JPH0322155B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to plasmids, and specifically relates to a novel plasmid collected from microorganisms belonging to Acetobacter. Plasmids, which are the main extrachromosomal genetic elements, are self-replicating genetic elements and are therefore widely used as useful vectors (carriers of foreign heterologous DNA) in genetic recombination technology. Genetic recombination technology is a technology that creates new organisms by manipulating genes, and has a wide range of applications, including biotechnology, biomedicine, industry, and agriculture. The present inventors have conducted various studies with the aim of developing a genetic manipulation system using Acetobacter, and as part of this research, they have now succeeded in isolating a new Acetobacter plasmid from a microorganism belonging to the Acetobacter family. That is, the present invention provides a plasmid collected from a microorganism belonging to Acetobacter, which has a molecular weight of
1.7±0.05 megadaltons (× ¹⁰⁶ daltons),
It has the following restriction enzyme cleavage map, and Eco RI,
It provides a circular plasmid that is not degraded by Bam HI, Hin f I, Pst I and Xho I restriction enzymes. The present invention will be explained in detail below. As a donor bacterium for the plasmid of the present invention, Acetopacter aceti K1005 gold strain (Feikoken Bacterial Serial No. 7219
No., September 7, 1981) is used. This strain has the general mycological properties exhibited by microorganisms belonging to Acetobacter aceti except for the points (1) and (2) below, such as Virgies manual.
Bergey's Manual of Determinative Bacteriology
Bacteriology) 8th edition, pages 276-278 (1974)
It has the properties described in . (1) On wort agar medium, it forms yellow-brown colonies with a flat center and somewhat jagged edges. (2) In static culture using a wort liquid medium, unlike known strains of the same species, a phenomenon such as the culture solution climbing up the side wall of the container was not observed, and the culture solution was cloudy and the liquid surface The bacterial membrane that forms is also thin and fragile. Examples of methods for isolating plasmids from the above-mentioned bacterial strains include () alkaline lysis method and () cleared lysate method. The outline of the above two methods is as follows. () Alkaline lysis method Inoculate the cells of Acetobacter aceti K1005 strain from a preserved slant medium into a liquid complete medium, and collect the cells after shaking culture. After thoroughly washing the resulting bacterial cells with a pH 8.0 buffer containing EDTA (ethylenediaminetetraacetic acid),
Suspend in the same buffer, then add to this suspension
Add SDS (sodium dodecyl sulfate) solution adjusted to pH 12.45 with NaOH to lyse. After removing impurities such as proteins from the eluted substances by phenol extraction method, approximately 2 to
Add 3 times the volume of ethanol and leave to stand overnight at approximately -20°C to precipitate the DNA. The precipitate collected by centrifugation is subjected to 0.8% agarose gel electrophoresis. Each separated on the basis of molecular weight difference
The DNA migration position is detected using 365 nm ultraviolet light. Each DNA emits fluorescence when irradiated with ultraviolet rays after being immersed in an ethidium bromide solution, so the migration position can be easily determined. As a result, chromosomal DNA and five plasmid DNAs
The classification is confirmed. Then these plasmids
The section of plasmid DNA that migrates at the fastest speed is cut out and isolated, and used as a standard. () Cleared lysate method Collect bacterial cells according to the method () above, and then
Lysozyme and SDS after washing with pH8.0 buffer
Lyse the bacterium using After removing impurities in the lysate by phenol extraction or the like, DNA is precipitated by ethanol precipitation. This method is used to isolate plasmids in a closed circular DNA state.
The DNA precipitate is subjected to cesium chloride-ethidium bromide density gradient centrifugation to band the DNA. The plasmid DNA band portion is collected by an appropriate method, such as by pulling it out from the side of the centrifuge tube with a syringe needle or sucking it up using a Pasteur pipette, etc., and then extracting ethidium bromide using isoamyl alcohol. After removal, the DNA is purified by dialysis or ethanol precipitation. The purified DNA is subjected to 0.8% agarose gel electrophoresis analysis, and the plasmid segment that migrates at the fastest rate is isolated according to method () above and used as a standard. In both the above-mentioned methods () and (), plasmid segments showing similar mobilities were observed in agarose gel electrophoresis. Each of these plasmid segments was isolated and used as a standard, and their molecular weight was determined by comparing the mobility in agarose gel electrophoresis using a plasmid with a known molecular weight as an internal marker. ) × 10 ⁶ Daltons, and it was confirmed that they were the same. In the present invention, this preparation is referred to as pQP54 plasmid.
Note that (±0.05) in the above indicates the measurement error range, but is omitted in the description of the molecular weight below. In addition, various restriction enzymes (Hin c, Hin d, Eco RI, Bam HI,
The decomposition characteristics by Hin fI, Pst I and Xho I were investigated according to conventional methods, and the results were as follows. Hin c with a molecular weight of 1.7 x 10 ⁶ Daltons
DNA fragments each have a molecular weight of 1.6 × Hin d.
10 ⁶ Daltons and two pieces of 0.1×10 ⁶ Daltons
Digested into DNA fragments, Eco RI, Bam HI, Hin
Not degraded by fI, Pst I and Xho I. As mentioned above, the plasmid of the present invention has a relatively low molecular weight as a plasmid, and is difficult to break down when collected from bacterial cells, so it is very easy to extract, and there are no restriction enzyme cleavage sites. Because it is small in number, it is easy to handle in actual genetic recombination operations, etc.
It has various advantages. Hereinafter, the present invention will be explained in more detail with reference to Examples. In the present invention, all % means weight/volume %. Example A: Isolation and purification () Alkaline lysis method A loopful of Acetobacter aceti K1005 bacterial cells was inoculated from a preserved slant medium into 5 ml of liquid complete medium, and this was incubated at 30°C until a bacterial film formed on the surface of the liquid. It was left undisturbed. The liquid complete medium used in this example contains 3% ethanol, 1% acetic acid, 1% polypeptone, and 1% yeast extract.
%, glucose 1% and balance water. The bacterial membrane on the surface of the thus obtained culture solution was broken by shaking to form a suspension, and then 1 ml of this solution was transferred to 100 ml of a liquid complete medium previously placed in a 500 ml Sakaguchi flask. The cells were cultured with shaking at 30°C for 15 hours. After culturing, collect the bacterial cells (wet weight 0.15
g) in TE buffer [Tris (tris(hydroxymethyl)aminotamene) 50mM, EDTA
After washing thoroughly with a solution with a concentration of 20 mM and adjusted to pH 0.8 with hydrochloric acid, it was suspended in 0.5 ml of the same buffer. Add lysis buffer (Tris
(50mM EDTA, 20mM EDTA, 1% SDS, adjusted to pH 12.45 with NaOH) 9.5ml
was added and stirred well, and then left to stand at 26.5°C for 90 minutes to lyse the bacteria. Add 0.6ml of 2M to this lysate.
1.2ml 5M with Tris HCl buffer (PH7.0)
A NaCl solution was added and the mixture was allowed to stand at 0° C. for 30 minutes to precipitate proteins, which were then centrifuged and the supernatant liquid was removed from which impurities were further removed by phenol extraction. After removing residual phenol by ether extraction method, 2
~2.5 times the volume of ethanol was added and the mixture was allowed to stand overnight at -20°C to precipitate DNA. The DNA precipitate was dissolved in TE buffer and subjected to 0.8% agarose gel electrophoresis to separate each plasmid, and the migration position was detected using 365 nm ultraviolet light. Cut out the gel of the DNA segment that migrated the fastest using a sterilized razor blade, dissolve it in 3 times the volume of sodium perchlorate solution (8M), and filter this solution through a glass filter (Whatman GF/
C, diameter 2.4 cm, average particle size 1.3 µm) was suctioned through a Buchner funnel, and the DNA fragments were adsorbed onto the glass filter.
After washing this glass filter with a sodium perchlorate solution (6M) and further washing with a 75% ethanol solution at -20°C, DNA was eluted from the glass filter in a TE buffer. Then, ethanol was removed under reduced pressure to obtain a pQP54 plasmid preparation. () Cleared lysate method Collect cells according to method () above, wash the cells (wet weight 2g) with 50mM Tris HCl buffer (PH8.0), and suspend them in 4ml of the same buffer. and ice-cold. Add 0.8ml of lysozyme solution (10mg/ml, 50mM Tris HCl buffer, PH8.0) to this and leave it for 5 minutes at 0°C, then add 1.6ml of 40mM EDTA solution (PH8.0) and add it again. Let stand for 5 minutes, then add 6.4ml of 2
% SDS solution (in TE buffer) was added and allowed to stand at 0°C for an additional 2 hours. The treated solution was centrifuged at 30,000 rpm for 1 hour, the supernatant was treated with phenol according to the method () above, and then DNA was precipitated using ethanol. The obtained DNA precipitate was dissolved in TE buffer solution to a weight of 6 g, 6 g of cesium chloride was added thereto and dissolved well, and 0.4 ml of ethidium bromide solution (10 mg/ml) was added.
Centrifugation was performed at 39,000 rpm for 40 hours. Of the two upper and lower DNA bands generated, the lower plasmid band was recovered using a Pasteur pipette. Ethidium bromide was removed from the recovered plasmid section by isoamyl alcohol extraction, and cesium chloride was further removed by repeating ethanol precipitation. The purified DNA precipitate was subjected to 0.8% agarose gel electrophoresis according to method () above.
A pQP54 plasmid preparation was obtained. B: Molecular weight measurement The molecular weight of the pQP54 plasmid obtained by the above methods () and () was measured by agarose gel electrophoresis analysis.
The molecular weight of the pQP54 plasmid was found to be 1.7×10 ⁶ Daltons. This agarose gel electrophoresis analysis was carried out as described below, and the internal markers used were plasmids with known molecular weights shown in Table 1 below. Agarose gel electrophoresis analysis (1) Equipment Vertical slab gel electrophoresis device (2) Gel 0.8% agarose [manufactured by Sigma] (3) Voltage 80V (4) Buffer E buffer (40mM Tris, 20mM acetic acid,
2mM EDTA, PH8.1) Under the above operating conditions, first dilute 1/2 of the standard sample.
~1/3 amount of staining marker [50% sucrose, 25
After thoroughly mixing with mM EDTA, 0.05% BPB (bromophenol blue), and 0.05% xylene cyanol FF], it was placed on the top of the gel and electrophoresis was started. 0.5μg/ml after electrophoresis according to the usual method
Soak in ethidium bromide solution for at least 10 minutes,
Next, the object was photographed after being exposed to 365 nm ultraviolet light to generate fluorescence. Table 1 Name of plasmid used as internal marker Nominal molecular weight (× ¹⁰⁶ Daltons) pSC138 9.6 pBR313 5.8 pBR322 2.6 C: Degradation characteristics with restriction enzymes pQP54 plasmid preparation was
c, Hin d, Eco RI, Bam HI, Hin
fI, Pst I and Xho I, all commercial products)
As a result of treating the plasmid with Hin c and investigating its degradation characteristics, this plasmid was converted into one DNA fragment with a molecular weight of 1.7 × 10 ⁶ Daltons by Hin c, and a molecular weight of 1.6 × 10 ⁶ Daltons and 0.1 × 10 ⁶ Daltons by Hin d, respectively. Bam is broken down into two DNA fragments.
It was found that it was not degraded by HI, Hin fI, Pst I and Xho I. In addition, the restriction enzyme cleavage map of the pQP54 plasmid is as shown above. At this time, the digestion is carried out in the reaction buffer shown in Table 2 below depending on the restriction enzyme. In each case, the reaction is first carried out at 37°C for 2 hours, and then at 65°C.
The reaction was stopped by heating for 10 minutes, and the mixture was rapidly cooled on ice. Molecular weight measurement after degradation is performed using λDNA as Hin
Using the fragment of known molecular weight degraded in step d as an internal marker, the measurement was carried out according to the method for measuring the molecular weight in B above. The results are shown in Table 3 below.

【table】

[Table] Decomposed into

[Table] From Table 3 above, pQP54 plasmid was Hin d
When treated with Hin c, only a DNA fragment of 1.6 × 10 ⁶ daltons was detected by agarose gel electrophoresis, but when this plasmid was treated with Hin c, a DNA fragment of 1.7 × 10 ⁶ daltons was obtained.
And as a result of molecular weight measurement, this plasmid has a molecular weight of 1.7×10 ⁶
Due to the fact that it is a Dalton, this plasmid
It is estimated that Hin d degrades the DNA into two DNA fragments of 1.6×10 ⁶ Daltons and 0.1×10 ⁶ Daltons, but the smaller DNA fragment was not detected by agarose gel electrophoresis. The above assumption was confirmed by the results of the following study in which a composite plasmid was constructed using the pQP54 plasmid of the present invention and a known E. coli vector using Hin d cleavage. That is, as an E. coli vector, it has ampicillin resistance and tetracycline resistance, and has a molecular weight of 2.6.
Using the known pBR322 E. coli plasmid of ×10 ⁶ daltons, this Hin d cleavage fragment (cut at the site of the tetracycline resistance gene) was ligated with the Hin d cleavage fragment of the pQP54 plasmid using T-4 ligase. A known host strain, E. coli C-600nal ^r strain (which has been confirmed not to retain the plasmid), was transformed using the composite plasmid obtained. Here, this complex plasmid contains Hin d of pBR322 plasmid.
Because it has a fragment, it has ampicillin resistance but has lost tetracycline resistance (that is, it is sensitive to tetracycline), and its molecular weight is 2.6 × 10 ⁶ Daltons or more. Be expected. Therefore, when we searched for plasmids held in E. coli transformed strains showing genetic traits of ampicillin resistance and tetracycline sensitivity, we found that 4.2×10 ⁶ Daltons and 2.7×
A plasmid with a molecular weight of 10 ⁶ Daltons was detected. From each molecular weight of these two types of plasmids,
If we subtract the molecular weight of pBR322 plasmid, which is 2.6× ¹⁰⁶ Daltons, we get 1.6× ¹⁰⁶ Daltons and 0.1×
10 ⁶ Daltons, which means that the pQP 54 plasmid was degraded by Hin d into a fragment of 1.6 × 10 ⁶ Daltons and a fragment of 0.1 × 10 ⁶ Daltons. be interpreted. In the above research, the transformation of E. coli and the search for plasmids were performed using "molecular cloning".
(Molecular Cloning, T.Maniatis, EFFritsh,
and J. Sambrook, eds., Cold Spring Harbor.
Laboratory Publishing, 1982). Usefulness of the pQR54 plasmid of the present invention The plasmid of the present invention is an acetic acid bacterium plasmid that has advantages such as being a small molecule and having limited cleavage with restriction enzymes. This Hin
d Creation of a composite plasmid using the cleaved fragment;
It has high potential for use in the field of genetic recombination technology, such as for the transformation of acetic acid bacteria or Escherichia coli.

Claims (1)

[Scope of Claims] 1. A plasmid collected from a microorganism belonging to Acetobacter, which has a molecular weight of 1.7±0.05 megadaltons, has the following restriction enzyme cleavage map, and has Eco RI, Bam HI, and Hin fI. , a circular plasmid that is not degraded by Pst I and Xho I restriction enzymes. 2. The circular plasmid according to claim 1, wherein the microorganism is Acetobacter aceti K1005 strain (Feikoken Bibori No. 7219).