CN102649967B - Method for transforming wild-type bacillus subtilis - Google Patents
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Abstract
本发明公开了转化野生型枯草芽孢杆菌的方法。本发明提供的方法包括制备感受态细胞和电击转化两个步骤；所述制备感受态细胞依次包括如下步骤：（1）将野生型枯草芽孢杆菌接种至生长培养基，使野生型枯草芽孢杆菌的初始浓度为1.8×107-7.7×107CFU/mL，35-37℃振荡培养2-3.5h；（2)将1体积份完成步骤(1)的培养体系与0.8-1.2体积份诱导培养基混匀，35-37℃振荡培养1.5-3.5h；所述电击转化包括如下步骤：将待转化的质粒与所述感受态细胞混匀并冰浴，然后采用1.2-1.8kV的电压进行电击，得到导入所述待转化质粒的重组枯草芽孢杆菌。本发明提供的方法，转化效果好，操作简便。The invention discloses a method for transforming wild-type Bacillus subtilis. The method provided by the present invention includes two steps of preparing competent cells and electric shock transformation; the preparation of competent cells includes the following steps in turn: (1) inoculating wild-type Bacillus subtilis into the growth medium, so that the wild-type Bacillus subtilis The initial concentration is 1.8×10 7 -7.7×10 7 CFU/mL, shake culture at 35-37°C for 2-3.5 hours; (2) Mix 1 volume part of the culture system that completed step (1) with 0.8-1.2 volume parts of induction culture mix well, shake culture at 35-37°C for 1.5-3.5h; the electric shock transformation includes the following steps: mix the plasmid to be transformed with the competent cells and put them in an ice bath, and then use a voltage of 1.2-1.8kV for electric shock , to obtain the recombinant Bacillus subtilis introduced into the plasmid to be transformed. The method provided by the invention has good conversion effect and simple operation.
Description
Technical field
The present invention relates to bacterium and transform field, be specifically related to a kind of method that transforms wild-type subtilis.
Background technology
Subtilis is a kind of gram positive bacterium, extensively be present in occurring in nature, a lot of plant endogenesis and soil are practised and occupied subtilis is the potential biocontrol of plant disease factor, set up corresponding transformation system and be to bacterial strain carry out fluorescent mark with in-situ monitoring its surely grow, the prerequisite of the regularity of distribution.Conversion refers to the process of DNA in bacterium picked-up external environment, and bacterium can adapt to complicated external environment by Natural Transformation, and the generation of conversion may cause the phenomenons such as gene clone, mutant generation and gene mapping.
Subtilis is shifted to new management mechanisms to study and shows, after cell induction outer signals, through a series of regulatory pathway induction competence, form the generation of factor ComK, ComK regulates and controls a series of later stage competence again and forms gene (late competence genes) expression, on these genes encoding cytolemma, identify and accept in the complex body of foreign DNA and born of the same parents the products such as protective enzyme, the generation of current all conversions all needs cell in impression state, so just may transform successfully, therefore the good competent cell of processability is to realize the committed step that subtilis transforms.
Tradition method for transformation is difficult to realize the artificial conversion of wild-type subtilis under laboratory condition, and many bacterial strains that success transforms are through genetic improvement or have adapted to the bacterial strain of laboratory condition.For wild-type subtilis is artificial, transform, existing method is mainly Natural Transformation method, protoplast transformation method and tradition electric shock conversion method etc.Natural Transformation method is simple to operate, but success ratio is extremely low, and it is very crucial that the substratum inducing cell in the method enters this step of impression state, adds that the probability of Natural Transformation occurs after plasmid is very low.Protoplast transformation method transformation efficiency is relatively high, but complex steps, adds the steps such as the regeneration of the treatment process of the amount of N,O-Diacetylmuramidase and treatment time, polyoxyethylene glycol (PEG) and treatment time, cell walls and transformant screening very crucial, is difficult to hold.Tradition electric shock conversion method is used more, operates relatively easyly, but transformation efficiency is not high, and voltage (setting of electric capacity and resistance) and time constant are key parameters.
Summary of the invention
The object of this invention is to provide a kind of method that transforms wild-type subtilis.
The method of conversion wild-type subtilis provided by the invention, comprises and prepares competent cell and two steps of electric shock conversion;
The described competent cell of preparing in turn includes the following steps:
(1) wild-type subtilis is seeded to growth medium, the starting point concentration that makes wild-type subtilis is 1.8 * 10
7-7.7 * 10
7cfu/mL(is as 1.8 * 10
7-5 * 10
7cFU/mL or 5 * 10
7-7.7 * 10
7cFU/mL), 35-37 ℃ (as 35-36 ℃ or 36-37 ℃) shaking culture 2-3.5h(is as 2-3h or 3-3.5h); The solvent of described growth medium is water (as distilled water), and solute and concentration thereof are as follows: ammonium sulfate 1.8-2.2g/L(is as 1.8-2g/L or 2-2.2g/L), dipotassium hydrogen phosphate 14-16g/L(is as 14-14.8g/L or 14.8-16g/L), trisodium citrate 1.5-2.5g/L(is as 1.5-1.9g/L or 1.9-2.5g/L), magnesium sulfate 0.09-0.1g/L(is as 0.09-0.098g/L or 0.098-0.1g/L), glucose 4.8-5.2g/L(is as 4.8-5g/L or 5-5.2g/L) and casein hydrolysate 0.18-0.22g/L(as 0.18-0.2g/L or 0.2-0.22g/L);
(2) culture system of 1 parts by volume completing steps (1) and 0.8-1.2 parts by volume (as 0.8-1 parts by volume or 1-1.2 parts by volume) inducing culture are mixed, 35-37 ℃ of (as 35-36 ℃ or 36-37 ℃) shaking culture 1.5-3.5h(is as 1.5-3h or 3-3.5h); The solvent of described inducing culture is water (as distilled water), and solute and concentration thereof are as follows: ammonium sulfate 1.8-2.2g/L(is as 1.8-2g/L or 2-2.2g/L), dipotassium hydrogen phosphate 14-16g/L(is as 14-14.8g/L or 14.8-16g/L), trisodium citrate 1.5-2.5g/L(is as 1.5-1.9g/L or 1.9-2.5g/L), magnesium sulfate 0.09-0.1g/L(is as 0.09-0.098g/L or 0.098-0.1g/L) and glucose 4.8-5.2g/L(as 4.8-5g/L or 5-5.2g/L);
Described electric shock transforms and comprises the steps: plasmid to be transformed and described competent cell to mix also ice bath, then adopt 1.2-1.8kV(as 1.2-1.5kV or 1.5-1.8kV) voltage shock by electricity, obtain importing the recombined bacillus subtilis of described plasmid to be transformed.
The proportioning of described plasmid to be transformed and described competent cell is: 100ng-5 μ g(is as 100ng-2 μ g or 2-5 μ g) as described in plasmid to be transformed: 0.2 * 10
9cFU-0.49 * 10
9cFU(is as 0.2 * 10
9cFU-0.31 * 10
9cFU or 0.31 * 10
9cFU-0.49 * 10
9cFU) competent cell.
The described competent cell of preparing also comprises the steps (3): will complete the culture system ice bath 30min of described step (2), then 4 ℃ of centrifugal collecting cells.
Described centrifugal condition specifically can be: the centrifugal 10min of 5000r/min.
The described competent cell of preparing has also comprised the steps: after described step (3), use successively cell described in the distilled water of 4 ℃ of precoolings and the HEPES buffer solution for cleaning of 4 ℃ of precoolings, then use 4 ℃ of precooling HEPES glycerine damping fluid re-suspended cells, be competent cell.
The preparation method of HEPES damping fluid (1mM, pH7.0): 0.238g HEPES is dissolved with distilled water and be settled to 1L.
The preparation method of HEPES glycerine damping fluid (1mM, pH7.0): 0.238g HEPES pressed powder, 100mL glycerine and distilled water are fully mixed and be settled to 1L with distilled water.
During described electric shock transforms, the time of described ice bath specifically can be 30min.
During described electric shock transforms, the resistance of described electric shock specifically can be 200 Ω, electric capacity and specifically can be 25 μ F.
Described electric shock transforms and also comprise the described step being handled as follows after electric shock: in electric shock cup, add 700-1000 μ L(as 700-800 μ L or 800-1000 μ L) the LB liquid nutrient medium of 30-37 ℃ preheating mixing, then 30-37 ℃ of shaking culture 3h.
The preparation method of LB liquid nutrient medium (pH7.2): 10g Tryptones, 5g yeast extract, 10g sodium-chlor and distilled water are fully mixed and be settled to 1L with distilled water.
Described plasmid to be transformed specifically can be shuttle vectors pBE2, shuttle vectors pS4GFP or shuttle vectors pMarA.
Described wild-type subtilis can be and not pass through genetically engineered subtilis.Described subtilis specifically can be subtilis 9407.
Arbitrary described vibration specifically can adopt following condition: 150-220r/min above, as 150-180r/min or 180-220r/min.The radius of described vibration specifically can be 12mm.
Subtilis enters competence at can the have an appointment cell of 10-20% of exponential growth later stage, especially in oligotrophic substratum, easily forms competent cell, the minimal medium that the glucose of for example take is sole carbon source.Existing complex operation while the present invention is directed to conventional subtilis method for transformation for wild-type subtilis, the problem such as success ratio is low, the cycle is long, transformation efficiency is low, a kind of simple and easy to do wild-type subtilis rapid conversion novel method is provided, the method is relied on the Natural Transformation principle of subtilis, clear thinking, easy and simple to handle, changing effect is good, in order bacterial strain is carried out to fluorescent mark, with in-situ monitoring, it is grown surely, the regularity of distribution lays a good foundation, for further genetic manipulation lays the first stone.The present invention can apply on wild-type subtilis transforms, and has a extensive future.
Accompanying drawing explanation
Fig. 1 is the structural representation of shuttle vectors pBE2.
Fig. 2 is the EcoR I single endonuclease digestion qualification result in embodiment 1; 1 is shuttle vectors pBE2(positive control), 2 and 3 are respectively the plasmid extracting from two different pure culture bacterial strains, and M is 1kb DNA ladder.
Fig. 3 is the structural representation of shuttle vectors pMarA.
Fig. 4 is the Pst I in embodiment 2, Kpn I and EcoR I single endonuclease digestion qualification result; 3,6,9 is shuttle vectors pMarA(positive control), 1,4,7 is certain plasmid to be verified, and 2,5,8 is distilled water (blank), and M is 1kb DNA ladder.
Fig. 5 is the fluorescence microscope result in embodiment 3.
Fig. 6 is EcoR I single endonuclease digestion and the EcoR I/Hind III double digestion qualification result in embodiment 3; 1,4 is carrier pS4GFP(positive control), 2 and 5 is a plasmid to be measured, and 2 identify for single endonuclease digestion, and 4 identify for double digestion, and 3 and 6 is an another plasmid to be measured, and 3 identify for single endonuclease digestion, and 6 identify for double digestion, and M is 1kb DNA ladder.
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 routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times." h " in this patent all represents hour." min " in this patent all represents minute.
Subtilis (Bacillus subtilis) 9407: and the preservation separated with microecology laboratory by China Agricultural University's biocontrol of plant disease, public Ke Cong China Agricultural University obtains; Reference: round, Fu Xuechi, Chen Xinyi, Wang Qi. the control of genus bacillus biological prevention and control agent to apple disease. Plant Pathology .2011.41 (ZK): 135-140.
Shuttle vectors pBE2(structural representation is shown in Fig. 1): public Ke Cong China Agricultural University obtains; Reference: Tian Tao, QiXue Chen, Wang Qi, Mei Ruhong. genus bacillus Green Fluorescent Protein and the pre-test of surely growing at wheat body surface thereof. Plant Pathology .2004.34 (4): 346-351.
Shuttle vectors pS4GFP(genus bacillus and shuttle vehicle are transformed and are obtained on pGFP4412 plasmid basis, and pGFP4412 plasmid is transformed and obtained on shuttle vectors pBE2 basis): public Ke Cong China Agricultural University obtains; Reference: Fan Xiaojing, Qiu Sixin, Wu little Ping, Hong Yongcong, Cai Xueqing, Hu Fangping. labeled with green fluorescent protein gene endophytic Bacillus subtilis. application and environmental organism journal .2007.13 (4): 530-534.
Shuttle vectors pMarA(genus bacillus and shuttle vehicle, structural representation is shown in Fig. 3): public Ke Cong China Agricultural University obtains; Reference: Yoann Le Breton, Nrusingh Prasad Mohapatra, W.G.Haldenwang.In Vivo Random Mutagenesis of Bacillus subtilis by Use of TnYLB-1, a mariner-Based Transposon.Applied and Environmental Microbiology.2006.72 (1): 327-333..
The preparation method of LB liquid nutrient medium (pH7.2): 10g Tryptones, 5g yeast extract, 10g sodium-chlor and distilled water are fully mixed and be settled to 1L with distilled water; 121 ℃ of high-temperature sterilization 20min.
The preparation method of LB solid medium (pH7.2): 10g peptone, 5g yeast extract, 10g sodium-chlor, 15g agar powder and distilled water are fully mixed and be settled to 1L with distilled water; 121 ℃ of high-temperature sterilization 20min.
The preparation method of 5 * inorganic salt mother liquor (pH7.0): 10g ammonium sulfate, 74g dipotassium hydrogen phosphate, 9.5g trisodium citrate and 1.0g bitter salt are dissolved with distilled water and be settled to 1L; 121 ℃ of high-temperature sterilization 20min.
The preparation method of 20% glucose solution: 20g glucose is dissolved with distilled water and be settled to 100mL; With 0.22 μ m biofilter filtration sterilization.
The preparation method of 2% casein hydrolysate solution: 2g casein hydrolysate (Casamino acids) is dissolved with distilled water and be settled to 100mL, with 0.22 μ m biofilter filtration sterilization.
The preparation method of HEPES damping fluid (1mM, pH7.0): 0.238g HEPES is dissolved with distilled water and be settled to 1L; With 0.22 μ m biofilter filtration sterilization, be stored in 4 ℃.
The preparation method of HEPES glycerine damping fluid (1mM, pH7.0): 0.238g HEPES pressed powder, 100mL glycerine and distilled water are fully mixed and be settled to 1L with distilled water; With 0.22 μ m biofilter filtration sterilization, be stored in 4 ℃.
The conversion of embodiment 1, wild-type subtilis
One, the preparation of growth medium and inducing culture
The preparation method of growth medium (1L): 5 * inorganic salt mother liquor, 20% glucose solution, 2% casein hydrolysate solution and distilled water are mixed, obtain growth medium; The solvent of growth medium is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 2g/L, dipotassium hydrogen phosphate 14.8g/L, trisodium citrate 1.9g/L, magnesium sulfate 0.098g/L, glucose 5g/L, casein hydrolysate 0.2g/L.
The preparation method of inducing culture (1L): 5 * inorganic salt mother liquor, 20% glucose solution and distilled water are mixed, obtain inducing culture; The solvent of inducing culture is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 2g/L, dipotassium hydrogen phosphate 14.8g/L, trisodium citrate 1.9g/L, magnesium sulfate 0.098g/L, glucose 5g/L.
Two, the preparation of competent cell
1, take out the subtilis 9407 preserving in-80 ℃ of refrigerators, on LB solid medium, activate.
2, with the single bacterium colony after the activation of sterilizing toothpick picking, be inoculated in 5mL growth medium 37 ℃, 180r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 10h.
3, the bacterium liquid of getting 2mL step 2 is inoculated in that in 15mL growth medium, (starting point concentration of subtilis 9407 is 5 * 10
7cFU/mL), 37 ℃, 180r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3h.
4, the culture system of 1 parts by volume completing steps 3 and 1 parts by volume inducing culture are mixed to 37 ℃, 180r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 1.5h.
5, by the culture system ice bath 30min of completing steps 4, then add in the 50mL centrifuge tube of 4 ℃ of precoolings, 4 ℃, 5000r/min are centrifugal, and (centrifugal radius is 7.07cm, instrument model is Himac CR22e, Japan Hitachi company product) 10min, abandons supernatant, collecting cell.
6, the cell obtaining by liquid-transfering gun compressing cleaning step 5 with the distilled water of 4 ℃ of precoolings of 30mL, 4 ℃, 5000r/min centrifugal (centrifugal radius is 7.07cm, and instrument model is Himac CR22e, Japanese Hitachi company product) 10min, abandon supernatant, collecting cell.
7, the cell obtaining by liquid-transfering gun compressing cleaning step 6 with the distilled water of 4 ℃ of precoolings of 30mL, 4 ℃, 5000r/min centrifugal (centrifugal radius is 7.07cm, and instrument model is Himac CR22e, Japanese Hitachi company product) 10min, abandon supernatant, collecting cell.
8, the cell obtaining by liquid-transfering gun compressing cleaning step 7 with the 30mL HEPES damping fluid of 4 ℃ of precoolings, 4 ℃, 5000r/min centrifugal (centrifugal radius is 7.07cm, and instrument model is Himac CR22e, Japanese Hitachi company product) 10min, abandon supernatant, collecting cell.
9, the cell obtaining by the resuspended step 8 of HEPES glycerine damping fluid of 4 ℃ of precoolings of 2mL, is sub-packed in that to drop at once liquid nitrogen middling speed after the 1.5mL centrifuge tube (200 μ L/ pipe) of 4 ℃ of precoolings cold.Every pipe is containing having an appointment 0.31 * 10
9cFU competent cell.
Above-mentioned steps 5 to 9 is all carried out on ice.
Three, electric shock transforms
1, get 5 μ g shuttle vectors pBE2, add in the competent cell that 1 pipe step 2 obtains, on ice, by liquid-transfering gun, slightly tell to inhale shuttle vectors pBE2 and competent cell are fully mixed, ice bath 30min.
2, the system of completing steps 1 is transferred in the electric shock cup of precooling on ice (2mm), with BioRad Gene PulserXcell electroporation apparatus, carries out pulse electric shock (resistance 200 Ω, electric capacity 25 μ F, voltage 1.2kV).
3,, after completing steps 2, to the LB liquid nutrient medium that adds 37 ℃ of preheatings of 800 μ L in electric shock cup, by liquid-transfering gun, slightly tell persorption even immediately.
4, after completing steps 3, by the whole sucking-offs of bacterium liquid in electric shock cup to centrifuge tube, 37 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3h.
5, the culture system of completing steps 4 is coated containing on the LB solid medium of 50 μ g/mL kantlex to 37 ℃ of standing cultivations.
Four, the checking of changing effect
The form single bacterium colony close with subtilis 9407 of 1, growing on the flat board of random picking step 3, carrying out purifying cultivation containing on the LB solid medium of 50 μ g/mL kantlex, obtains the bacterial strain of 8 strain pure cultures altogether.
The bacterial strain of the pure culture 2, step 2 being obtained is containing in the LB liquid nutrient medium of 50 μ g/mL kantlex, and 37 ℃, 180r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) spend the night.
3, get the culture system of completing steps 2, a part is extracted genomic dna and is identified that 16S rDNA fragment (adopts the primer of 63F and 1387R composition to carry out pcr amplification, then pcr amplification product checked order; 63F:5 '-CAGGCCTAACACATGCAAGTC-3 '; 1387R:5 '-GGGCGGWGTGTACAAGGC-3 '), another part extracts plasmid and carries out single endonuclease digestion evaluation by restriction enzyme EcoR I, if 16S rDNA sequencing fragment is consistent with 16S rDNA fragment sequence (as shown in the sequence 1 of sequence table) the EcoR I single endonuclease digestion qualification result consistent and EcoR I single endonuclease digestion qualification result and shuttle vectors pBE2 of subtilis 9407, this bacterial strain is successfully shuttle vectors pBE2 to be imported to the recombinant bacterium of subtilis 9407.Part EcoR I single endonuclease digestion the results are shown in Figure 2.
The 8 strain pure culture bacterial strains that step 1 obtains are the recombinant bacterium that successfully shuttle vectors pBE2 is imported to subtilis 9407, and transformation efficiency is 100%.
The conversion of embodiment 2, wild-type subtilis
One, the preparation of growth medium and inducing culture
The preparation method of growth medium (1L): 5 * inorganic salt mother liquor, 20% glucose solution, 2% casein hydrolysate solution and distilled water are mixed, obtain growth medium; The solvent of growth medium is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 1.8g/L, dipotassium hydrogen phosphate 14g/L, trisodium citrate 1.5g/L, magnesium sulfate 0.09g/L, glucose 4.8g/L, casein hydrolysate 0.18g/L.
The preparation method of inducing culture (1L): 5 * inorganic salt mother liquor, 20% glucose solution and distilled water are mixed, obtain inducing culture; The solvent of inducing culture is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 1.8g/L, dipotassium hydrogen phosphate 14g/L, trisodium citrate 1.5g/L, magnesium sulfate 0.09g/L, glucose 4.8g/L.
Two, the preparation of competent cell
1, with 1 of the step 2 of embodiment 1.
2, with the single bacterium colony after the activation of sterilizing toothpick picking, be inoculated in 5mL growth medium 30 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 8h.
3, the bacterium liquid of getting 2mL step 2 is inoculated in that in 15mL growth medium, (starting point concentration of subtilis 9407 is 1.8 * 10
7cFU/mL), 35 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 2h.
4, the culture system of 1 parts by volume completing steps 3 and 1.2 parts by volume inducing cultures are mixed to 35 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3.5h.
5, with 5 of the step 2 of embodiment 1.
6, with 6 of the step 2 of embodiment 1.
7, with 7 of the step 2 of embodiment 1.
8, with 8 of the step 2 of embodiment 1.
9, with 9 of the step 2 of embodiment 1.Every pipe is containing having an appointment 0.2 * 10
9cFU competent cell.
Above-mentioned steps 5 to 9 is all carried out on ice.
Three, electric shock transforms
1, get 2 μ g shuttle vectors pMarA, add in the competent cell that 1 pipe step 2 obtains, on ice, by liquid-transfering gun, slightly tell to inhale shuttle vectors pMarA and competent cell are fully mixed, ice bath 30min.
2, the system of completing steps 1 is transferred in the electric shock cup of precooling on ice (2mm), with BioRad Gene Pulser Xcell electroporation apparatus, carries out pulse electric shock (resistance 200 Ω, electric capacity 25 μ F, voltage 1.8kV).
3,, after completing steps 2, to the LB liquid nutrient medium that adds 30 ℃ of preheatings of 700 μ L in electric shock cup, by liquid-transfering gun, slightly tell persorption even immediately.
4, after completing steps 3, by the whole sucking-offs of bacterium liquid in electric shock cup to centrifuge tube, 30 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3h.
5, the culture system of completing steps 4 is coated containing on the LB solid medium of 50 μ g/mL kantlex to 30 ℃ of standing cultivations.
Four, the checking of changing effect
The form single bacterium colony close with subtilis 9407 of 1, growing on the flat board of random picking step 3, carrying out purifying cultivation containing on the LB solid medium of 50 μ g/mL kantlex, obtains the bacterial strain of 8 strain pure cultures altogether.
The bacterial strain of the pure culture 2, step 2 being obtained is containing in the LB liquid nutrient medium of 50 μ g/mL kantlex, and 37 ℃, 180r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) spend the night.
3, get the culture system of completing steps 2, a part is extracted genomic dna and is identified that 16S rDNA fragment (adopts the primer of 63F and 1387R composition to carry out pcr amplification, then pcr amplification product checked order, 63F:5 '-CAGGCCTAACACATGCAAGTC-3 ', 1387R:5 '-GGGCGGWGTGTACAAGGC-3 '), another part extracts plasmid and uses respectively restriction enzyme Pst I, Kpn I and EcoR I are carried out single endonuclease digestion evaluation, if the consistent and Pst I of 16S rDNA fragment sequence (as shown in the sequence 1 of sequence table) of 16SrDNA sequencing fragment and subtilis 9407, Kpn I and EcoR I single endonuclease digestion qualification result all with the Pst I of shuttle vectors pMarA, Kppn I is consistent with EcoR I single endonuclease digestion qualification result, this bacterial strain is successfully shuttle vectors pMarA to be imported to the recombinant bacterium of subtilis 9407.The Pst I of certain plasmid, Kppn I and EcoR I single endonuclease digestion the results are shown in Figure 4.
In the 8 strain pure culture bacterial strains that step 1 obtains, 6 strains are successfully shuttle vectors pMarA to be imported to the recombinant bacterium of subtilis 9407, and transformation efficiency is 75%.
The conversion of embodiment 3, wild-type subtilis
One, the preparation of growth medium and inducing culture
The preparation method of growth medium (1L): 5 * inorganic salt mother liquor, 20% glucose solution, 2% casein hydrolysate solution and distilled water are mixed, obtain growth medium; The solvent of growth medium is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 2.2g/L, dipotassium hydrogen phosphate 16g/L, trisodium citrate 2.5g/L, magnesium sulfate 0.1g/L, glucose 5.2g/L, casein hydrolysate 0.22g/L.
The preparation method of inducing culture (1L): 5 * inorganic salt mother liquor, 20% glucose solution and distilled water are mixed, obtain inducing culture; The solvent of inducing culture is distilled water, and solute and concentration thereof are as follows: ammonium sulfate 2.2g/L, dipotassium hydrogen phosphate 16g/L, trisodium citrate 2.5g/L, magnesium sulfate 0.1g/L, glucose 5.2g/L.
Two, the preparation of competent cell
1, with 1 of the step 2 of embodiment 1.
2, with the single bacterium colony after the activation of sterilizing toothpick picking, be inoculated in 5mL growth medium 37 ℃, 220r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 12h.
3, the bacterium liquid of getting 2mL step 2 is inoculated in that in 15mL growth medium, (starting point concentration of subtilis 9407 is 7.7 * 10
7cFU/mL), 36 ℃, 220r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3.5h.
4, the culture system of 1 parts by volume completing steps 3 and 0.8 parts by volume inducing culture are mixed to 36 ℃, 220r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3h.
5, with 5 of the step 2 of embodiment 1.
6, with 6 of the step 2 of embodiment 1.
7, with 7 of the step 2 of embodiment 1.
8, with 8 of the step 2 of embodiment 1.
9, with 9 of the step 2 of embodiment 1.Every pipe is containing having an appointment 0.49 * 10
9cFU competent cell.
Above-mentioned steps 5 to 9 is all carried out on ice.
Three, electric shock transforms
1, get 100ng shuttle vectors pS4GFP, add in the competent cell that 1 pipe step 2 obtains, on ice, by liquid-transfering gun, slightly tell to inhale shuttle vectors pS4GFP and competent cell are fully mixed, ice bath 30min.
2, the system of completing steps 1 is transferred in the electric shock cup of precooling on ice (2mm), with BioRad Gene Pulser Xcell electroporation apparatus, carries out pulse electric shock (resistance 200 Ω, electric capacity 25 μ F, voltage 1.5kV).
3,, after completing steps 2, to the LB liquid nutrient medium that adds 37 ℃ of preheatings of 1000 μ L in electric shock cup, by liquid-transfering gun, slightly tell persorption even immediately.
4, after completing steps 3, by the whole sucking-offs of bacterium liquid in electric shock cup to centrifuge tube, 37 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3h.
5, the culture system of completing steps 4 is coated containing on the LB solid medium of 50 μ g/mL kantlex to 37 ℃ of standing cultivations.
Four, the checking of changing effect
The form single bacterium colony close with subtilis 9407 of 1, growing on the flat board of random picking step 3, carrying out purifying cultivation containing on the LB solid medium of 50 μ g/mL kantlex, obtains the bacterial strain of 16 strain pure cultures altogether.
2, the bacterial strain of pure culture is carried out to fluorescent microscope detection, can send the positive bacterial strain of bacterial strain of green fluorescence, obtain altogether 12 strain positive strains, partial results is shown in Fig. 5, can observe bacterial strain and send stronger fluorescence.
3, get the positive strain that step 2 obtains, containing in the LB liquid nutrient medium of 50 μ g/mL kantlex, 37 ℃, 180r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) spend the night.
4, get the culture system of completing steps 3, a part is extracted genomic dna and is identified that 16S rDNA fragment (adopts the primer of 63F and 1387R composition to carry out pcr amplification, then pcr amplification product checked order, 63F:5 '-CAGGCCTAACACATGCAAGTC-3 ', 1387R:5 '-GGGCGGWGTGTACAAGGC-3 '), another part extracts plasmid and carries out respectively EcoR I single endonuclease digestion and the evaluation of EcoR I/Hind III double digestion, if the 16S rDNA fragment sequence (as shown in the sequence 1 of sequence table) of 16S rDNA sequencing fragment and subtilis 9407 is consistent and EcoR I single endonuclease digestion and EcoR I/Hind III double digestion qualification result all consistent with EcoR I single endonuclease digestion and the EcoR I/Hind III double digestion qualification result of shuttle vectors pS4GFP, this bacterial strain is successfully shuttle vectors pS4GFP to be imported to the recombinant bacterium of subtilis 9407.The EcoR I single endonuclease digestion of part plasmid and EcoR I/Hind III double digestion the results are shown in Figure 6.
In the 16 strain pure culture bacterial strains that step 1 obtains, 12 strains are successfully shuttle vectors pS4GFP to be imported to the recombinant bacterium of subtilis 9407, and transformation efficiency is 75% (note: the pure culture bacterial strain that can send green fluorescence under 12 strain fluorescent microscopes is all accredited as target recombinant bacterium).
The conversion of comparative example 1, wild-type subtilis
One, the preparation of growth medium and inducing culture
Two, the preparation of competent cell
Three, electric shock transforms
Adopt respectively the voltage of 1.0kV and 2.0kV, other is all with the step 3 of embodiment 1.
Four, the checking of changing effect
While adopting the voltage of 1.0kV, step 1 obtains 5 strain pure culture bacterial strains, and wherein only having 1 strain is successfully shuttle vectors pBE2 to be imported to the recombinant bacterium of subtilis 9407, and transformation efficiency is 20%.
While adopting the voltage of 2.0kV, step 1 obtains 5 strain pure culture bacterial strains, and wherein only having 1 strain is successfully shuttle vectors pBE2 to be imported to the recombinant bacterium of subtilis 9407, and transformation efficiency is 20%.
The conversion of comparative example 2, wild-type subtilis
One, the preparation of growth medium and inducing culture
Two, the preparation of competent cell
Three, transform
1, get 5 μ g shuttle vectors pBE2, add in the competent cell that 1 pipe step 2 obtains, on ice, by liquid-transfering gun, slightly tell to inhale pBE2 plasmid and competent cell are fully mixed, ice bath 30min.
2, after completing steps 1, the system of step 1 is transferred in the electric shock cup of precooling on ice (2mm), to the LB liquid nutrient medium that adds 37 ℃ of preheatings of 800 μ L in electric shock cup, by liquid-transfering gun, slightly tells persorption even immediately.
3, after completing steps 2, by the whole sucking-offs of bacterium liquid in electric shock cup to centrifuge tube, 37 ℃, 150r/min shaking culture (vibration radius is 12mm, and instrument model is QDZ5-HZS-H) 3h.
4, the culture system of completing steps 3 is coated containing on the LB solid medium of 50 μ g/mL kantlex to 37 ℃ of standing cultivations.
Four, the checking of changing effect
Claims (7)
1. transform a method for wild-type subtilis, comprise and prepare competent cell and two steps of electric shock conversion;
The described competent cell of preparing in turn includes the following steps:
(1) wild-type subtilis is seeded to growth medium, the starting point concentration that makes wild-type subtilis is 1.8 * 10
7-7.7 * 10
7cFU/mL, 35-37 ℃ of shaking culture 2-3.5h; The solvent of described growth medium is water, and solute and concentration thereof are as follows: ammonium sulfate 1.8-2.2g/L, dipotassium hydrogen phosphate 14-16g/L, trisodium citrate 1.5-2.5g/L, magnesium sulfate 0.09-0.1g/L, glucose 4.8-5.2g/L and casein hydrolysate 0.18-0.22g/L;
(2) culture system of 1 parts by volume completing steps (1) and 0.8-1.2 parts by volume inducing culture are mixed to 35-37 ℃ of shaking culture 1.5-3.5h; The solvent of described inducing culture is water, and solute and concentration thereof are as follows: ammonium sulfate 1.8-2.2g/L, dipotassium hydrogen phosphate 14-16g/L, trisodium citrate 1.5-2.5g/L, magnesium sulfate 0.09-0.1g/L and glucose 4.8-5.2g/L;
Described electric shock transforms and comprises the steps: plasmid to be transformed and described competent cell to mix also ice bath, then adopts the voltage of 1.2-1.8kV to shock by electricity, and obtains importing the recombined bacillus subtilis of described plasmid to be transformed;
During described electric shock transforms, the resistance of described electric shock is that 200 Ω, electric capacity are 25 μ F.
2. the method for claim 1, 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.
3. method as claimed in claim 1 or 2, is characterized in that: the described competent cell of preparing also comprises the steps (3): will complete the culture system ice bath 30min of described step (2), then 4 ℃ of centrifugal collecting cells.
4. method as claimed in claim 1 or 2, is characterized in that: during described electric shock transforms, the time of described ice bath is 30min.
5. method as claimed in claim 1 or 2, it is characterized in that: described electric shock transforms and is also included in the step being handled as follows after described electric shock: in electric shock cup, add the LB liquid nutrient medium of L30-37 ℃ of preheating of 700-1000 μ and mix, then 30-37 ℃ of shaking culture 3h.
6. method as claimed in claim 1 or 2, is characterized in that: described plasmid to be transformed is shuttle vectors pBE2, shuttle vectors pS4GFP or shuttle vectors pMarA.
7. method as claimed in claim 1 or 2, is characterized in that: described wild-type subtilis is not for passing through genetically engineered subtilis.
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| CN110747151B (en) * | 2019-12-06 | 2021-06-15 | 华南农业大学 | A kind of medium combination for promoting the natural growth and transformation of Bacillus subtilis and its transformation method |
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| CN102010876A (en) * | 2010-10-21 | 2011-04-13 | 山东省医学科学院基础医学研究所 | Recombinant bacillus subtilis EV71-VP1 expression vector and preparation method and application thereof |
| CN102242142A (en) * | 2011-04-27 | 2011-11-16 | 南京农业大学 | Method for increasing output of bacillus subtilis antibacterial peptide through knock-out of phrC gene |
| CN102676570A (en) * | 2012-04-26 | 2012-09-19 | 山东省医学科学院基础医学研究所 | Recombinant bacillus subtilis immunoglobulin binding protein functional-domain expression vector and application thereof |
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| WO2009117843A1 (en) * | 2008-03-24 | 2009-10-01 | 清华大学 | An amidase gene knock-out engineered strain for nitrile hydratase production, its construction and application |
| CN102010876A (en) * | 2010-10-21 | 2011-04-13 | 山东省医学科学院基础医学研究所 | Recombinant bacillus subtilis EV71-VP1 expression vector and preparation method and application thereof |
| CN102242142A (en) * | 2011-04-27 | 2011-11-16 | 南京农业大学 | Method for increasing output of bacillus subtilis antibacterial peptide through knock-out of phrC gene |
| CN102676570A (en) * | 2012-04-26 | 2012-09-19 | 山东省医学科学院基础医学研究所 | Recombinant bacillus subtilis immunoglobulin binding protein functional-domain expression vector and application thereof |
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