle plasmid guide into yellow brevibacterium Download PDFInfo
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- CN100374570C CN100374570C CNB2004100602002A CN200410060200A CN100374570C CN 100374570 C CN100374570 C CN 100374570C CN B2004100602002 A CNB2004100602002 A CN B2004100602002A CN 200410060200 A CN200410060200 A CN 200410060200A CN 100374570 C CN100374570 C CN 100374570C
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- brevibacterium flavum
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- shuttle plasmid
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Abstract
The present invention discloses a rapid and effective electrotransformation method for leading recombinant plasmid capable of shuttle expressing in colibacillus and yellow brevibacterium into the yellow brevibacterium. The present invention is characterized in that when the colibacillus-DH5 alpha which carries the recombinant plasmid is cultured in a special culture medium, the yellow brevibacterium is treated to be in a dormant state. When the recombinant plasmid which is capable of shuttle expressing in the colibacillus and the yellow brevibacterium is led into the yellow brevibacterium in the form of electrotransformation, and when the colibacillus-DH5 alpha is used as host bacteria to obtain the recombinant plasmid, methylation modification action from the colibacillus to shuttle plasmid is reduced, and restriction enzyme cutting action from the yellow brevibacterium to the shuttle plasmid is also reduced. Therefore, the shuttle plasmid can be led into the yellow brevibacterium effectively by means of electrotransformation and can be duplicated and transmitted stably with electrotransformation efficiency of 2.0*10 <3>.
Description
Technical field
The present invention relates to a kind of biomaterial and import the method that known microorganisms host bacterium carrier obtains recombinant plasmid, be specifically related to a kind of efficient electric method for transformation that recombinant shuttle plasmid is imported brevibacterium flavum.
Background technology
Brevibacterium flavum belongs to brevibacterium sp, is usually used in each seed amino acid of fermentative production.Because its Gram-positive can't import foreign DNA by making competence, thereby must foreign DNA be imported brevibacterium flavum, to make up the reorganization bacterium by methods such as electricity conversions.Daily, people can electric the conversion in the process that imports brevibacterium flavum of the recombinant plasmid of shuttling expressing find in intestinal bacteria and brevibacterium flavum a kind of, the recombinant plasmid that obtains for the host bacterium with the bacillus coli DH 5 alpha, owing to be subjected to the modification that methylates, after importing some brevibacterium flavum bacterial classification, can and cut into fragment by these endobacillary restriction enzyme identifications, thereby be difficult to obtain to contain the bacterial strain of complete recombinant plasmid.In order to overcome these difficulties, people needing to demand a kind of effective means of invention urgently, can reduce the methylate modification of intestinal bacteria to shuttle plasmid, and brevibacterium flavum is to the restriction enzyme digestion effect of shuttle plasmid, this shuttle plasmid can be imported in the brevibacterium flavum efficiently by electric method for transformation, realize stablizing and duplicate and transmit.
Summary of the invention
Transform in the process that imports brevibacterium flavum at the recombinant plasmid of shuttling expressing is electric in intestinal bacteria and brevibacterium flavum at present, the recombinant plasmid that obtains for the host bacterium with the bacillus coli DH 5 alpha, owing to be subjected to the modification that methylates, after importing some brevibacterium flavum bacterial classification, can and cut into fragment by these endobacillary restriction enzyme identifications, thereby be difficult to obtain to contain the problem of recombinant plasmid.
The present invention's research and grasp can transform the method fast and effectively that imports brevibacterium flavum by the recombinant plasmid electricity of shuttling expressing with a kind of in intestinal bacteria and brevibacterium flavum.Promptly
The invention provides a kind of special substratum.
The invention provides a kind of method that the brevibacterium flavum bacterium is handled.
The invention provides a kind of special substratum, culture medium prescription:
Ammonium sulfate 1.8～2.2g/l
Potassium primary phosphate 13～14g/l
Ferrous sulfate 0.45～0.65mg/l
Bitter salt 0.2～0.3g/l
In the process that the bacillus coli DH 5 alpha that carries recombinant plasmid is cultivated, reduce the methylate modification of intestinal bacteria to shuttle plasmid.
The present invention also provides a kind of method that the brevibacterium flavum thalline is suitably handled, promptly utilize 0.5～2 μ g/ml penbritin and 0.1～1.5% tween 80 that the thalline of brevibacterium flavum is handled, can on somatic cells wall and cytolemma, form aperture, cause the intracellular matter outflow, thereby cell is grown normally and metabolism is affected, make it be in dormant state, to reduce the restriction enzyme digestion effect of brevibacterium flavum to shuttle plasmid.This shuttle plasmid can be imported in the brevibacterium flavum efficiently by electric method for transformation, and can stablize and duplicate and transmit.
By above technological invention, can reach following effect:
1,, can obviously reduce the methylate modification of intestinal bacteria to shuttle plasmid by adopting special culture medium provided by the invention that the bacillus coli DH 5 alpha that carries recombinant plasmid is cultivated.Simultaneously, make brevibacterium flavum to be transformed be in dormant state.
2, this shuttle plasmid can be imported in the brevibacterium flavum efficiently by electric method for transformation, and can stablize and duplicate and transmit.The electricity transformation efficiency reaches 2.0 * 10
3And adopt general electric method for transformation to be difficult to obtain to contain the bacterial strain of recombinant plasmid.
Description of drawings
Slightly
Embodiment
For realizing above-mentioned technique effect, the specific embodiment of the present invention:
1, the recombinant shuttle plasmid pECA1 that selects for use of the present invention contains replication orgin and the exogenous genetic fragment of plasmid pBL1 and pUC18, can shuttle back and forth to duplicate and express in brevibacterium flavum and bacillus coli DH 5 alpha.When adopting rich medium that the bacillus coli DH 5 alpha that carries shuttle plasmid is cultivated, plasmid DNA is owing to be subjected to the modification that methylates, after importing some brevibacterium flavum bacterial classification, can and cut into fragment by these endobacillary restriction enzyme identifications, thereby be difficult to obtain to contain the bacterial strain of complete recombinant plasmid.
The present invention cultivates the bacillus coli DH 5 alpha that carries recombinant plasmid by adopting following substratum, can obviously reduce the methylate modification of intestinal bacteria to shuttle plasmid.
Culture medium prescription:
Ammonium sulfate 2g/l
Potassium primary phosphate 13.6g/l
Ferrous sulfate 0.5mg/l
Bitter salt 0.25g/l
2, be 7.0 with above-mentioned substratum with the potassium hydroxide adjust pH, be distributed into the bottle sterilization, adding aseptic glucose solution to final concentration before the inoculation is 2g/l; Adding aseptic thiamine hydrochloride cellulose solution to final concentration is 0.5mg/l; Adding chloromycetin solution to final concentration is 20 μ g/ml.
3, will carry the pECA1 bacillus coli DH 5 alpha and be seeded in the substratum shaking culture 12～14 hours, harvested cell, the alkaline hydrolysis method is extracted plasmid DNA, CsCl-ethidium bromide equilibrium gradient centrifuging plasmid DNA purification, and be dissolved in that to make DNA concentration in an amount of sterilized water be 8～10 μ g/ml.
4, the preparation of brevibacterium flavum C-12 to be transformed.This bacterial classification can be purchased by Chinese microorganism management committee common micro-organisms preservation center, also can purchase by market, as long as the plasmid DNA in bacillus coli DH 5 alpha source is had the bacterial classification of strong restriction enzyme digestion effect.
5, the triangular flask that thalline will be housed was placed on ice 10 minutes, poured in the centrifuge tube of precooling 4 ℃ into and 6000 left the heart 10 minutes down, carefully removed supernatant liquor, 10% glycerine washing 4 times, and it is standby that thalline is dissolved in 300 μ l10% glycerine.
6, adopt Bio-Rad MicroPulser
TMType electric shock instrument, the 0.2cm cup that shock by electricity is drawn the bacteria suspension of 20 μ l prepared fresh, adds the plasmid DNA of 1～2 μ l, behind the mixing in the electric shock cup of adding precooling.Regulate the electric shock instrument, use the Ec2 standard program, start electricimpulse, instrument should demonstrate the strength of electric field that has 12.5kV/cm in the 6ms.
7, take out the electric shock cup, the sucking-off suspension adds 1ml SOC nutrient solution, 30 ℃ of soft shaking culture 2 hours.
8, draw 200 μ l electric shock transformant, be laid on the LB agar plate that contains 20 μ g/ml paraxin, be inverted for 30 ℃ and cultivated 3～5 days, visible transformed clone occurs.
Embodiment one: culture medium prescription one:
Ammonium sulfate 2g/l
Potassium primary phosphate 13.6g/l
Ferrous sulfate 0.5mg/l
Bitter salt 0.25g/l
Utilize above-mentioned substratum, by the bacillus coli DH 5 alpha that carries recombinant plasmid is cultivated, reach and reduce the methylate modification of intestinal bacteria shuttle plasmid, make brevibacterium flavum to be transformed be in dormant state, shuttle plasmid can be imported in the brevibacterium flavum efficiently by electric method for transformation, and can realize stablizing and duplicate and transmit.
Embodiment two: culture medium prescription two:
Ammonium sulfate 1.8g/l
Potassium primary phosphate 13g/l
Ferrous sulfate 0.45mg/l
Bitter salt 0.2g/l
Utilize above-mentioned substratum, by the bacillus coli DH 5 alpha that carries recombinant plasmid is cultivated, reach and reduce the methylate modification of intestinal bacteria shuttle plasmid, make brevibacterium flavum to be transformed be in the dormancy attitude, shuttle plasmid can be imported in the brevibacterium flavum efficiently by electric method for transformation, and can realize stablizing and duplicate and transmit.
Embodiment three: culture medium prescription three:
Ammonium sulfate 2.1g/l
Potassium primary phosphate 13.9g/l
Ferrous sulfate 0.64mg/l
Bitter salt 0.3g/l
Utilize above-mentioned substratum, by the bacillus coli DH 5 alpha that carries recombinant plasmid is cultivated, reach and reduce the methylate modification of intestinal bacteria shuttle plasmid, make brevibacterium flavum to be transformed be in dormant state, shuttle plasmid can be imported in the brevibacterium flavum efficiently by electric method for transformation, and can realize stablizing and duplicate and transmit.
Embodiment four: the preparation of brevibacterium flavum C-12 to be transformed
Brevibacterium flavum C-12 to be transformed can purchase by Chinese microorganism management committee common micro-organisms preservation center, also can purchase by market, as long as the plasmid DNA in bacillus coli DH 5 alpha source is had the bacterial classification of strong restriction enzyme digestion effect.
New activatory bacterial classification in 6 days is seeded in earlier in the 10ml liquid LB substratum, cultivated 8 hours for 30 ℃, its O.D.550 is about about 0.2, get 1ml be inoculated in in the 500ml Erlenmeyer flask of dress 100ml liquid LB 30 ℃ continue to be cultured to O.D.550 and be about 1.0.Add final concentration and be the penbritin of 0.5 μ g/ml and final concentration and be 1.5% tween 80, slightly vibrated 1 hour.Penbritin and tween 80 all have certain destruction to the cell walls of brevibacterium flavum, can on somatic cells wall and cytolemma, form aperture, cause the intracellular matter outflow, thereby cell is grown normally and metabolism is affected, but bacterium is not dead, but be in a kind of dormant state, and when treating that condition suits, can the continued growth breeding.
Claims (1)
1. one kind will transform the method that imports brevibacterium flavum by the recombinant plasmid electricity of shuttling expressing in intestinal bacteria and brevibacterium flavum, it is characterized in that,
A. design the substratum of forming by 1.8～2.2g/l ammonium sulfate, 13～14g/l potassium primary phosphate, 0.45～0.65mg/l ferrous sulfate, 0.2～0.3g/l bitter salt;
B. with the described substratum of step a the bacillus coli DH 5 alpha that carries recombinant plasmid is cultivated, reduced the modification that methylates of intestinal bacteria shuttle plasmid;
C. brevibacterium flavum being carried out dormant state handles, promptly the cell walls destruction of brevibacterium flavum is handled with penbritin and 0.1～1.5% tween 80 of final concentration 0.5～2 μ g/ml, on somatic cells wall and cytolemma, form aperture, cause the intracellular matter outflow, normal growth of cell and metabolism are affected, make it be in dormant state, to reduce the restriction enzyme digestion of brevibacterium flavum to shuttle plasmid, this shuttle plasmid is imported in the brevibacterium flavum by electric method for transformation, and can stablize and duplicate and transmit.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100602002A CN100374570C (en) | 2004-07-07 | 2004-07-07 | Electric conversion method of making shuttle plasmid guide into yellow brevibacterium |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2004100602002A CN100374570C (en) | 2004-07-07 | 2004-07-07 | Electric conversion method of making shuttle plasmid guide into yellow brevibacterium |
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| CN1718732A CN1718732A (en) | 2006-01-11 |
| CN100374570C true CN100374570C (en) | 2008-03-12 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1995362B (en) * | 2006-12-28 | 2010-06-23 | 浙江工业大学 | Gibberella fujikuroi electroporation genetic transformation method |
| CN101864441A (en) * | 2010-05-19 | 2010-10-20 | 江南大学 | Method for knocking out mycobacteria ksdD gene and application thereof |
| CN103160535A (en) * | 2011-12-16 | 2013-06-19 | 江南大学 | Electrotransformation method for introducing shuttle plasmid into corynebacterium acetoacidophilum |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001083786A2 (en) * | 2000-05-01 | 2001-11-08 | Midwest Research Institute | Stable zymomonas mobilis xylose and arabinose fermenting strains |
| CN1361286A (en) * | 2000-09-15 | 2002-07-31 | 北京金赛狮生物制药技术开发有限责任公司 | Construction and application of recombinant probiotics strain to express human endothelial cell inhibin |
| CN1407108A (en) * | 2001-09-06 | 2003-04-02 | 胡放 | Genetic engineering recombination strain expression of lactoferritin and use thereof |
| WO2003064653A2 (en) * | 2002-02-01 | 2003-08-07 | Schering Aktiengesellschaft | Method for overexpressing dehydrogenases |
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001083786A2 (en) * | 2000-05-01 | 2001-11-08 | Midwest Research Institute | Stable zymomonas mobilis xylose and arabinose fermenting strains |
| WO2001083786A3 (en) * | 2000-05-01 | 2002-06-20 | Midwest Research Inst | Stable zymomonas mobilis xylose and arabinose fermenting strains |
| CN1361286A (en) * | 2000-09-15 | 2002-07-31 | 北京金赛狮生物制药技术开发有限责任公司 | Construction and application of recombinant probiotics strain to express human endothelial cell inhibin |
| CN1407108A (en) * | 2001-09-06 | 2003-04-02 | 胡放 | Genetic engineering recombination strain expression of lactoferritin and use thereof |
| WO2003064653A2 (en) * | 2002-02-01 | 2003-08-07 | Schering Aktiengesellschaft | Method for overexpressing dehydrogenases |
| WO2003064653A3 (en) * | 2002-02-01 | 2003-12-24 | Schering Ag | Method for overexpressing dehydrogenases |
Non-Patent Citations (1)
| Title |
|---|
| 大肠杆菌pheA 基因在黄色短杆菌中的克隆与表达. 雷呈祥等.生物工程学报，第12 (增刊)期. 1996 * |
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Granted publication date: 20080312 Termination date: 20100707 |