JPH0141311B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel plasmid. In recent years, various plasmids that can be used as vectors have become known.
The development of even more useful plasmids is expected. The present inventor has discovered that a new plasmid has a molecular weight of about 3.5 megadaltons (Md), which is suitable for use, has a large number of copies, has a restriction enzyme cleavage point, and is resistant to chloramphenicol and tetracycline. Bacillus subtilis
The present invention was completed by confirming that it exists stably in A. subtilis. That is, the plasmid from Corynebacterium xerosis
After isolating pTP10 and transforming B. subtilis with it, a plasmid (pTP11) was isolated again, and this was combined with staphylococcus-derived plasmid pNS1 (Gene 21 ,
105-112 (1983)) with the restriction enzyme Hind and ligation to reduce the molecular weight to approx.
Plasmid pTP1102 with 4.3 Md is obtained, but
This material is further cut with the restriction enzyme Xba and then ligated to reduce the molecular weight to approx.
Plasmid pTP1103 with 3.5 Md could be obtained. As the processing means in these steps, known general methods can be used, for example, the following methods are used. Corynebacterium culture and bacteriolysis were carried out using the method of Shiller et al. (Antimicrob. Agents Chemother. 18 ,
814-821 (1980)), and DNA extraction by the alkaline denaturation method may be performed according to the method of Hansen and Olsen (J. Bacteriol. 135 , 227-238 (1978)). Transformation of Bacillus subtilis was performed using the protoplast method of Chang and Cohen (Molec.gen.Genet. 168 , 111-115).
(1979)). Common methods such as agarose gel electrophoresis and density gradient centrifugation can be used to isolate plasmids, and plasmid DNA can be cut with restriction enzymes and DNA strands can be ligated with enzymes (ligase).
Ligation can also be performed by a conventional method. Next, the method for producing the plasmid of the present invention will be explained in detail using experimental examples.
The xerosis M82B strain is a strain isolated by the present inventor, and has a plasmid with a molecular weight of 30.4±2.7 Md, and this plasmid contains erythromycin, kanamycin, chloramphenicol, and tetracycline resistance genes. It also has a streptomycin resistance gene on its chromosome. Example 1: Isolation of plasmid DNA from Corynebacterium xerosis M82B strain Trypto soy broth medium (tryptone 17g,
A 1/100 volume fresh overnight culture of the M82B strain containing 3 g of soy peptone, 2.5 g of glucose, 2.5 g of potassium monohydrogen phosphate, 5 g of sodium chloride, 1 g of distilled water, PH7.3) was inoculated into the same fresh medium, and the culture was incubated at 37°C. After 3 hours of shaking culture, add penicillin G to a final concentration of 5 μg/ml.
and cultured with shaking for an additional 2 hours. This culture solution was cooled and centrifuged, and the resulting pellet was
TE buffer (10mM trisaminomethane,
After washing twice with 0.5mMEDTA, PH8.0), the cells were suspended in 0.5M sucrose dissolved in TE buffer.
Add lysozyme (Sigma) to this suspension at the final concentration
It was added at a concentration of 10 μg/ml and incubated at 37° C. for 2 hours. 10% SDS (sodium dodecyl sulfate) in this solution
Add the solution to a final concentration of 1% and incubate at 55℃ for 30 minutes.
It was left standing for a minute. Next, a freshly prepared 3N sodium hydroxide solution was added to adjust the pH to 12.1 to 12.3, and the mixture was mixed gently, and then a 2M Tris solution (PH7.0) was added in twice the amount of the sodium hydroxide solution. Further, 5M sodium chloride solution was added to give a final concentration of 1M, and the mixture was left at 4°C overnight. Thereafter, the supernatant was collected by refrigerated centrifugation at 10,000 rpm for 30 minutes, and an equal amount of redistilled phenol saturated with TE buffer was added and mixed well. The aqueous layer was separated by refrigerated centrifugation at 10,000 rpm for 20 minutes, 3 times the volume of -20°C ethanol was added, and the mixture was left at -20°C overnight. The pellet was collected by refrigerated centrifugation at 10,000 rpm for 30 minutes, dissolved in TE buffer, and the DNA
It was made into a solution. Example 2: Plasmid pTP10 extracted from M82B strain
Transformation of Bacillus subtilis by DNA Bacillus subtilis 168LMAH761222 (leu ^- , met ^- , ade ^- ,
hir ^- ) strain Bacto-Pen Atsei Broth (Difco: Bacto-beef extract 1.5 g, Bacto-yeast extract 1.5 g, Bacto-peptone 5 g, Bacto-dextrose 1 g, sodium chloride 3.5 g, dipotassium phosphate 3.68 g, Monopotassium phosphate 1.32g,
Inoculate 1/100 amount of the overnight culture in distilled water (1, PH7.0±0.1) into a new same medium and measure the optical density (OD) at 37℃.
Shaking culture was performed until 0.4 (600 nm). The bacterial cells were collected by centrifugation and suspended in SMMP buffer (0.5M sucrose, 0.02M disodium maleate, 1M magnesium chloride dissolved in 2x concentration of Bacto-Pen Atseipros) to a final concentration of 2 mg/ml.
Lysozyme was added so that After incubation at 37°C for about 1 hour, the bacteria were collected by centrifugation at 3000 rpm for 15 minutes.
After washing once with SMMP buffer, remove 6 ml of bacterial cells.
It was suspended in SMMP buffer to make a protoplast solution. 50μ of DNA solution obtained in Example 1 (approximately 0.5μg of DNA)
with the same amount of twice the concentration of SMM buffer (1M sucrose, 0.04M disodium maleate,
After adding 2M magnesium chloride), 1 ml of the protoplast solution described above was added. Furthermore, add 3 ml of 40% polyethylene glycol 6000 solution and
After letting it stand for a minute, add 5ml of SMMP buffer.
Bacteria were collected by centrifugation at 3000 rpm for 10 minutes. The collected protoplast pellets were suspended in 1 ml of SMMP buffer and cultured at 30°C for 1.5 hours, and then 0.1 ml was added to DM-3 regeneration medium containing 25 μg/ml of chloramphenicol (3.5 g of dipotassium phosphate, monobasic phosphate). Potassium 1.5g, Casamino Acid 5g, Magnesium Chloride
4.06g, sodium succinate 135g, yeast extract 5g, glucose 5g, agar 8g, bovine serum albumin 0.05g, distilled water 11, PH7.3).
By culturing at ℃ for 2 to 3 days, transformed strains were obtained at a frequency of 1 in 10 ⁹ to 10 ¹⁰ strains. Example 3: Isolation of plasmid DNA from a transformed strain The obtained chloramphenicol-resistant transformed strain was inoculated into 10 ml of Brain Heart Infusion medium (BHI medium) manufactured by Daif Collaborative Laboratories, and cultured overnight. did. Transfer this culture solution to CY medium (dipotassium phosphate 0.7%, monopotassium phosphate 0.3%).
%, ammonium sulfate 0.1%, sodium citrate 0.05%, casamino acids 0.2%, yeast extract 0.1%,
Magnesium sulfate 0.025%, glucose 0.5%, PH
7.0) Added to 10ml so that the absorbance was 0.1 (600nm). The culture was incubated with shaking at 37°C, and when the absorbance was 0.3 (600 nm), deoxyadenosine was added to a final concentration of 250 μg/ml. After 3 minutes, tritium-labeled thymidine was added to a final concentration of 10 μCi/ml. At absorbance of 0.8 (600 nm), growth was stopped on ice, centrifuged at 10,000 x g for 15 minutes at 4°C to separate the bacterial cells from the broth, and the bacterial pellet was added to TES buffer (0.05 M sodium chloride, The cells were resuspended in Tris-HCl buffer (PH8.0) containing 0.005M EDTA. Next, 0.2 ml of 0.5 M EDTA solution and 0.1 ml of lysozyme solution with a concentration of 10 mg/ml were added, and the mixture was incubated at 37 °C for 10 min.
It was cultured in This includes Bridge-58 (Kao Atlas Co., Ltd.)
was added to a final concentration of 0.5% and incubated at 37°C for 10 minutes. Next, add N-lauroyl sarcosinate sodium to a final concentration of 2.5% and heat at 50°C.
The cells were incubated for 30 minutes to perform bacteriolysis. After the lysate was made viscous and softened with a pipette, it was mixed with cesium chloride and ethidium bromide to form a solution with a density of 1.553. Add this solution to 126,000
Centrifugation was carried out at xg until equilibrium (approximately 40 hours). The solution was fractionated from the lower end of the centrifuge tube, 10μ of each fraction was adsorbed onto Watzmann filter paper GF/A, and the radioactivity of tritium adsorbed on this filter paper was measured using a liquid scintillation counter (Patz Card).
3300) and expressed in cpm, the chromosome
Two large peaks were obtained, a fraction showing high activity derived from DNA and a small peak derived from plasmid DNA. A fraction showing this small peak was collected and treated twice with n-octanol to extract and remove ethidium bromide. The aqueous layer was then dialyzed against a 10-fold dilution of SSC buffer (0.15M sodium chloride, 0.015M sodium citrate, PH7.0) to isolate the plasmid. The molecular weight of the isolated plasmid DNA was measured using an electron microscope and was found to be 4.7 Md. This plasmid is pTP11
shall be. In addition, the strain possessing this plasmid was named Bacillus subtilis LMAH (pTP11) strain. The copy number of this plasmid was approximately 20. Example 4: Transformation of Bacillus subtilis strain RM125 with plasmid pTP11 Plasmid from Bacillus subtilis LMAH (pTP11) strain
DNA was prepared by the following method. Bacillus subtilis LMAH (pTP11) strain is inoculated into 100 ml of BHI medium and cultured with shaking at 37°C for about 18 to 24 hours. This cell suspension is used to inoculate 20 flasks of the same medium at a rate of 1%. After shaking culture at 37°C for 18 to 24 hours, centrifugation was performed at 10,000 xg for about 15 minutes at 4°C, and the supernatant was removed by slanting. The obtained bacterial pellet
After washing twice with TES buffer, resuspend in 100 ml of TES buffer containing 25% sucrose. Next, 40 ml of 0.5M EDTA solution and 20 ml of lysozyme solution at a concentration of 10 mg/ml are added and the mixture is incubated at 37° C. for 30 minutes. Next, 20ml of Brisi-58 with a concentration of 50mg/ml
and incubate the mixture at 37 °C for 10 min. moreover
Add 20 ml of sodium N-lauroylsarcosine at a concentration of 250 mg/ml and incubate at 50°C for 10 to 30 minutes (cell disruption). After cooling and centrifuging the lysate at 26,000 x g for 30 minutes, add 50 ml of 5M sodium chloride solution to the supernatant and leave it overnight.
Polyethylene glycol 6000 was added to a final concentration of 10%, and the mixture was further left overnight at 4°C. This mixture was centrifuged at 10,000 xg for 15 minutes, and the resulting pellet was dissolved in TES buffer. This solution is mixed with cesium chloride and ethidium bromide to make a solution with a density of 1.553. Centrifuge this solution at 126,000 xg until equilibrium (approximately 40 hours).
Under UV illumination (365 nm) linear chromosomes and plasmid DNA are visible in the centrifuge tube as intensely fluorescent bands. This plasmid DNA was removed from the density gradient and
Ethidium bromide was removed by extraction twice with n-octanol, and the aqueous layer was then dialyzed against 10-fold diluted SSC buffer to yield pTP11. The obtained pTP11 DNA was used as a restriction-deficient and modification-deficient strain of Bacillus subtilis by the protoplast method described above.
The RM125 strain was transformed. Using one of the obtained chloramphenicol-resistant colonies, plasmid detection was performed using the tritium-labeled thymidine labeling method, and pTP11 was detected.
We were able to detect a plasmid with exactly the same molecular weight. This experiment confirmed that the chloramphenicol resistance gene was present on plasmid pTP11. Example 5: Preparation of restriction enzyme cleavage point map of plasmid pTP11 Digestion of plasmid DNA with restriction enzymes was performed under the following conditions. In addition, when reacting two types of restriction enzymes in combination, one enzyme reaction is stopped with phenol, and after the phenol is extracted with ether, twice the amount of ethanol is added, and the precipitated DNA is collected by centrifugation and TE. After dissolving in buffer, the following enzymatic reaction was performed. EcoR 10mM Tris-HCl buffer (PH7.2),
5mM magnesium chloride, 2mM mercaptoethanol, 50mM sodium chloride,
37℃ Hpa 20mM Tris-HCl buffer (PH7.4),
7mM magnesium chloride, 1mM dithiothreitol (DTT), 37°C Xba 6mM Tris-HCl buffer (PH7.4),
100mM sodium chloride, 6mM magnesium chloride, 37℃ Bcl 12mM Tris-HCl buffer (PH7.4),
12mM magnesium chloride, 12mM sodium chloride, 0.5mMDTT, 50°C Hind 20mM Tris-HCl buffer (PH7.4),
7mM magnesium chloride, 60mM sodium chloride, 37°C Digestion mixture was prepared by Sharp et al.
% agarose gel (J. Sharp et al,
Biochemistry, 12 3055-3063 (1973)). EcoR
Digested λ DNA was used as a molecular weight control (Thomas et al., J. Mol. Biol., 91 315-328
(1975)). The number of restriction enzyme cleavage points of pTP11 and the molecular weight of the cleavage fragment are shown in the table.

[Table] Example 6: Production of plasmid pTP1102 Bacillus subtilis RM125 (pNS1) strain (Gene 21 , 105~
112 (1983)) was extracted from plasmid pNS1 DNA in the same manner as described above (Example 4).
After mixing with pTP11DNA, it was cut with restriction enzyme Hind. That is, 1 μg of pTP11DNA and 11 μg of pNS
20mM Tris-HCl buffer (PH7.4), 7mM magnesium chloride, 60mM sodium chloride, Hind10
Cleavage is performed by reaction for 1 hour at 37°C in a unit-containing solution. The reaction was stopped by adding phenol and the phenol was removed by ether extraction.
Double the amount of ethanol was added to precipitate the DNA. The precipitated DNA pellet was added to 66mM Tris-HCl buffer (PH7.6), 6.6mM magnesium chloride, 10mM
After dissolving in 100μ of dithiothreitol and 4mM adenosine triphosphate solution, 0.01 unit of T4 DNA ligase was added and the DNA was ligated by reaction at 16°C for 16 hours. The reaction was stopped with phenol, and after removing the phenol by ether extraction, twice the amount of ethanol was added to precipitate the DNA. precipitated
Dissolve the DNA in 50μ of sterile distilled water for transformation.
It was used as a DNA sample. Transformation was carried out by the protoplast method described in Example 2. Bacillus subtilis strain RM125 was used as the recipient bacterium. Plasmids for colonies with resistance to both chloramphenicol and tetracycline
I conducted a DNA search. That is, the obtained colonies were mixed with lysis buffer (lysozyme 2 μg/ml,
RNase 0.1mg/ml, dissolved in TE buffer (25% sucrose), suspended in 40μ and incubated at 37℃.
The reaction was allowed to proceed for 2 hours. Add 10μ of 5% SDS solution to this solution.
was added and after complete lysis, 50μ of phenol was added and the aqueous layer was separated after centrifugation at 10,000 rpm for 10 minutes. Plasmid DNA was confirmed in this aqueous layer by agarose gel electrophoresis, and several transformants having plasmid DNA with the smallest molecular weight were selected. Plasmids were extracted from each transformed strain using the method described above.
Prepare DNA, EcoR, Hind and Kpn
A restriction enzyme cleavage point map was created for the following. Kpn
The reaction conditions were 6mM Tris-HCl buffer (PH7.5),
6mM sodium chloride, 6mM magnesium chloride,
6mM 2-mercaptoethanol, 37°C. As a result, it was confirmed that there was a transformed strain having plasmid pTP1102 in which the Hind-A fragment (1.5 Md) derived from pNS1 and the 2.8 Md fragment derived from pTP11 were combined. This strain is Bacillus subtilis
The strain was named RM125 (pTP1102). Example 7: Preparation of plasmid pTP1102 DNA Bacillus subtilis RM125 (pTP1102) strain is inoculated into 100 ml of Brain Heart Infusion (BHI) medium manufactured by Difco Laboratories. Check that the pH is 7.4±0.2. Pre-sterilize the medium in a 500 ml Sakaguchi flask. After inoculation, keep the flask at 37℃, approx.
Incubate with shaking for 24 hours. This cell suspension is used to inoculate 20 flasks of the same medium at a rate of 1%. After shaking culture at 37°C for 18-24 hours, the culture is separated from the broth at 10,000 xg for about 15 minutes at 4°C, and the supernatant is decanted from the bacterial pellet. The supernatant is discarded and the pellet is washed twice with TES buffer and then resuspended in 100 ml of TES buffer containing 25% sucrose. Next, 40ml of 0.5M-EDTA solution,
Add 20 ml of lysozyme solution with a concentration of 10 mg/ml and incubate the mixture for 30 minutes at 37°C. Next, 20 ml of Brij-58 at a concentration of 50 mg/ml is added and the mixture is incubated at 37°C for 10 minutes. Furthermore, add 20 ml of N-lauroylsarcosinate sodium at a concentration of 250 mg/ml, and
Incubate for minutes (cell disruption). After centrifuging the lysate at 26,000 x g for 30 minutes, add 50 ml of 5M sodium chloride solution to the supernatant, leave it overnight, and add polyethylene glycol 6,000 to the final concentration.
The solution was added to a concentration of 10% and left at 4°C overnight. This mixture was centrifuged at 10,000 x g for 15 minutes,
The pellet was dissolved in TES buffer. This solution is mixed with cesium chloride and ethidium bromide to form a solution with a density of 1.553. This solution
Centrifuge at 126,000 xg until equilibrium (approximately 40 hours). Under UV illumination (365 nm) linear chromosomes and plasmid DNA are visible in the centrifuge tube as intensely fluorescent bands. This plasmid
DNA was removed from the density gradient and ethidium bromide was removed by extraction twice with n-octanol, then the aqueous layer was diluted 10 times in SSC buffer (0.15M sodium chloride, 0.015M sodium citrate, PH
7.0) to obtain pTP1102DNA. Example 8: Preparation of DNA fragment deletion plasmid DNA from plasmid pTP1102 2 μg of plasmid pTP1102 DNA was added to 6 mM Tris-HCl buffer (PH7.4), 100 mM sodium chloride,
Cleavage is carried out by reaction at 37°C for 1 hour in a solution containing 6mM magnesium chloride and 10 units of Xba. The reaction was stopped by adding phenol, and after removing the phenol by ether extraction, twice the amount of ethanol was added to precipitate the DNA. The precipitated DNA pellet was added to 66mM Tris-HCl buffer (PH7.6), 6.6mM magnesium chloride, 10mM
After dissolving in 100μ of dithiothreitol and 4mM adenosine triphosphate solution, 0.01 unit of T4 DNA ligase was added and the DNA was ligated by reaction at 16°C for 16 hours. The reaction was stopped with phenol, the phenol was removed by ether extraction, and then twice the amount of ethanol was added to precipitate the DNA. precipitated
Dissolve the DNA in 50μ of sterile distilled water for transformation.
It was used as a DNA sample. Example 9: Transformation A 1/100 amount of an overnight culture of Bacillus subtilis strain RM125 in Bacto-ben atsei broth was inoculated into a new same medium, and cultured with shaking at 37°C until OD 0.4 (600 nm).
The bacterial cells were collected by centrifugation and suspended in SMMP buffer (0.5M sucrose, 0.02M disodium maleate, 1M magnesium chloride dissolved in double concentration Bacto-Pen assay broth).
Lysozyme was added to a final concentration of 2 mg/ml. After culturing at 37℃ for about 1 hour, collect bacteria by centrifugation at 3000 rpm for 15 minutes, wash once with SMMP buffer,
The bacterial cells were suspended in 6 ml of SMMP buffer to prepare a protoplast solution. 50μ of DNA solution obtained in Example 8 (approximately 0.5μg of DNA)
After adding the same amount of 2×SMM buffer, 1 ml of the above-mentioned protoplast solution was added. an additional 40%
Add 3ml of polyethylene glycol 6000 solution,
After standing at 0°C for 2 minutes, 5 ml of SMMP buffer was added and bacteria were collected by centrifugation at 3000 rpm for 10 minutes. The collected protoplast pellets were suspended in 1 ml of SMMP buffer and cultured for 1.5 hours at 30°C, then 0.1 ml of the pellet was spread on DM-3 regeneration medium containing 25 μg/ml of chloramphenicol and cultured at 37°C for 2 to 3 days. A transformed strain was obtained by doing this. The obtained chloramphenicol-resistant colonies were treated with lysis buffer (lysozyme 2 μg/ml, RNase 0.1
mg/ml, 25% sucrose dissolved in TE buffer) was suspended in 40μ and reacted at 37°C for 2 hours.
To this solution, 10μ of 5% SDS solution was added, and after complete lysis, 50μ of phenol was added, and after centrifugation at 10,000 rpm for 10 minutes, the aqueous layer was separated. Plasmid DNA was confirmed in this aqueous layer by agarose gel electrophoresis, and several transformed strains containing plasmid DNA with the smallest molecular weight were investigated in more detail. That is, plasmid DNA was prepared from each transformed strain by the method described above, Xba cleavage was performed by the method described above, and agarose gel electrophoresis revealed that 0.8 Md of plasmid pTP1102 was obtained.
The existence of plasmid pTP1103 lacking the Xba-fragment was confirmed. The strain containing pTP1103 was named Bacillus subtilis RM125 (pTP1103). pTP1103 has a molecular weight of 3.5Md and a copy number of approximately
It was 100 copies. The microorganism used in the above example has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and has the following number. Corynebacterium xerosis M82B FERM P-6971 Bacillus subtilis 168LMAH761222 FERM P-6972 Bacillus subtilis RM125 FERM P-6973 Bacillus subtilis RM125 (pNS1) FERM P-7008

[Brief explanation of drawings]

FIG. 1 is a restriction enzyme cleavage point map of the plasmid, and FIG. 2 is a flowchart.

Claims (1)

[Claims] 1. Has a tetracycline resistance gene and a chloramphenicol resistance gene, and has a molecular weight of about 3.5
Plasmid pTP1103, which is a megadalton and has the following restriction enzyme cleavage point map: