CN103966251A - Conversion method for introducing shuttle plasmids into brevibacterium flavum (or corynebacterium glutamicum) - Google Patents
Conversion method for introducing shuttle plasmids into brevibacterium flavum (or corynebacterium glutamicum) Download PDFInfo
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- CN103966251A CN103966251A CN201310033531.6A CN201310033531A CN103966251A CN 103966251 A CN103966251 A CN 103966251A CN 201310033531 A CN201310033531 A CN 201310033531A CN 103966251 A CN103966251 A CN 103966251A
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- brevibacterium flavum
- corynebacterium glutamicum
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Abstract
The invention discloses a rapid and effective method for converting recombinant plasmids that can perform shuttle expression in escherichia coli and brevibacterium flavum (or corynebacterium glutamicum) into corynebacterium glutamicum and auxotroph brevibacterium flavum. The method comprises two steps namely competent cell preparation and high-temperature culture and conversion: (1) inoculating auxotroph brevibacterium flavum (or corynebacterium glutamicum) into a specially-prepared culture medium to enable the auxotroph brevibacterium flavum (or corynebacterium glutamicum) to carry out rapid proliferation, and then transforming the auxotroph brevibacterium flavum (or corynebacterium glutamicum) to a culture medium with inefficient nutrients to prepare competent cells; (2) evenly mixing plasmids to be converted and the prepared competent cells, placing the mixture into an ice bath, culturing under a constant temperature, and finally introducing the plasmids into brevibacterium flavum (or corynebacterium glutamicum) to be converted. The method provided by the invention can reduce the restrictive enzyme digestion effect of brevibacterium flavum on shuttle plasmids, the shuttle plasmids can be stably copied and transmitted, and thus the method has the advantages of good conversion effect and convenient operation.
Description
Technical field
The present invention relates to bacterium and transform field, be specifically related to a kind of method importing in brevibacterium flavum (or Corynebacterium glutamicum) that plasmid is transformed, comprise wild-type and auxotroph.
Background technology
Brevibacterium flavum (or Corynebacterium glutamicum) is a kind of gram-positive microorganism, is extensively present in occurring in nature.Due to the characteristic of brevibacterium flavum (or Corynebacterium glutamicum), the standby competent cell of general chemical legal system is difficult to plasmid to import brevibacterium flavum (or Corynebacterium glutamicum) cell, or conventional conversion method is that electric shock transformation ratio is easier to plasmid transfered cell in construction of genetic engineering, but culture medium raw material prepared by electric shock conversion brevibacterium flavum (or Corynebacterium glutamicum) competent cell used is more expensive, operate relatively simple, but transformation efficiency is not too high, voltage (setting of electric capacity and resistance) and time parameter are key parameters.And for the simpler and cruder laboratory of condition, electricity turns in the situation that equipment lacks, prepare competent cell by this chemical process, and plasmid is transformed and entered in bacterium, after 37 DEG C of constant temperature culture for some time, plasmid can be in bacterium genetic stability, be a cost-effective good method.
Summary of the invention
A kind of method that the object of this invention is to provide easier special culture medium cultivation is prepared brevibacterium flavum (or Corynebacterium glutamicum) competent cell, and constant temperature culture imports plasmid the short-cut method of its cell energy genetic stability.
The method of preparing brevibacterium flavum (or Corynebacterium glutamicum) competent cell and transforming provided by the invention:
(1) described competent cell preparation process is as follows:
The mono-colony inoculation of auxotroph brevibacterium flavum AN78 of picking one fresh culture is in 2.5 ml GM I substratum, and 30 DEG C, under the condition of 130-150 rpm, shaking culture is spent the night; Overnight culture is transferred in the GM I that 2.5ml is fresh to 37 DEG C of 200-230 rpm shaking culture 3.5h by 10% inoculum size; Again culture is carried out to second pass generation, be inoculated in 5 ml GMII substratum, carry out second pass generation by 5% inoculum size, 37 DEG C of 200-230 rpm shaking culture 90 min, get 1 ml culture, centrifugal 5 min of 5000 rpm room temperature, with 1/10 volume supernatant liquor Eddy diffusion bacterial precipitation, are auxotroph brevibacterium flavum competent cell.In like manner, the competent cell that Corynebacterium glutamicum ATCC 13032 is prepared into, method is similar to above-mentioned preparation auxotroph brevibacterium flavum competent cell, auxotrophic bacterial strain, added reagent is amino acid, and non-auxotrophic bacterial strain is used amino acid instead sterilized water and replaced.The solvent of described GM I growth medium is water (as distilled water), and solute and concentration thereof are as follows: 15% K
2hPO
43H
2o, 6% KH
2pO4,2% (NH
4)
2sO4,0.2% MgSO
4, 1% Trisodium Citrate, is dissolved in above-mentioned each composition in distilled water 6.6 × 10
4pa autoclaving, room temperature preservation; 10% yeast powder, 1% caseinhydrolysate, 25mM MgCl
2(MgCl
26H
2o, molecular weight is 203), autoclaving, yeast powder solution is preferably now with the current, caseinhydrolysate and MgCl
2solution can room temperature storage; 20% glucose, 0.25% amino acid needed His(Histidine), Pro(proline(Pro)), Met(methionine(Met)), 0.1M CaCl
2(CaCl
22H
2o, molecular weight is 147), with the millipore filtration degerming of 0.22 μ m, glucose solution and CaCl
2room temperature storage, amino acid solution-20 DEG C preservation.
(2) described shuttle plasmid being imported to auxotroph brevibacterium flavum AN78(or Corynebacterium glutamicum ATCC 13032) step of cell is as follows:
Get the each 10 μ l of plasmid pXMJ19, pXMJ19-argH and sterilized water and add in auxotroph brevibacterium flavum AN78 competent cell suspension, to final concentration 1 μ g/ml, plasmid volume is no more than 1/20 of competent cell suspension volume, mixes.In 37 DEG C of water-baths, leave standstill 30-60 min, 37 DEG C of 200 rpm shaking culture 2-4 h, coats on the LB solid medium that contains paraxin (30 μ g/ml), and every 100 μ l transformation systems are coated with 1 flat board, the transformation system of each brevibacterium flavum is coated with 5 flat boards, is inverted overnight incubation for 37 DEG C.Calculate average conversion (transformant number/μ g DNA).Because brevibacterium flavum is auxotroph, in LB solid medium, add 3% amino acid mixing liquid (0.25% His, Pro, Met), auxotroph brevibacterium flavum AN78 well-grown.In like manner, Corynebacterium glutamicum 13032 transformation experiment flow processs are similar to brevibacterium flavum AN78, because Corynebacterium glutamicum 13032 is wild-type reference cultures, in the time of the LB of coating transformation system flat board, do not add amino acid mixing liquid.
The preparation method of LB liquid nutrient medium (PH 7.2): 10g Tryptones, 5g yeast extract, 10g sodium-chlor and distilled water are fully mixed and be settled to 1L with distilled water.
The LB substratum preparation of auxotroph brevibacterium flavum AN78: by 10 g Tryptoness, 5 g yeast extracts, 10g sodium-chlor with distilled water fully mixes and be settled to 1L with distilled water, add 0.25% His, Pro, Met amino acid mixing liquid 3ml.
Described plasmid to be transformed specifically can be shuttle plasmid pXMJ19 and its recombinant plasmid.
The auxotroph brevibacterium flavum AN78 of described wild-type Corynebacterium glutamicum or process mutagenesis can be the Corynebacterium glutamicum (or brevibacterium flavum) without genetic engineering bacterium transformation.The bacterial strain of described Corynebacterium glutamicum is ATCC 13032, and auxotrophic strain is brevibacterium flavum AN78.
The proportioning of described conversion and described competent cell is: 100 ng-5 μ g(as 100 ng-2 μ g or 2-5 μ g) as described in conversion plasmid: 0.2 Χ 10
9cFU-0.49 Χ 10
9cFU(is as 0.2 × 10
9cFU-0.31 × 10
9cFU or 0.31 × 10
9cFU-0.49 × 10
9cFU competent cell).
Described centrifugal condition specifically can be: 5000 r/min, centrifugal 10 min.
In the whole conversion process of described plasmid transfered cell, all under the condition in ice bath.
Arbitrary described oscillating condition can adopt as follows above: 150-220 rpm, as 150-180 rpm or 180-220 rpm, the radius of described vibration specifically can be 12 mm.
Brevibacterium flavum has part cell in exponential phase of growth and enters competence, especially in oligotrophic substratum, easily forms competent cell, for example minimal medium taking glucose as sole carbon source.The present invention is directed to conventional brevibacterium flavum (or Corynebacterium glutamicum) method for transformation, rely on the Natural Transformation principle of brevibacterium flavum or Corynebacterium glutamicum, clear thinking, easy and simple to handle, changing effect is good, for the further genetic manipulation of bacterial strain is laid the first stone.The present invention can apply on excellent bacillus class transforms, and has a extensive future.
Brief description of the drawings
Fig. 1 .pXMJ19 plasmid structural representation.
Fig. 2 .pXMJ19-argH plasmid structural representation.
Fig. 3. for the plasmid pXMJ19 of the extracting in embodiment 1 and
ecoRi or
bamHi single endonuclease digestion qualification result.1 swimming lane is
ecoRthe plasmid pXMJ19 of I single endonuclease digestion; 2 swimming lanes are
bamHthe plasmid pXMJ19 of I single endonuclease digestion; 3 swimming lanes are the plasmid pXMJ19 that the not enzyme of extracting is cut; 4 swimming lanes are Marker, are respectively from top to bottom 10000 bp, 7000 bp, 4000 bp, 2000 bp, 1000 bp, 500 bp, 250 bp.
Fig. 4. for the recombinant plasmid pXMJ19-argH of extracting in embodiment 2 and
ecoRi and
bamHi double digestion qualification result.1 swimming lane is Marker, is respectively from top to bottom 10000 bp, 7000 bp, 4000 bp, 2000 bp, 1000 bp, 500 bp, 250 bp; 2 swimming lanes are
ecoRthe plasmid pXMJ19-argH of I single endonuclease digestion; 3 swimming lanes are
ecoRi and
bamHi double digestion recombinant plasmid pXMJ19-argH; 4 swimming lanes are the plasmid of extracting, and 5 swimming lanes are
bamHthe plasmid pXMJ19-argH of I single endonuclease digestion.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from conventional reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.In this patent " h " all representatives hour." min " in this patent all represents minute.
Corynebacterium glutamicum ATCC 13032: purchased from Fu Xiang bio tech ltd, Shanghai; Auxotroph brevibacterium flavum AN78 is that preserve in this laboratory.
Fig. 1 is shown in shuttle plasmid pXMJ19(structural representation): purchased from Shaoxing Institute of Technology of College of Engineering, Peking University.
The recombinant plasmid pXMJ19-argH that shuttles back and forth, this laboratory builds, and structural representation is shown in Fig. 2.
The preparation method of LB liquid nutrient medium (PH 7.2): by 10 g Tryptoness, 5 g yeast extracts, 10 g sodium-chlor with distilled water fully mixes and be settled to 1L with distilled water; 121 DEG C of high-temperature sterilization 20 min.
The preparation method of LB solid medium (PH 7.2): by 10 g Tryptoness, 5 g yeast extracts, 10 g sodium-chlor, 15g agar powder with distilled water fully mixes and be settled to 1L with distilled water; 121 degree high-temperature sterilization 20 min.
The preparation method of 10 × inorganic salt mother liquor (PH 7.0): 10 g ammonium sulfate, 60 g dipotassium hydrogen phosphates, 10 g trisodium citrates and 2g bitter salt are dissolved with distilled water and be settled to 1L, 121 DEG C of high-temperature sterilization 20 min.
The preparation method of 20% glucose solution: 20g glucose is dissolved and is settled to 100 ml with distilled water, used the filtration sterilization of 0.22um biofilter, be stored in 4 DEG C of refrigerators.
The preparation method of 2% casein hydrolysate solution: by 2g casein hydrolysate, dissolve and be settled to 100ml with distilled water, with 0.22 μ m biofilter filtration sterilization.
Embodiment 1 shuttle plasmid imports the method for transformation of wild-type Corynebacterium glutamicum ATCC 13032
The preparation of growth medium and inducing culture
The preparation method of growth medium (1L): by 10 × inorganic salt mother liquor, 20% glucose solution, 2% casein hydrolysate solution and distilled water mix, and obtain growth medium GM I; The solvent of growth medium is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 2 g/L, dipotassium hydrogen phosphate 14.8 g/L, trisodium citrate 1.9 g/L, magnesium sulfate 0.098 g/L, glucose 5 g/L, casein hydrolysate 0.2 g/L.
The preparation method of inducing culture (1L): by 10 × inorganic salt mother liquor, 0.5 ml 0.1M CaCl
2, 1ml 25 mM MgCl
2, 20 % glucose solutions and distilled water mix, and obtain inducing culture GM II; The solvent of inducing culture is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 2 g/L, dipotassium hydrogen phosphate 14.8 g/L, trisodium citrate 1.9 g/L, magnesium sulfate 0.098 g/L, glucose 5 g/L.
The preparation of competent cell is as being shown in foregoing invention content.
Shuttle plasmid is transformed and imports Corynebacterium glutamicum, and experiment flow is as being shown in foregoing invention content.
The checking of changing effect
Random picking transforms the form single bacterium colony close with Corynebacterium glutamicum ATCC 13032 of dull and stereotyped upper growth, carrying out purifying cultivation containing random picking 10 strains on the LB solid medium of 30 μ g/ml paraxin, wherein transform bacterial strain each 5 strains respectively of pXMJ19-argH and pXMJ19 plasmid, obtain altogether the bacterial strain of 10 strain pure cultures.
By the pure culture bacterial strain obtaining, containing in the LB liquid nutrient medium of 30 μ g/ml paraxin, 37 DEG C, 180rpm shaking culture spend the night.
Take into culture system extracting plasmid, a part of recombinant plasmid pXMJ19-argH is go forward side by side performing PCR amplification of template, and electrophoresis detection result, then checks order amplified production; The plasmid pXMJ19 of another part extracting and recombinant plasmid pXMJ19-argH electrophoresis detection are also used restriction enzyme
ecoRi or
bamHi carries out single endonuclease digestion qualification, and recombinant plasmid pXMJ19-argH carries out
ecoRi and
bamHthe checking of I double digestion.This bacterial strain is for successfully importing shuttle plasmid pXMJ19-argH and pXMJ19 in Corynebacterium glutamicum ATCC 13032 cells.
ecoRi and
bamHi single endonuclease digestion checking plasmid pXMJ19(is shown in Fig. 3), result shows, successful shuttle plasmid transforms and has imported in the cell of Corynebacterium glutamicum ATCC 13032.
The 10 strain pure culture bacterial strains that obtain, detect through extracting plasmid, agarose gel electrophoresis, (cell that wherein imports pXMJ19-argH is two strains 6 strains, be four strains and import the cell of pXMJ19) be the recombinant bacterium that successfully shuttle vectors pXMJ19-argH or pXMJ19 is imported to Corynebacterium glutamicum 13032, transformation efficiency is 60%.
Embodiment 2 imports shuttle plasmid the method for transformation of auxotroph brevibacterium flavum AN78
The preparation of growth medium GM I and inducing culture GM II
The preparation method of growth medium (1 L) GM I: by 10 Χ inorganic salt mother liquors, 20% glucose solution, 2% casein hydrolysate solution and distilled water mix, and obtain growth medium; The solvent of growth medium is distilled water, solute and concentration thereof are as follows: ammonium sulfate 2 g/L, dipotassium hydrogen phosphate 14.8 g/L, trisodium citrate 1.9 g/L, magnesium sulfate 0.098 g/L, glucose 5 g/L, casein hydrolysate 0.2 g/L, 0.25% amino acid mixing liquid (His, Met, Pro).
The preparation method of inducing culture (1 L) GM II: by 10 × inorganic salt mother liquor, 20% glucose solution and distilled water mix, and obtain inducing culture; The solvent of growth medium is distilled water, solute and concentration thereof are as follows: ammonium sulfate 2g/L, dipotassium hydrogen phosphate 14.8 g/L, trisodium citrate 1.9 g/L, magnesium sulfate 0.098 g/L, glucose 5 g/L, 0.25% amino acid mixing liquid (His, Met, Pro).
The preparation of competent cell is as being shown in foregoing invention content.
Shuttle plasmid is transformed and imports auxotroph brevibacterium flavum AN78, and experiment flow is as being shown in foregoing invention content.
The checking of changing effect
The form single bacterium colony close with auxotroph brevibacterium flavum AN78 of growing on the flat board of random picking step 3, carrying out purifying cultivation containing random picking 10 strains on the LB solid medium of 30 μ g/ml paraxin, obtains the bacterial strain of 10 strain pure cultures altogether.
By the pure culture bacterial strain obtaining, containing in the LB liquid nutrient medium of 30 μ g/ml paraxin, 37 DEG C, 180 rpm shaking culture spend the night.
Take into culture system extracting plasmid, a part of recombinant plasmid pXMJ19-argH is go forward side by side performing PCR amplification of template, and electrophoresis detection result, then checks order amplified production; The plasmid pXMJ19 of another part extracting and recombinant plasmid pXMJ19-argH electrophoresis detection are also used restriction enzyme
ecoRi or
bamHi carries out single endonuclease digestion qualification, and recombinant plasmid pXMJ19-argH carries out
ecoRi and
bamHthe checking of I double digestion.This bacterial strain is for successfully importing shuttle plasmid pXMJ19-argH and pXMJ19 in auxotroph brevibacterium flavum AN78 cell, and the recombinant plasmid pXMJ19-argH of extracting is through double digestion
ecoRi and B
amHin I, result has the band of two treaty 6600bp and 1400bp to occur (the results are shown in Figure 4), proves that shuttle plasmid successfully transforms to have imported in auxotroph brevibacterium flavum AN78.
The 10 strain pure culture bacterial strains that above-mentioned steps obtains are successfully shuttle vectors pXMJ19-argH and pXMJ19 are imported in auxotroph brevibacterium flavum AN78 cell, and transformation efficiency is 100%.
Claims (6)
1. on special substratum, make under the competent situation of bacterial cell, the plasmid of shuttling expressing in intestinal bacteria and brevibacterium flavum (or Corynebacterium glutamicum) can be transformed to the method that enters into auxotroph brevibacterium flavum (or Corynebacterium glutamicum), comprise and prepare competent cell and transform two steps.
2. the substratum that prepared by competent cell as claimed in claim 1, is characterized in that formula:
10ml GMI:
1ml 10×spizizen salts，
0.1ml 10% yeast powder,
0.25ml 20% glucose,
0.2ml 1% caseinhydrolysate,
0.2ml 0.25% is amino acid needed,
Supplementing sterile purified water to cumulative volume is 10ml;
10ml GMII:
1ml 10×spizizen salts，
0.05ml 10% yeast powder,
0.25ml 20% glucose,
0.04ml 1% caseinhydrolysate,
0.2ml 0.25% is amino acid needed,
0.05ml 0.1M CaCl
2，
1ml 25mM MgCl
2，
Supplementing sterile purified water to cumulative volume is 10ml;
10×spizizen salts（100ml）：
15% K
2HPO
4·3H
2O， 15g 3g
6% KH
2PO
4， 6g 1.2g
2% (NH
4)
2SO
4， 2g 0.4
0.2% MgSO
4， 0.2g 0.04
1% Trisodium Citrate, 1g 0.2
Be dissolved in distilled water 6.6 × 10
4pa autoclaving, room temperature preservation; Auxotrophic bacterial strain, added reagent is amino acid, non-auxotrophic bacterial strain is used amino acid instead sterilized water and is replaced; By first fast breeding on GMI substratum of auxotroph brevibacterium flavum (or Corynebacterium glutamicum), reach after logarithmic phase, GMII on the substratum that the nutrition of transferring lacks relatively, make cell in starvation, be equivalent under the competent condition of cell, after plasmid transfered cell, can reduce brevibacterium flavum (or Corynebacterium glutamicum) and the restriction enzyme of plasmid be cut to effect, genetic stability.
3. the method as described in as arbitrary in claim 1 to 2, is characterized in that: the proportioning of described plasmid to be transformed and described competent cell is: plasmid to be transformed described in 100ng-5 μ g: 0.2 × 10
9cFU-0.49 × 10
9cFU competent cell.
4. as the method as described in arbitrary in claims 1 to 3, it is characterized in that: described plasmid to be transformed is shuttle plasmid pXMJ19, pXMJ19-argH.
5. the method as described in as arbitrary in claim 1 to 4, is characterized in that: the whole experiment flow of described conversion is all under the condition in ice bath.
6. as the method as described in arbitrary in claim 1 to 4, it is characterized in that: described auxotroph brevibacterium flavum is through the Histidine of mutagenesis, proline(Pro), methionine(Met) auxotrophic strain; Described Corynebacterium glutamicum ATCC 13032 is reference cultures.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755056A (en) * | 2016-12-14 | 2017-05-31 | 吴银娣 | The Pichia pastoris that a kind of acid stress resistance is improved |
| CN110016450A (en) * | 2019-04-28 | 2019-07-16 | 江南大学 | One plant of brevibacterium flavum for producing L-PROLINE and its application |
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| JP2006067884A (en) * | 2004-09-01 | 2006-03-16 | Kao Corp | Recombinant bacterium of genus bacillus |
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2013
- 2013-01-29 CN CN201310033531.6A patent/CN103966251A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2006067884A (en) * | 2004-09-01 | 2006-03-16 | Kao Corp | Recombinant bacterium of genus bacillus |
Non-Patent Citations (3)
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| DAQING XU ET AL.: "Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer", 《JOURNAL OF MICROBIOLOGICAL METHODS》, vol. 80, 31 December 2010 (2010-12-31), XP026807602 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106755056A (en) * | 2016-12-14 | 2017-05-31 | 吴银娣 | The Pichia pastoris that a kind of acid stress resistance is improved |
| CN110016450A (en) * | 2019-04-28 | 2019-07-16 | 江南大学 | One plant of brevibacterium flavum for producing L-PROLINE and its application |
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Application publication date: 20140806 |