JPH0322154B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to plasmid pMV203, which has a small molecular weight and is extremely advantageous for genetic manipulation.

More specifically, the present invention relates to plasmid pMV203, which is isolated from Acetobacter acetic acid bacteria, has a small molecular weight, and has few cleavage sites due to limiting oxygen.

Until now, there have been very few reports on plasmids derived from acetic acid bacteria. For example, Acetobacter aceti
No. 1023 has a plasmid with a molecular weight of 17 × 10 ⁶ Daltons (17 megadaltons), and this
It was reported that it was named pTA5001 (Agric Biol
Chem 46(2)2) 381-389, 1982), and
Acetobacter aceti No. 1023 has a molecular weight of 17×10 ⁶
It has been reported that Gluconobacter has a plasmid several times the size of Dalton's (Japan Agricultural Chemistry Society 1981 Conference Abstracts, p. 6), and a plasmid search was conducted on 37 Gluconobacter type culture strains. , the presence of plasmids was observed in 27 strains, and the molecular weight was 2 to 72 megadaltons, and it was reported that some strains had multiple (up to 4 types) of plasmids (Fermentation Engineering, Vol. 61, No. 1, 15-18, ( 1983))).

The present inventors conducted intensive research based on the assumption that a better plasmid existed in acetic acid bacteria, and as a result, they were able to isolate plasmid pMV203 from Gluconobacter suboxidans IFO3130. I've reached it.

The present invention relates to plasmid pMV203 shown in the restriction enzyme map of FIG.

The plasmid pMV203 of the present invention has Xho, Acc
/Sal/Hind and Sma cleavage sites, Aat, BamH, Bbe, Bcl, Bgl
, BstE, Cla, EcoR, EcoR, Hind
, Hpa, Kpn, Mlu, Nde, Mlu,
It is distinctive in that it does not have cleavage sites for Nde, Pst, Pvu, Sac, and Xba.

Furthermore, the molecular weight of plasmid pMV203 is 1.6 megadaltons (Md).

Plasmid pMV203 of the present invention can be isolated from cultured cells of Gluconobacter suboxidans IFO 3130.

Gluconobacter suboxidans IFO
For example, 3130 is cultured in A medium to obtain a large amount of bacterial cells, washed with a buffer solution, lysed with a lytic enzyme,
A supernatant is collected by centrifugation, polyethylene glycol is added to the supernatant, and the mixture is left at 4°C overnight, followed by centrifugation to obtain a precipitate.

The obtained precipitate was dissolved in a buffer solution, ethidium bromide was added, and cesium chloride was further added to make the density 1.57. Density gradient centrifugation was performed, and each chromosomal band was fractionated by ultraviolet irradiation in a centrifugal tube. Purify the fraction and use it as a plasmid.
pMV203 can be isolated.

The plasmid pMV203 of the present invention has not been previously reported and is a new plasmid. Furthermore, the plasmid has an extremely small molecular weight of 1.6 megadaltons, is extremely easy to purify, and has at least several restrictions. They can be said to be useful plasmids for genetic manipulation, as they each have one enzyme recognition site and are suitable for producing chimeric plasmids.

Next, examples of the present invention will be shown.

Example 1 (Plasmid separation and purification method) Gluconobacter suboxidans IFO
3130 in 5 ml of A medium [2% glycerol, 0.2%
Ammonium chloride, 0.05% monopotassium phosphate,
0.05% dipotassium phosphate, 0.02% magnesium sulfate, 0.2% yeast extract, 0.2% polypeptone, PH
6.5] and cultured with shaking at 30°C for 24 hours.
Thereafter, the cells were subcultured into 500 ml of new A medium at a concentration of 1%, and cultured with shaking at 30°C for 36 hours. 20m after collecting bacteria
50mM Tris-HCl buffer (PH
8.0) twice. Obtained wet bacterial cells 2
7 ml of TES buffer (50mM Tris-HCl,
Add 20mM EDTA, 25% sucrose, PH8.0),
The bacterial cells were suspended, and 4 ml of lysotium solution (0.25M Tris-HCl, 20 mg/ml of lysotium, PH8.0) was further added, and the mixture was allowed to stand at 0°C for 5 minutes. Next, add 4 ml of 0.25 MEDTA solution (PH8.0), leave it at 0℃ for 5 minutes, and then
The reaction was allowed to take place at ℃ for 20 minutes. After the reaction, 3 ml of 10% sodium lauryl sulfate was added and left to stand at 37°C for 20 minutes, then 5 ml of 5M saline was added and left to stand at 0°C overnight. Centrifuge at 48,200 x g for 60 minutes at 0°C.
The supernatant was collected. Then add this supernatant to a final concentration of 10%.
Polyethylene glycol 6000 was added to the mixture to give a precipitate. After standing overnight at 4°C, centrifugation was performed at 3000 x 10 minutes to obtain a precipitate. This precipitate was mixed with 7 ml of UC buffer (50mM Tris-HCl, 5mM EDTA,
After dissolving in 50mM NaCl, pH 7.8), add ethidium bromide to a final concentration of 500μg/ml, and then add cesium chloride to adjust the density.
Adjusted to 1.57. This solution was heated at 15℃ and 100,000×g.
Density gradient centrifugation was performed for 40 hours. After centrifugation, the centrifugation tube was irradiated with 365 nm ultraviolet light using an ultraviolet lamp, and the band appearing below the chromosome band was separated as a plasmid fraction. The fraction was then treated with isopropanol,
After removing ethidium bromide, TE buffer (10mM Tris-HCl, 1mM EDTA, PH7.5)
Dialyzed against. Gluconobacter suboxidans IFO 3130 has three plasmids with different molecular weights. After dialysis, the plasmid fraction was subjected to vertical 0.8% agarose gel electrophoresis, and the gel was diluted with 1 μg/ml ethidium bromide. After immersion staining, the corresponding plasmid band was fractionated on the gel under ultraviolet irradiation and isolated from other plasmids. Sodium perchlorate solution (6M sodium perchlorate, 25mM disodium phosphate, 25mM
monosodium phosphate, 10mM EDTA, PH7.0)
was added and left to stand at 37°C for 1 hour to dissolve the agarose, and then transferred to a Whattmann GFC filter.
DNA was adsorbed. Elute the adsorbed DNA with TE buffer, add 200μ of this eluate to 100μ of 99.5
After adding % ethanol and keeping it at -80℃ for 2 hours,
The DNA was precipitated by centrifugation at 15,000 rpm for 5 minutes, and the plasmid was separated by vacuum drying.
pMV203 was obtained. (Cleavage specificity and molecular weight measurement method using various restriction enzymes) The plasmids prepared above were dissolved in TE buffer, and reacted with at least a 5-fold excess of restriction enzymes under optimal conditions for each restriction enzyme. After the reaction, it was analyzed by vertical agarose gel electrophoresis. That is, 1
% agarose gel using Tris acetate buffer (40%
mM Tris, 20mM acetic acid, 2mM EDTA,
PH8.1). Thereafter, the gel was stained by immersing it in a 1 μg/ml solution of ethidium bromide.
This gel was irradiated with ultraviolet rays, the number of generated fragments was determined, and the molecular weight of each fragment was calculated from the migration distance of each fragment. The molecular weight was calculated based on a standard line created from the migration distance of each fragment of known molecular weight generated by Hind cleavage of lambda phage DNA that was electrophoresed simultaneously on the same agarose.

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme map of plasmid pMV203.

Claims (1)

[Claims] 1 Plasmid shown in the restriction enzyme map below
pMV203.