JPH0139751B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a plasmid production method and a new plasmid, and aims to provide a plasmid that can be used as a vector. Progress is being made in the development of technology to endow microorganisms with new abilities through genetic manipulation and use them to produce various useful substances. The plasmid (plasmid DNA) that is
Various things have become known. However, it is expected that many more useful and safe plasmids will be developed to fully meet a wide range of uses. The present inventor has discovered that a plasmid derived from Streptococcus faecalis, which resides in the intestine and has no problem with pathogenicity, stably exists in Bacillus subtilis, and has a restriction enzyme cleavage point. The present invention was completed by confirming that the vector has drug resistance and can be used as a vector. That is, the present invention provides a method for producing a plasmid by isolating a plasmid from the bacterial cells of Streptococcus faecalis, transferring it to Bacillus subtilis, and isolating the plasmid again from this, and a method for producing a plasmid thus obtained. It concerns plasmid pTP62. Streptococcus faecalis is known as an intestinal resident streptococcus, and it is considered preferable to use it as a plasmid donor from the viewpoint of safety. Although many strains of this bacterial species are known, it is convenient to have some sort of marker for processing operations, and for example, strains with low resistance (resistance) to drugs are preferred. In particular, tetracycline-resistant strains and chloramphenicol-resistant strains are considered suitable for obtaining plasmids. As a representative example, the present inventor has developed wild-type strains that are resistant to tetracycline, chloramphenicol, streptomycin, kanamycin, and erythromycin.
KSf2 strain and KSf24m, a strain without drug resistance
The KSf2-2 strain obtained by conjugative transfer of the strain can be mentioned, and this strain is kept at the Microbial Technology Research Institute (Accession number: FAIKEN BIYORI) No. 5772. Streptococcus faecalis may be cultured using the culture methods normally used for this bacterial species, such as Brain Heart Infusion Medium (BHI medium), CY medium, etc. containing meat extract, peptone, inorganic salts, etc. 25-40℃ in normal medium preferably 37
Around 10 to 10 hours at around ℃, preferably 18~
Incubate for 24 hours. The bacterial cells are collected by centrifugation, washed if necessary, suspended in an appropriate buffer, and lysed by the action of lysozyme in the presence of a surfactant. As a surfactant, Bridge-58
(Brij-58, Kao Atlas Co., Ltd.) (Final concentration in bacterial suspension 0.2-1%, especially about 0.7% (w/v)), N-
Sodium lauroyl sarcosinate (Hani Chemicals
Co., Ltd.) (final concentration 1-10%, especially about 2.5%) and sodium dodecyl sulfate (SDS, final concentration 1-10%)
%, particularly about 2.5%), and is appropriately selected depending on the processing to be performed later. Lysozyme may be added to the bacterial suspension at a concentration of several tens of μg to several hundred μg/ml, and the lysozyme may be added to the bacterial suspension at temperatures ranging from 0°C to 37°C for several tens of minutes to several hours, for example, 30 minutes to 3 hours. It lyses the bacteria. After lysis, raise the temperature to around 50℃.
It is appropriate to prevent DNA degradation by DNA degrading enzymes (DNase). A common method may be used to separate the plasmid from the lysis solution; for example, density gradient centrifugation using cesium chloride is suitable, and it is more preferably carried out in the presence of ethidium bromide. The resulting desired fraction is extracted with an alcohol such as isoamyl alcohol or n-octanol to remove ethidium bromide, and dialyzed using an appropriate buffer to remove cesium, thereby obtaining an aqueous solution of the plasmid. The amount of this plasmid can be measured by UV absorption, and it can also be stored frozen or refrigerated. The length of the plasmid can be measured by electron microscopy and the molecular weight calculated. Next, the obtained Streptococcus fuecalis plasmid is transferred to Bacillus subtilis to transform Bacillus subtilis. At this time, since the heterologous plasmid may be destroyed by Bacillus subtilis, restriction enzymes are used. It is suitable to use strains deficient in and modification enzymes. Restriction-defective and modification-defective Bacillus subtilis can also be created by artificially mutating them (Uzumi et al.
See also Molec .
It is also possible to use a known strain such as the RM125 strain, which is kept at the Stock Center as BGSC No. 1A253 and is available to anyone. When transferring plasmids, it is necessary to introduce DNA into Bacillus subtilis.
It is advantageous to impart uptake ability and make the cells competent, and for this purpose the method of Anagnostopoulos et al. (C.Anagnostopoulos &J.Spizizen;
J. Bacteriol. 81 741-746 (1961)), and cultured in a synthetic medium containing casamino acids, magnesium, glutamate, etc. In addition, the transformation method using protoplasts by S.Chang et al (Molec.gen.Genet. 168 111−
115 (1979) may also be used. Bacillus subtilis, which has been transformed into competent cells, is combined with a plasmid solution and placed in an appropriate medium, such as BHI medium, at 37°C.
Bacillus subtilis is transformed by culturing for several minutes to several hours. At this time, it is convenient to add an appropriate antibiotic or the like to the culture medium, culture it, and isolate and obtain the desired bacterial strain based on the drug resistance transmitted by the plasmid. It is possible to produce a large amount of plasmid by growing the transformed Bacillus subtilis in an appropriate medium, but it is more advantageous for industrialization to use strains that are easy to grow. Many strains can be used, including those used for the production of acids, adenylic acid, etc. Therefore, the plasmid is isolated from the transformed Bacillus subtilis and transferred to another strain of Bacillus subtilis, but this procedure is the same as the isolation of the plasmid from Streptococcus faecalis and transfer to Bacillus subtilis described above. Just do it. From the standpoint of safety, it is preferable to select a strain that cannot grow without specific nutrients and to use one that will not proliferate except through intentional cultivation. Examples of this type of strain include arginine, methionine, histidine, adenine, leucine, tryptophan,
There are strains that require one or more types of tyrosine, etc., and representative examples include Bacillus subtilis 168 adenine-requiring strain, which is widely used in laboratories, and Bacillus subtilis.
Examples include 168 leucine, methionine, adenine, and histidine auxotrophs, and Bacillus subtilis 168 adenine and guanine auxotrophs. When such a strain is transformed and grown in a medium containing necessary nutrients and the plasmid is isolated in the same manner as described above, a plasmid that can be used as a vector can be obtained. In an embodiment of the present invention, for example, a plasmid obtained from the KSf2-2 strain is used to transform Bacillus subtilis RM125 (r-M, m
-M, arg15, leuA8) (hereinafter abbreviated as RM125) and cultured on a brain heart infusion agar plate containing chloramphenicol to isolate the plasmid (strain KBS14), the molecular weight was approximately 9.1 M Dalton (Md) plasmid and approx.
A plasmid of 18.4 Md is obtained. When Bacillus subtilis 168 was transformed with these and the resulting plasmid was isolated from the resulting chloramphenicol-resistant strain, the molecular weight was approx.
Plasmid pTP62 of 9.1 Md is obtained. The number of cleavage sites of this product with restriction enzymes and the molecular weight of the resulting fragments are as follows.

【table】

[Table] Example 1 (1) Wild strain of Streptococcus fuecalis
Conjugative transfer of tetracycline, chloramphenicol, streptomycin, kanamycin, and erythromycin resistant KSf2 strain derived from KSf2 strain to KSf24m strain Streptococcus faecalis KSf2 strain and resistant to fusidic acid and rifampicin, but not resistant to other drugs The KSf24m strain, which also does not have a plasmid, is inoculated into a brain heart infusion (BHI) liquid medium, and cultured with shaking at 37°C. When the absorbance reaches 0.8 (600 nm), mix 0.1 ml of the culture solution of the KSf2 strain and 0.01 ml of the culture solution of the KSf24m strain and spread it on a BHI plate. 37℃,
After 20 hours of incubation, 0.85% sodium chloride solution 1.0
ml and scrape off the bacterial moss to make a bacterial suspension. Dilute this bacterial suspension with 0.85% sodium chloride solution to make 10x and 100x solutions. Rifuampicin (25 μg/ml), fusidic acid (25 μg/ml), tetracycline (3.13 μg/ml)
ml) of the bacterial suspension stock onto a BHI plate containing
Apply 0.1ml each of the 10x diluted solution and the 100x diluted solution. After incubation at 37°C for 48 hours, BHI of the same composition was added to the colonies generated on this BHI plate.
Perform single colony isolation three times using plates. Regarding the colonies obtained here, tetracycline, erythromycin, chloramphenicol, streptomycin, kanamycin,
Susceptibility tests to rifampicin and fusidic acid were conducted. Colonies that were resistant to all drugs were designated as KSf2-2 strain. (2) Isolation of plasmid DNA from strain KSf2-2 Strain KSf2-2 was inoculated into 10 ml of BHI 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%, Deifco Casamino Acid 0.2%, Deifco Yeast Extract 0.1%, Magnesium Sulfate 0.025%,
Absorbance 0.1 (600n) in 10ml of glucose 0.5%, PH7.0
m). Culture was carried out 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 the final concentration.
It was added at a concentration of 10 μCi/ml. 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. The pellet was resuspended in TES buffer (Tris-HCl buffer (PH 8.0) containing 0.05M sodium chloride, 0.005M EDTA). next,
0.2 ml of 0.5 MEDTA 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. Brizy 58 was added to a final concentration of 0.5% and incubated at 37°C for 10 minutes. Next, add sodium N-lauroyl sarcosinate to a final concentration of 2.5%.
and cultured at 50°C for 30 minutes to perform bacteriolysis. The lysate was made viscous with a pipette, softened, and then mixed with cesium chloride and ethidium bromide to form a solution with a density of 1.553. This solution
Centrifugation was performed at 126,000×g until equilibrium (approximately 40 hours). Fractionate the solution from the lower end of the centrifuge tube, adsorb 10μ of each fraction onto Watzmann filter paper GF/A, and measure the radioactivity of tritium adsorbed on this filter paper using a liquid scintillation counter (Patsucard 3300). It is measured and expressed in cpm.
It gives two large peaks, a highly active fraction derived from chromosomal 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 converted to SSC (0.15M sodium chloride,
The plasmid was isolated by dialysis against a 10-fold dilution of 0.015M sodium citrate (PH7.0).
Molecular weight: 37.5 Md (3) Transformation of Bacillus subtilis strain RM125 with plasmid DNA obtained from strain KSf2-2 A primary culture medium was prepared as follows. K ₂ HPO ₄・3H ₂ O 18.3 g KH ₂ PO ₄ 6.0 g Sodium citrate・2H ₂ O 1.0 g Ammonium sulfate 2.0 g Casamino acid 0.5 g Distilled water After dissolving 80 ml or more, sterilize at 121℃ for 15 minutes. Separately sterilized magnesium solution (20%
MgSO ₄ 7H ₂ O) 10ml, glutamate solution (100mg/ml) 1ml, tryptophan solution (5ml
mg/ml). Take 10ml of this mixture, 1ml of leucine solution (5mg/ml), 1ml of arginine solution (5mg/ml), 1ml of glucose solution (50%),
87 ml of sterile distilled water was added to serve as a primary culture medium. This is a restriction skeleton-deficient/modification-deficient strain of Bacillus subtilis.
Inoculate with RM125 strain. The turbidity of the primary culture medium at the time of bacterial inoculation was set to 10 Klett units (Klett-Samerson absorbance meter, filter number 54). After inoculation, culture with shaking at 37℃, and when it reaches 100 Kretz units, dispense 2ml into small test tubes containing 0.1ml of glycerin, quickly freeze with dry ice and acetone, and store at -80℃. did. The entire amount of the stored primary culture was inoculated into 10 ml of a secondary culture medium having the following composition. The secondary culture medium was prepared as follows. K ₂ HPO ₄・3H ₂ O 18.3 g KH ₂ PO ₄ 6.0 g Sodium citrate・2H ₂ O 1.0 g Ammonium sulfate 2.0 g Distilled water 80 ml After dissolving and sterilizing, add separately sterilized magnesium solution (20% MgSO ₄・7H ₂ O) 1 ml, glutamate solution (100 mg/ml) 0.1 ml, tryptophan solution (5 mg/ml) 0.1 ml, histidine solution (5 mg/ml) 0.1 ml, 5% casamino acid solution
Add 0.2 ml and 0.5 ml of sterile distilled water. Main mixture 1
Separately sterilized leucine solution (5 mg/ml) 0.1 ml
ml, 0.1 ml of arginine solution (5 mg/ml), 0.1 ml of glucose (50%), and 8.7 ml of sterile distilled water were added to prepare a secondary culture medium. The entire volume of the stored primary culture was inoculated into this 10 ml. After shaking culture at 37°C, 0.9ml of competent culture with 110 Kretz units was added.
Plasmid DNA solution obtained from KSf2-2 strain
Add 0.1 ml and incubate at 37°C for 1 hour. Next, add 4 ml of BHI medium and culture with shaking at 37°C for 24 hours. 0.1 ml of the main culture was applied to a BHI agar plate containing 6.25 μg/ml of chloramphenicol.
After culturing at 37°C for 24 hours, chloramphenicol-resistant strains were obtained at a frequency of 1 in 10 ⁹ to 10 ¹⁰ strains. The obtained chloramphenicol-resistant strain
The KBS strain was 14. KBS14 strain has a molecular weight of 9.1 and 18.4
It contained two types of megadalton plasmid DNA. How to extract plasmid DNA from Bacillus subtilis KBS14 strain Streptococcus faecalis
Plasmid DNA was isolated from the KSf2-2 strain in the same manner. (4) Plasmid obtained from Bacillus subtilis KBS14 strain
Transformation of Bacillus subtilis LMAH strain by DNA
subtilis 168LMAH761222) was competently cultured by the same method as Bacillus subtilis RM125 strain.
However, instead of the arginine solution in the primary culture medium, 1 ml of methionine solution (5 mg/ml), 1 ml of histidine solution (5 mg/ml), and 1 ml of adenine solution (2
mg/ml) was added to make 85 ml of distilled water. Similarly, for the secondary culture medium, instead of the arginine solution, 0.1 ml of methionine solution (5 mg/ml), 0.1 ml of histidine solution (5 mg/ml), and 0.1 ml of adenine solution (5 mg/ml) were used.
mg/ml) was added to make 8 ml of distilled water. In addition to this, plasmid DNA obtained from Bacillus subtilis KBS14 strain
was added and cultured in the same manner as in (4) to obtain chloramphenicol-resistant colony KBS15. (5) Streptococcus fuecalis KSf2−2
Plasmids from Bacillus subtilis strain KBS15 possessing a chloramphenicol resistance plasmid derived from the strain
Isolation of pTP62 Bacillus subtilis strain KBS15 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 1000 ml 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%. 18~ at 37℃
After shaking culture for 24 hours, incubate at 10,000 x g for about 15 minutes.
Separate from the broth at °C and decant the supernatant from the bacterial pellet. Discard the supernatant and remove the pellets.
After washing twice with TES buffer, 100 ml of 25%
Resuspend in TES buffer containing sucrose. then 0.5M
- Add 40 ml of EDTA solution and 20 ml of Lyzuzyme 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. moreover
Add 20 ml of N-lauroylsarcosine sodium 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, leave it overnight, add polyethylene glycol 6,000 to a final concentration of 10%, and leave it overnight at 4°C. did. 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. This solution was heated at 126,000 x g until equilibrium (approximately 40
time) Centrifuge. Under UV irradiation (365nm)
Linear chromosomes and plasmid DNA are visible in the centrifuge tube as intensely fluorescent bands. The plasmid DNA was removed from the density gradient, extracted twice with n-octanol to remove ethidium bromide, and the aqueous layer was then dialyzed against 10-fold diluted SSC buffer to yield pTP62. (6) Restriction enzyme digestion and agarose gel electrophoresis All restriction enzymes used were those manufactured by Bethesta Research Laboratories, Inc. Digestion of plasmid DNA was performed under the following conditions. Hinc 10mM Tris-HCl buffer (PH7.9),
60mM sodium chloride, 6.6mM magnesium chloride, 1mM dithiothreitol (DTT), 37°C Tac 10mM Tris-HCl buffer (PH7.4),
10mM magnesium chloride, 0.1mM DTT,
60℃ 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 DTT,
37℃ Hind 20mM Tris-HCl buffer (PH7.5),
7mM magnesium chloride, 60mM sodium chloride, 37°C Pst 20mM Tris-HCl buffer (PH7.5),
10mM magnesium chloride, 5mM ammonium sulfate, 100μg/ml BSA, 30°C Hinf 6mM Tris-HCl buffer (PH7.5),
6mM magnesium chloride, 100mM sodium chloride, 6mM 2-mercaptoethanol,
37℃ Taq 10mM Tris-HCl buffer (PH8.4),
6mM magnesium chloride, 6mM 2-mercaptoethanol, 65°C Sma 15mM Tris-HCl buffer (PH8.0),
6mM magnesium chloride, 15mM potassium chloride, 32°C Hpa 20mM Tris-HCl buffer (PH7.4),
10mM magnesium chloride, 1mM DTT,
6mM potassium chloride, 37°C. Digestion mixtures were analyzed in 1% agarose gels prepared by Sharp et al. (J.Sharp
et al “Reaction of two restriction enzyme activities in Haemophilus parainfluenza using agarose-ethidium bromide electrophoresis analysis”
Biochemistry, 12 , 3055–3063 (1973))
EcoRI-digested λDNA was used as a molecular weight control. (Thomas et al., 1975 “Bacteriophage lambda DNA with EcoRI restriction enzymes”)
The number of restriction enzyme cleavage points of pTP62 and the molecular weight of the cleavage fragment are as shown above. Example 2 (1) Bacillus subtilis 168AG strain [Bacillus subtilis405-113-B6] was used instead of Bacillus subtilis LMAH strain in (4) of Example 1.
(ATCC No. 19163)] and cultured in the same manner to obtain the KBS15b strain. However, instead of the leucine solution and arginine solution in the primary culture medium, use 1 ml of adenine solution (2 mg/ml) and guanine (5 mg/ml) as the medium.
ml), 0.5 ml of adenine solution (5 mg/ml) and guanine (5 mg/ml) instead of leucine solution and arginine solution in the secondary culture medium.
0.1 ml was added. (2) The Bacillus subtilis strain 15b obtained in (1) above was treated in the same manner as in (4) of Example 1 to generate the same plasmid as in Example 1.
pTP62 was obtained.

Claims (1)

[Claims] 1. A plasmid isolated from Streptococcus faecalis is transferred to Bacillus subtilis, and the plasmid isolated from the plasmid has a molecular weight of about 9.1×10 ⁶ daltons, the number of cleavage sites with a restriction enzyme, and the resulting fragment. Plasmid pTP62 with the following molecular weight: [Table] 2 Isolate a plasmid from Streptococcus fuecalis, transfer it to a restriction-deficient or modification-deficient strain of Bacillus subtilis, isolate a plasmid from this, further transfer this plasmid to Bacillus subtilis and multiply it. A method for producing plasmid pTP62, which comprises isolating the plasmid from it.