JPS6066983A - Plasmid - Google Patents

Abstract

PURPOSE:A novel acetic acid bacterium cyclic plasmid, collected from a microorganism belonging to the genus Acetobacter, and having specific decomposition characteristics for restriction enzymes and a specific molecular weight. CONSTITUTION:A cyclic plasmid (pQP54 plasmid), separated from a microorganism, e.g. Acetobacter aceti K1005 strain (FERM-P No.7219), by the alkali lytic or cleared lysate method, and having 1.7 (+ or -0.05)X10<6> daltons molecular weight. The plasmid is decomposed into one DNA fragment having 1.7X10<6> daltons molecular weight with a restriction enzyme HincII, and into two DNA fragments having 1.6X10<6> daltons and 0.1X10<6> daltons molecular weights with a restriction enzyme HindIII but will not be decomposed with restriction enzymes EcoR I , BamH I , Hinf I , Pst I and Xho I .

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to cilasmids, and more particularly to a novel plasmid collected from microorganisms belonging to Acetobacter.

Cilasmids, which are the main extrachromosomal genetic elements, are self-replicating genetic elements and are therefore widely used as useful vectors (carriers of foreign foreign fffl DNA) in gene recombination technology. Genetic recombination technology is a technology that creates new organisms by manipulating genes, and it has a wide range of applications, including biological technology, biomedicine, construction equipment, and agriculture.

The present inventors have conducted various studies with the goal of developing a genetic manipulation system using Acetobacter, and as part of that research, they have now succeeded in isolating a new Acetobacter plasmid from a microorganism similar to Acetobacter.

That is, the present invention provides a cyclic plasmid F with a maximum molecular weight of 1.7 (Shiho 05) x 106 daltons, which has been discovered from microorganisms that infect Acetobacter and exhibits the following decomposition characteristics against restriction enzymes. It is something to do.

It is decomposed into one fragment by single all, two fragments by Bind+n, and not decomposed by αtomoe R1, Tango HI, Hin fl, Dandan Ioyo uXho I.

Hereinafter, one aspect of the present invention will be explained in detail.

As a donor bacterium for the cilasmid of the present invention, Acetobacter
The 1005 strain (Ikoken Bacterial Report No. 7219, submitted on September 7, 1939) is used in Aceci.

This i7 strain is based on the general mycological properties of microorganisms that prey on Acetobacter aceti except for the following points (1) and (2). terminative pacteriorono (Be
rgey's Manual of ])etermi
Native Bacteriology 8th Edition No. 27
6-278 N (1974).

(No. 11 On a wort agar medium, a noble brown colony is formed with a flat center and a somewhat jagged periphery.

(2) In static culture using a wort liquid medium, unlike known strains of the same species, a phenomenon in which the culture solution rises close to the wall of the container is not observed, and the culture solution is cloudy and liquid. Direct shedding on the surface is also thin and fragile.

Examples of methods for isolating plasmids from the above-mentioned bacterial strains include the (X) alkaline lysis method and the (■Liclear F. Lysade method).

The four methods of the above three methods are as follows.

(tl alkaline butybetic method Aceto/ζphta-aceti KI005 cup strain was inoculated from a preserved slant medium into a liquid complete medium, and the cells were harvested after one standing culture.

The obtained bacterial cells were treated with EDTA (ethylenediamine 4N1).
After thorough washing with a buffer solution of pH 8,9 containing
Add 5DS (F-decyl sodium sulfate) solution to H12,45 and incubate. 5th floor of the material to be displayed
-) After removing impurities such as proteins by phenol extraction, add about 2 to 3 times the volume of ethanol to the lysate and leave it at about -10°C overnight to wash out the DNA. The sediment collected by centrifugation is subjected to 0.8% agarose gel electrophoresis. The migration position of each DNA, which is separated based on the difference in molecular weight, is detected using 365nrn ultraviolet light. After being immersed in an ethidium bromide solution, it emits fluorescence when irradiated with ultraviolet rays, so the migration position can be easily determined.

As a result, division of chromosomal DNA and five plasmid DNAs was confirmed. Then, of these plasmid DNAs, the section of plasmid DNA that migrates at the fastest speed is cut out and isolated, and used as a standard sample.

(1) Collect bacterial cells according to the above (11) method, pH 8, 0.
After washing with a buffer solution, the tubes are coated with lysozyme and SDS. After removing impurities in the bacterial solution by phenol extraction or the like, the DNA is precipitated by ethanol precipitation. In order to separate the plasmid in a closed circular DNA state, this DNA precipitate is subjected to cephram chloride-ethidium zolomide density gradient centrifugation to form a band of lt-CDNA. Among these, the plasmid DNA band portion is collected by an appropriate method, for example, by pulling it out from the side of the centrifuge tube at the injection side or sucking it up using a noso stool pipette, etc., and then adding ethidium bromide using isoamyl alcohol. After removal, the DNA is purified by side analysis or ethanol precipitation. Purified DNA was added to 0.8% agaro.
The plasmid fraction that migrates at the fastest speed is isolated by subjecting it to Sgelmi pneumothoresis analysis and using it as a standard according to method (1) above.

In both methods (4) and (11) described above, plasmid segments showing similar mobilities were observed in agarose gel electrophoresis. Each of these cilasmids was isolated and used as a standard, and their molecular papers were placed on a plasmid with a known molecular weight as an internal marker, and the mobility was compared. Good quality, 7 (-1=0.05
)
The product is called pQP54 plasmid. In addition, although (±0-05) in "-" accepts the 11 fixed difference range, 1'lri is omitted in the following description of the number of molecules.

In addition, each pQP54 plasmid library! 17 Restriction enzyme ()tin clI, I (in dllll, Eco R
I, Bam, Ifin fl.

The decomposition characteristics due to two
The results were as follows.

One DNA fragment with a molecular weight of 1.7 x 106 daltons was obtained using Hinc■, and each DNA fragment with a molecular weight of 1.6 x [
06 Dalton and 0.06 Dalton. lx106 Dalton's two D's
NA @ disassembled into pieces, Eco R11Bam Hl
, H7n fl , and Pst I have relatively low molecular weight and are difficult to break down when collected from bacterial cells, so they are very easy to extract, and they have few cleavage sites with restriction enzymes, so they can be used in real life. In genetic recombination operations, it has various advantages such as ease of handling.

The present invention will be explained in more detail below with reference to Examples. In the present invention, all % means N amount/capacity %.

Example A: Isolation and purification (I) Alkaline bacteriostatic method A loopful of bacterial cells of Acetopufter aceti KIO05 was inoculated from a preserved slant medium into five liquid complete mediums, and the mixture was inoculated until a bacterial film formed on the surface of the liquid. The liquid complete medium used in this example had a composition of 3% ethanol, 1% acetic acid, 1% polypeptone, 1% IWffl extract, 1% glucose, and the remaining water. It is something.

IflJI on the surface of the culture solution thus obtained was shaken to form a suspension, and then 1 ml of this solution was
1 was transferred to 100 mA of a liquid complete medium previously stored in a 500 mA Sakalo flask, and this was cultured with shaking at 15°C for 15 hours.

After culturing, collect the bacterial cells (wet Tsume Junho 15g).
After thorough washing with a mild M solution [Tris(hydrogyzomethyl)aminomethane) 50mM, EDTA at atrnM iQ degree, adjusted to pT40, 8 with salt 1j2].

It was suspended in the same buffer 0.5++6. Add to this the lysis buffer ('l'ris at 50rnM, EDTA at 20nM).
After adding 9.5 m6 of a solution containing 1% mM and SDS and adjusting the pH to 12.45 with NaOH and stirring well, the mixture was incubated at 26.5°C for 90 minutes to incubate. To this lysate was added 0.6 mt of 2M'l'ris HCI buffer (pH 7,0) and 1.2 ml of 5M NaCl (i7.
The solution was added and the mixture was allowed to stand at 0° C. for a minute to precipitate proteins, etc. This was centrifuged and the supernatant was removed, and the impurities contained therein were further removed by phenol extraction. After removing residual phenol by ether extraction method, add 2 to 2
Five 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, subjected to 0.8% agarose gel electrophoresis to separate each plasmid, and the electrophoresis position was determined at 355 nm.
Detected using ultraviolet light.

Cut out the gel of the DNA section that migrated the fastest using a sterilized razor blade, and add 3 times the volume of sodium perchlorate solution (8M
), and this solution was filtered through a glass filter (What
The DNA fragments were adsorbed onto the glass filter by vacuum filtration using a Soffner funnel equipped with a man GF/C, diameter 2.4 cTn, average particle size 1.3 μm. After washing this glass filter with a sodium perchlorate solution (6M) and further washing with a 75% ethanol solution at -20°C, the DNA was eluted from the glass filter in a TE buffer. Then, the ethanol was removed under reduced pressure, and one QP-specific cilasmid was removed.

(IT) Clear-Lysade method Collect bacteria according to method (r) above, and weigh the bacteria with a wet weight of t.
el) in 5QmM Tris HCI relaxation solution (pH 8,
After washing with 0), the suspension was suspended in 4me of the same buffer and cooled on ice.

Add 0.8 ml to this, Norizozyme solution (10 rng/m
e, 50 mM'l'ris HCI buffer, pH
8,0) and let it stand at 0°C for 5 minutes, then added 1.5 hours of 40mM EDTA solution (PI+8.0) and incubated again at 0°C.
Leave to stand for minutes, then 6.4 mb of 2% SDS solution (TE
buffer solution) was added thereto, and the mixture was allowed to stand at 0°C for an additional 2 hours. The treated solution was centrifuged for 1 hour at 3o and ooorpm, and the supernatant was treated with phenol according to the fl method described above, and then DNA was precipitated using ethanol.

The obtained DNA precipitate was dissolved in TE buffer and il'c
Add 6g of cesium chloride to it, dissolve it well, and add ethidium bromide liquid resistant (10m9/day) to 0.
．． 4. After adding ml, centrifugation was performed at 39,000 rpm for 40 hours. Two DNA pans P separated into upper and lower parts
The lower plasmid band was collected using a Norstool pipette. Ethidium gromide was removed from the recovered plasmid fraction by isoamyl alcohol extraction, and cesium chloride was removed by repeating the ethanol precipitation method.

The purified DNA precipitate was subjected to 0.8% 7)fo-Sgelumi electrophoresis according to method (1) above to obtain a pQP54 cilasmid sample.

B: Molecular weight measurement pQ determined by method (1) and method (U) above
As a result of measuring the molecular weight of P54 Shirasumi P by agarose gel electrophoresis analysis, it was found that the molecular weight of this pQP54 plasmid was 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) Device Vertical slab gel electrophoresis device b1 (2) Gel 0.8% aga o-x [:/manufactured by Sigma] (3) Voltage 0V (4) Buffer E buffer Solution (40mM Tris, 20rr+M vinegar, 2-resistant MEDTA, pH 8,1) Under the above operating conditions, first, the specimen was coated with a staining marker [50% sucrose, Z5]. rnM
El) TA,.

0.05% BPB (bromophenol blue'I,
, 0.05% Gyzolene/Anol FF], and then placed on the top of the Kel and electrophoresis was started. After performing electrophoresis in accordance with the conventional method, it was immersed in 0.5 μg/me ethidium bromide solution for 10 minutes or more, and then exposed to a 365 nrn ultraviolet light to generate fireflies.
/i3 Shadow was performed.

Table 1 psc138 9. '(i pBR3135, 8 pBR3222, 6 C: Restriction enzyme separation M characteristics pQp 54 Plasmid standard various restriction enzymes (l1in
clI, Hindll, EcoRI, BamHI,
1) stl, Pst I, and Xho I, all commercially available products), and were decomposed into two DNA fragments of M tons.
and

At this time, the digestion is carried out in the reaction buffer shown in Table 2 below depending on the restriction enzyme.
After reacting for 2 hours at 65'C, the reaction was stopped by heating at 65'C for 10 minutes, and the mixture was rapidly cooled on ice. The molecule fl after decomposition is measured by using a fragment of known molecular weight obtained by decomposing λ DNA with Hin dll as an internal marker, and measuring the molecule fl of B above as an internal marker.
This was carried out according to the method specified by FllJ.

The results are shown in Table 3 below.

Table 2 PH7,4 Hin fl Bi1 pH7,4 Table 3 Cavity R1 decomposed / good bonito H (tt Kie full / Hitomi I 〃 Teri I 〃 From Table 3 above, I) QP5.4 cilasmid axis di
When treated with rt, only a 1.6XI06 dalton DNA fragment was detected by agarose gel electrophoresis, but when this plasmid was treated with 1('in CAT), a 1.7Xto6 dalton DNA fragment was detected. , and as a result of molecular engineering i″4111, this plasmid is 1.7
Due to the fact that it is x106 Daltons, this plasmid has 1.6
．． The smaller DNA fragment is separated into two DNA fragments by agarose gel electrolysis.
), it is presumed that it was not detected.

The above 4 (:) was confirmed by the results of the following ?i1 study in which a composite plasmid was created using the pQP 54 plasmid of the present invention and a known E. coli vector using single-mounted cleavage.

322 large) Using the Shukan plasmid, this Kokoroyama ■ Cut 1
Local fragment (cut at the site of the tetracycline-degradable gene [fT
t ) and HindlH-cut 1 of pqp 54 cilasmid
The analyte fragments were ligated with T-4 ligase, and the thus-prepared composite plasmid was used to infect a known host strain E-4.
E. coli C-600nal' strain (which has been confirmed not to harbor plasmids) was transformed. Here, this complex plasmid is pBR322
Since it has the Hin dllT fragment of the plasmid, it has ampicillin resistance but has lost tetrazycline resistance (that is, it is sensitive to tetracycline), and its molecular weight is 2.6 x 106 Daltons or more. It is expected that it will be a good thing. Therefore, when we searched for plasmids held in E. coli transformed strains showing the genetic traits of ampicillin resistance and tetracycline sensitivity, we found 4.2 x 106 Daltons and 2.7 x IO6 $.
A plasmid with a molecular weight similar to Luton was detected. If we subtract the molecular weight of the pBR322 plasmid, 2.6 x 106 Daltons, from the molecular weights of these two plasmids, we get 1.6 x 106 Daltons and 0. 1×106 melton, which is 1×106 melton.

The pQR54 plasmid was modified by dm to 1.6X10
6 Dalton sized fragments and 0. This is interpreted as not indicating that IXtO6 was degraded into dalton-sized fragments.

In addition, in the above-mentioned research, the transformation of large intestine and the search for cilasmids were carried out using "molecular cloning" (Mo1ec).
ular Cloning, T. Maniati
s, E+F, Fri tsh.

and J, Sambrook, ed., Cold Spri.
ng Harbor Laboratory Publishing, 19
1% to the method described in 1982)! I did it by H.

1. Usability 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. , for example, as mentioned above 5
This ff1n dllF truncated 11if fragment has potential applications in the field of genetic recombination technology, such as the production of composite cilasmids and its use in the transformation of acetic acid bacteria or Escherichia coli.

Applicant's representative Inomata/1'] Procedural amendment dated October 1989 q: Pi+, - Director-General Kazuo Wakasugi 1, Indication of case 4/14/1980 F, Application No. 172805 No. 2
, Title of the invention Plasmid 3, Person making the amendment Relationship with the case 4.5 Applicant Kewpie Corporation (one person) 8. Details of the amendment, page 417, line 11 and W, page 18, line 1 'I)
QB54.1 is corrected to r pQP 54 J.

Claims (1)

[Scope of Claims] 1. A molecule j411.7 collected from a microorganism belonging to Acetobacter and having the following decomposition characteristics with respect to restriction enzymes.
(±0.05) × 106 Dalton circular plasmid. Once in all is decomposed into one fragment, H4ndlll is decomposed into two fragments, μヱold, BamHI, [in fl
. It is not decomposed in Danjuku I and Einaka ■. 2. The circular plasmid according to claim 1, wherein the microorganism is Acetobacter aceti KIO05 strain (Kikoken Bacteria No. 7219).