JPH0139753B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel plasmid. In recent years, various types of plasmids (plasmid DNA) that can be used as vectors have become known, but it is expected that even more useful and safe plasmids will be developed to fully meet a wide range of uses. . The present inventor has discovered that Corynebacterium xerosis, which has no problem with pathogenicity.
The present invention was completed by taking advantage of the fact that a plasmid derived from B. xerosis exists stably in Bacillus subtilis, has a restriction enzyme cleavage point, and has drug resistance. Specifically, a plasmid (pTP10) was isolated from Corynebacterium xerosis, Bacillus subtilis was transformed with this, a plasmid (pTP11) was isolated again, and this was cut with the restriction enzyme XbaI and ligated to obtain a molecular weight of approximately 1.7 megabytes. A plasmid (pTP12) that is Dalton (Md) was obtained;
In addition, by cutting plasmid pTP11 and plasmid pNS1 with the restriction enzyme Hind and ligation, a plasmid with a molecular weight of approximately 4.3 Md was obtained.
pTP1101 and pTP1102 are obtained. As the processing means in these steps, known general methods can be used, for example, the following methods are used. Culture and lysis of Corynebacterium were performed using the method of Shiller et al. (Antimicrob. Agents Chemothef. 18
814-821 (1980)), and extraction of DNA 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 an R plasmid with a molecular weight of 30.4±2.7Md, and on this R plasmid, erythromycin, kanamycin, chloramphenicol,
Contains a tetracycline resistance gene. It also has a streptomycin resistance gene on its chromosome. Example 1 Isolation of plasmid DNA from strain M82B In other words, trypto soy broth medium (17 g of tryptone, 3 g of soy peptone, 2.5 g of glucose,
Potassium monohydrogen phosphate 2.5g, sodium chloride 5g
Inoculate 1/100 of the overnight culture of M82B strain (1/1 g, distilled water, pH 7.3) into the same fresh medium, culture with shaking at 37°C for about 3 hours, and then add penicillin G to the final concentration.
It was added at a concentration of 5 μg/ml and cultured with shaking for an additional 2 hours. The culture solution was collected by refrigerated centrifugation, and the pellet was washed twice with TE buffer (10mM tris-aminomethane, 0.5mMEDTA, PH8.0), and then TE
It was suspended in 0.5M sucrose dissolved in buffer. Lysozyme (Sigma) was added to this suspension at a final concentration of 10 μg/ml and incubated at 37° C. for 2 hours. A 10% SDS (sodium dodecyl sulfate) solution was added to this solution to give a final concentration of 1%, and the solution was allowed to stand at 55°C for 30 minutes. Next, add freshly prepared 3N sodium hydroxide solution to pH 12.1.
~12.3 and mix gently, 2M Tris solution (PH7.0)
was added in an amount twice that of the sodium hydroxide solution. moreover
A 5M sodium chloride solution was added to give a final concentration of 1M, and the mixture was left at 4°C overnight. after that
Collect the supernatant by refrigerated centrifugation at 10,000 rpm for 30 minutes.
An equal amount of redistilled phenol saturated with TE buffer was added and mixed well. After separating the aqueous layer by refrigerated centrifugation at 10,000 rpm for 20 minutes, the aqueous layer was collected and
Three times the amount of ethanol at 20°C 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 and dissolved in TE buffer to obtain a DNA solution. Example 2 Plasmid extracted from M82B strain
Transformation of B. subtilis with pTP10DNA i.e. B. subtilis 168LMAH761222 (leu ^- ,
met ^- , ade ^- , hir ^- ) strains of Bacto-pen Atsei Broth (Deihuiko: Bacto-Beef Extract 1.5g, Bacto-Yeast Extract 1.5g)
g, bacto peptone 5 g, bacto-dextrose 1 g, sodium chloride 3.5 g, dipotassium phosphate 3.68 g, monopotassium phosphate 1.32 g, distilled water 1
, PH7.0±0.1) was inoculated into a new same medium in 1/100 amount and cultured with shaking at 37°C until 0D0.4 (600 nm). Collect bacterial cells by centrifugation and use SMMP
Buffer (0.5M sucrose, 0.02M disodium maleate, 1M magnesium chloride)
Lysozyme was added to the final concentration of 2 mg/ml. After incubation at 37℃ for about 1 hour
Bacteria were collected by centrifugation at 3000 rpm for 15 minutes, washed once with SMMP buffer, and then suspended in 6 ml of SMMP buffer to prepare a protoplast solution. 50μ of DNA solution obtained in Example 1 (approximately 0.5μg of DNA)
After adding the same amount of 2×SMM buffer (1M sucrose, 0.04M disodium maleate, 2M magnesium chloride) to the solution, 1 ml of the above-mentioned protoplast solution was added. Further, 3 ml of a 40% polyethylene glycol 6000 solution was added, and after the mixture was allowed to stand at 0°C for 2 minutes, 5 ml of SMMP buffer was added and bacteria were collected by centrifugation at 3,000 rpm for 10 minutes. Suspend the collected protoplast pellets in 1 ml of SMMP buffer for 30 minutes.
After culturing at ℃ for 1.5 hours, 0.1 ml of the DM-3 regeneration medium containing 25 μg/ml of chloramphenicol (3.5 g of dipotassium phosphate, 1.5 g of monopotassium phosphate, 5 g of casamino acids, 4.06 g of magnesium chloride, succinic acid) Sodium 135g, Yeast Extract 5
g, glucose 5g, agar 8g, bovine serum albumin 0.05g, distilled water 11, PH7.3) and 37°C.
By culturing 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 the transformed strain The obtained chloramphenicol-resistant transformant was used as brain heart infusion (BHI).
The cells were inoculated into 10 ml of culture medium and cultured overnight. This culture solution was mixed with CY medium (dipotassium phosphate 0.7%, monopotassium phosphate 0.3%, ammonium sulfate 0.1%, sodium citrate 0.05%, deifcocasamino acid 0.2%,
Difco yeast extract 0.1g, magnesium sulfate
Absorbance in 10ml (0.025%, glucose 0.5%, PH7.0)
It was added so that the wavelength was 0.1 (600 nm). 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, add tritiated thymidine to a final concentration of 10 μCi/ml.
I added it so that it becomes . Growth was stopped on ice at absorbance 0.8 (600nm),
The cells were separated from the broth at 10,000×g for 15 minutes at 4° C., and the supernatant was decanted from the cell pellet.
Transfer the pellet to TES buffer (0.05M sodium chloride,
Tris-HCl buffer containing 0.005M EDTA (PH
8.0)). Then 0.5M EDTA
0.2 ml of lysozyme 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 minutes. Bridge 58
was added to a final concentration of 0.5% and incubated at 37°C for 10 minutes. Next, N-lauroylsarcosinate sodium was added to a final concentration of 2.5%, and the mixture was heated at 50°C.
Bacteriolysis was performed by culturing for 30 minutes. After removing the viscosity and softening the lysate with a pipette, add cesium chloride,
It was mixed with ethidium bromide to form a solution with a density of 1.553.
The solution was centrifuged at 126,000 xg until equilibrium (approximately 40 hours). The solution is fractionated from the lower end of the centrifuge tube, 10μ of each fraction is adsorbed on Watzmann filtration GF/A, and the radioactivity of the tritium adsorbed on this filtration is measured using a liquid scintillation counter (Patz card).
3300) and expressed in cpm. chromosome
It gives two large peaks, 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. Next, the water layer
Plasmids were isolated by dialysis against a 10-fold dilution of SSC (0.15M sodium chloride, 0.015M sodium citrate, PH7.0). Isolated plasmid
The molecular weight of the DNA was measured using an electron microscope and was found to be 4.7 Md. This plasmid is called pTP11. In addition, the bacterial strain possessing this plasmid was LMAH
(pTP11) strain. The copy number of this plasmid was approximately 20. Example 4 Retransformation of plasmid pTP11 into Bacillus subtilis strain RM125 Plasmid DNA was prepared from LMAH (pTP11) strain by the following method. Bacillus subtilis LMAH (pTP11) strain is inoculated into 100 ml of Brain Heart Infusion (BHI) medium manufactured by Deifico Laboratories. The composition of this medium is shown below. Calf Brain Infusion 20.0% Beef Heart Infusion 25.0% Proteose Peptone 1.0% Bacto-Dextrose 0.2% Sodium Chloride 0.5% Disodium Phosphate 0.25% Distilled Water 1000ml Confirm that the pH is 7.4±0.2. medium
Pre-sterilize in a 500 ml Sakaguchi flask. After inoculation, the flask is 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 18-24 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). 26000 x g of dissolved material,
After cooling and centrifuging for 30 minutes, add 50 ml of 5M sodium chloride solution to the supernatant, leave it overnight, then add polyethylene glycol 6000 to a final concentration of 10%.
Further, it was left at 4°C overnight. this mixture
The pellet was centrifuged at 10,000×g for 15 minutes and dissolved in TES buffer. This solution is mixed with cesium chloride and ethidium bromide to give 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. The plasmid DNA was removed from the density gradient, the ethidium bromide removed by extraction twice with n-octanol, and the aqueous layer was then diluted 10 times with SSC buffer (0.15M sodium chloride, 0.015M sodium citrate, PH7. 0) to obtain pTP11.
The obtained pTP11 DNA was transformed into Bacillus subtilis restriction defective = modification defective strain by the protoplast method described above.
The RM125 strain was transformed. When one of the obtained chloramphenicol-resistant colonies was used to detect a plasmid by the thymidine labeling method, it was possible to detect a plasmid with exactly the same molecular weight as pTP11. From this experiment, plasmid
It was confirmed that a chloramphenicol resistance gene was present on pTP11. Example 5 Preparation of restriction enzyme cleavage 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, the phenol is further extracted with ether, two volumes of ethanol are added, and the precipitated DNA is collected by centrifugation and TE After dissolving in buffer, an enzymatic reaction was then performed. EcoR 10mM Tris-HCl buffer (PH7.2),
5mM magnesium chloride, 2mM mercaptoethanol, 50mM sodium chloride, 37°C 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°C Bcl 12mM Tris-HCl buffer (PH7.4),
12mM magnesium chloride, 12mM sodium chloride, 0.5mM DTT, 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 and the molecular weight of the cleavage fragments of pTP11 are shown in the table, and the cleavage map is shown in FIG.

[Table] Example 6 Isolation of DNA fragment-deficient plasmid from plasmid pTP11 Plasmid pTP11 DNA was subjected to Xba
Alternatively, after cutting with Hind, ligation reaction was performed with T4 DNA ligase. Reaction conditions are 66mM
- Tris-HCl buffer (PH6.5), 6.6mM magnesium chloride, 10mM dithiothreitol, 0.4mM
The test was carried out at 15°C for 20 hours in the presence of ATP. After the reaction, Bacillus subtilis strain RM125 was transformed by the protoplast method described above. When the obtained chloramphenicol-resistant colonies were examined, it was found that Xba-
Plasmid pTP12 of 1.7 Md was obtained from colonies obtained from the T4 ligase combination. In addition, from the combination of Hind-T4 ligase,
2.8 Md of pTP13 was obtained. Example 7 Creation of restriction enzyme cleavage map and measurement of molecular weight and copy number of plasmids pTP12 and pTP13 Copy number of plasmids pTP12 and 13 was measured by ^3H -thymidine labeling method.
DNA was taken and its molecular weight was accurately measured using an electron microscope. In addition, restriction enzyme cleavage mapping was performed in exactly the same manner as described above. As a result, the plasmid
pTP12 has a small molecular weight of 1.7 Md and has two cleavage points for Hpa, but no cleavage points for Xba. This plasmid has a large copy number of approximately 200, and when Bacillus subtilis containing this plasmid was cultured in liquid culture, chloramphenicol acetyltransferase activity was observed in the medium. pTP13 was found to be a circularized version of a large Hind fragment (2.8Md) of pTP11. The copy number of this plasmid was approximately 70. Figure 2 shows the restriction enzyme cleavage point map of pTP12 and pTP13.
and shown in FIG. Example 8 DNA deletion plasmid from plasmid pTP5
Isolation of pNS1 Gene21: 105-112 for isolation of pNS1
(1983). i.e. Bacillus subtilis plasmid pTP5DNA (M.Kono et al Microbios
Letters 5:55-59 (1978))
168LMAH761222 (pTP5) was extracted by the method described in Example 4. 20 μg of this pTP5DNA
Cleavage is performed by reaction at 37°C for 1 hour in a solution containing mM Tris-HCl buffer (PH7.4), 7mM magnesium chloride, 60mM sodium chloride, and 10 Hind units. 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 dissolved in 66mM Tris-HCl buffer (PH7.6), 6.6mM magnesium chloride, 10mM
After dissolving in 100μ of M 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. The precipitated DNA was dissolved in 50μ of sterile distilled water and used as a DNA sample for transformation. Transformation was carried out by the protoplast method described in Example 2. Bacillus subtilis GSY908 strain was used as the recipient bacterium. The obtained Tc-resistant colonies were suspended in 40μ of a lysis buffer (lysozyme 2μg/ml, RNase 0.1mg/ml, 25% sucrose dissolved in TE buffer) and reacted at 37°C for 2 hours. 5% in this solution
After 10μ of SDS solution was added and the bacteria were completely lysed, 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. Specifically, plasmid DNA was prepared from each transformed strain by the method described above, Hind cleavage was performed by the method described above, and agarose gel electrophoresis was performed. As a result, deletion of the Hind-C fragment (0.35Md) of plasmid pTP5 The presence of plasmid pNS1 was confirmed. This plasmid pNS1DNA was extracted and Bacillus subtilis
Transform the RM125 strain by the protoplast method,
It was confirmed that the Tc resistance gene was present on plasmid pNS1. Bacillus subtilis for subsequent experiments
RM125 (pNS1) strain was used. The restriction enzyme cleavage map of pNS1 is shown in FIG. Example 9 Isolation of plasmids pTP1101 and pTP1102 and confirmation of orientation Plasmid from Bacillus subtilis RM125 (pNS1) strain
pNS1DNA was extracted by the method described above, mixed with plasmid pTP11DNA, and then extracted by the method described above (Example 8).
After cleavage with Hind according to the procedure, the product was ligated with T4 ligase and transformed into RM125 strain by the protoplast method. Plasmids for colonies with resistance to both chloramphenicol and tetracycline
I conducted a DNA search. The obtained colonies were lysed as in Example 8 and subjected to agarose gel electrophoresis, and several strains with the smallest molecular weight of plasmid DNA were selected. Plasmid DNA was prepared from these transformed strains, and EcoR, Hind, Kpn
A restriction enzyme cleavage point map was created for the following. In addition to EcoR and Hind, for the creation of restriction enzyme cleavage maps.
I used Kpn. The reaction conditions for Kpn are 6mM Tris-HCl buffer (PH7.5), 6mM sodium chloride, 6mM magnesium chloride, 6mM 2-mercaptoethanol, and 37°C. As a result, two types of plasmids were created in which the pNS1-derived Hind-A fragment (1.5Md) was ligated to the pTP11-derived 2.8Md fragment in the forward and reverse directions.
It was confirmed that there were two types of transformed strains having pTP1101 and pTP1102, respectively. Restriction enzyme cleavage maps of pTP1101 and pTP1102 are shown in FIGS. 4, 5, and 6. The number of copies was approximately 100 copies each. 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 P6972 Bacillus subtilis RM125 FERM P-6973 Bacillus subtilis 168LMAH761222 (pTP5)
FERM P-6974

[Brief explanation of drawings]

1 to 7 are restriction enzyme cleavage maps of plasmids.

Claims (1)

[Claims] 1. The molecular weight is approximately 1.7 megadaltons, and the restriction enzyme cleavage map is Plasmid pTP12, which is represented by