JPS59162883A - Plasmid pmvx group and pmvy group - Google Patents

Abstract

PURPOSE:To obtain novel plasmid pMVx group and pMVy group having remarkable advantages in genetic operation, by separating from acetobacters. CONSTITUTION:The novel plasmid pMVx group and pMVy group having a molecular weight of 0.9-3.6 M-Dalton and having one recognition site by at least one kind of restriction enzyme were obtained from 11 kinds of acetic bacteria strains such as Acetobacter pasteurianus IFO3223, Gluconobacter sclerodius IAM1842, etc. The plasmid pMVx group has only one recognition site by EcoR I or HindIII, and is composed of pMV106, pMV201 and pMV107. The plasmid pMVy group has one recognition site by Acc I , and is composed of pMV108, pMV202, pMV203, pMV204, pMV205, pMV109, pMV110, pMV111, pMV 113, pMV114 and pMV115.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the pMVx group and pMVy group, which have small molecular weights and are extremely advantageous for genetic manipulation.

More specifically, the present invention is directed to an acetic acid bacterium isolated from acetic acid bacteria, which possesses only one recognition site for at least one kind of restriction enzyme, has a molecular weight of 0.9 to 3.6 megadaltons, and is extremely advantageous for genetic manipulation. pMV x group and pMvy group.

Until now, there have been very few reports on plasmids derived from acetic acid bacteria. For example, it has been reported that Acetobacter aceti 41023 has a plasmid with a molecular weight of 117 x 10' daltons (17 megadaltons), which was named pTA5001 (Agri BioChem 46
(2) 581-389, 1982), and it was also reported that Acetobacter aceti A1023 has a plasmid with a molecular weight several times the molecular weight of 17 x 10' Daltons (Proceedings of the 1981 Conference of the Japanese Society of Agricultural Chemistry, p.
6) Furthermore, we searched for plasmids in 67 Gluconobacter-like bacterial type culture strains, and found the presence of plasmids in 27 strains.The molecular IT ranged from 2 to 72 megadaltons, and some strains had multiple plasmids (up to 4). Report that there is something 1 Fermentation Engineering Volume 61 No. 1 15-18,
(1983).

The present inventors conducted intensive research based on the assumption that there would be better plasmids in Acetobacter bacteria, and as a result, as shown in Table 1VC below, a large number of plasmids were found from 11 Acetobacter strains. It was isolated.

In the above table ta) indicates Fi megadaltons 2. Molecule t was measured by '1 pneumophoresis.

3. Images were plotted based on a standard line created based on the migration distance of circular double-stranded DNA of known molecular weight.

4. The DNA used was lambda phage DNA, pTA
5001, bacteriophage PM2DNA, R8F
'1o1o, pBR325, pACYC184, pA
CYC177゜pC194.

After further research in search of a more advantageous plasmid from the numerous plasmids derived from acetic acid ferment bacteria obtained here, we found a plasmid with a small molecular weight that is advantageous for genetic manipulation, and which has only one site recognized by a restriction enzyme. We have now established what can be called a group.

Among this group of plasmids, Be o RI or Hi
pM is the one that has only one recognition site by n d ■
This was designated as the Vx group. In the following Table 2, p belonging to the pMVx group
Mvio6. pMV201, pMV107O physicochemical properties and hosts are shown.

Furthermore, among the plasmid groups, those having only one m site recognized by AccI were classified as the pMVF group.

In the following Table 6, pMVypK belongs to pMVl 08
.

pMV202. pMV205. pMV204. pMV2
05. pMV109, pMVllo, pMVill,
pMVll5. The physicochemical properties and host of pPJV114°pM'V115 are shown.

The meanings of the abbreviations shown in Tables 2 and 3 above are as follows.

AccI: Ac1netobacter c
Restriction enzyme BamHI from B. alcoaceticus source: Bacillus amyloliq
ue#acie%s H source restriction enzyme Bst E H: Bacillus stea
lothermophilus ET source restriction enzyme C1a I: Caryophanon Latu
ml Source of restriction enzyme BcoRI: Escherichia coli R
Y13 source restriction enzyme Hindl[: Haemophilus 1nfl
uer+zae Rd source restriction enzyme Pst I: Providencia 5tua
Restriction enzyme 5ali from rtii source: 8treptomyces albu
s G source restriction enzyme There are no previously reported examples of the plasmids of the pMVx group and pMVy group of the present invention, and all of them are new plasmids, and the present cilasmid group has a molecular weight of 0.9 to 3.
It is extremely small at 6 megadaltons, making it extremely easy to make tn, and it is suitable for producing chimeric plasmids because it has only one recognition site for at least one restriction enzyme. It can be said that this is a group of plasmids that are useful for genetic manipulation.

Next, examples of the present invention will be shown.

Leprosy example 1 (Plasmid isolation and purification method) Acetobacter basturianus IF'03223 5
d A medium [2% glycerol, 0.2% ammonium chloride, 0.05% monopotassium phosphate, 0.05 dipotassium typhosphate, 0.02% magnesium sulfate.

0.2% yeast extract, 0.2% polypeptone, pH6
, 5] and cultured at 30°C for 24 hours. After that, subplant with new A medium 5ooyvc11,
The culture was shaken at 60°C for 36 hours [34]. After collecting bacteria, 20
The bacterial cells were washed twice with 50 m m ) Lis-HCl buffer (P) (B, 0) containing m M (1) EDTAk. 2 g of the obtained wet bacterial cells was mixed with 97 ml of TE8 buffer (5
411
A lysozyme solution (0.25M Tris-HCl, lysozyme 20~, pH 8.0) of Ll was further added, and the mixture was incubated at 0°C for 5 minutes. Next, add 4 d of 0.25 MEDTA solution (pH 8,0).
After addition, the mixture was incubated at 0°C for 5 minutes, and then reacted at 37°C for 20 minutes. After the reaction, 311 Ll of 10 sodium thirauryl sulfate was added, and after standing at 67°C for 20 minutes, 5N 5M saline was added, and the mixture was left standing at 0°C overnight. 60 for 48,200g
Centrifugation was performed for a minute, and the supernatant was collected. Next, polyethylene glycol 6 and ODD were added to this supernatant to a final concentration of 10%, and after standing at 4°C overnight,
The mixture was centrifuged at g for 10 minutes to obtain a precipitate.

, this precipitate was mixed with 7 ml of UC buffer (50 mM) Lis-HCl, 5 mM EDTA, 50 mM NaCl, p
After dissolving in H7,8), ethidium bromide was added to give a final concentration of 500 μg/p, and cesium chloride was further added to adjust the density to 1.57. This melt Q
Density gradient centrifugation was performed at fc15° C. and 100.200 g for 40 hours. After centrifugation, the centrifuge tube was irradiated with 365 nm ultraviolet light using an ultraviolet lamp, and the band that appeared below the chromosome band was separated as a plasmid fraction. Next, the fractionated solution was treated with Impropatool to completely remove ethidium bromide, and then treated with TE buffer (10mM Tris-HCl, 1mM EDTA,
Pti 7.5). Acetobacter basturianus IFO6226 has eight cilasmids with different molecular weights. After dialysis, the plasmid fraction was subjected to vertical 0.8 thiagarose gel electrophoresis, and the gel was stained by soaking it in ethidium bromide at 1 kg/kg. Thereafter, the corresponding plasmid band was fractionated on the gel under ultraviolet irradiation and isolated from other plasmids. Add 7M sodium perchlorate solution (6M) to the separated gel in an amount 6 times the weight of the gel.
Sodium perchlorate, 25mM disodium phosphate, 2
5mM monosodium phosphate, 10mM BDTA,
pH 70) was added, and the mixture was allowed to stand at 67°C for 1 hour to dissolve the agarose, and then DNA tl-adsorbed onto a Whatma/a [MGpc filter].

The adsorbed DNA was eluted with TE buffer, and 200 μl of 995% ethanol was added to 100 μl of this eluate.
After leaving at -80℃ for 2 hours, 15. [The DNA was centrifuged at 100 g for 5 minutes to precipitate the DNA, which was further vacuum-dried to obtain plasmid pvvio6.

(Cleavage specificity and molecular weight measurement method using various restriction enzymes) The plasmid prepared above was dissolved in TE buffer, and the restriction enzyme (AccI) was added in excess of at least 5 times.

BamHI, )lind*, Psi I and 8al I are manufactured by Takara Shuzo Co., Ltd., BstEII, C1aXI
tl Boehringer Mannheim, BcoR4, Bethesda Research, were all used. ) were reacted under optimal conditions for each restriction enzyme. After the reaction, it was analyzed by vertical agarose gel electrophoresis. That is, using a 1% agarose gel, Tris acetate buffer (40mM Tris, 20mM
The gel was run in acetic acid, 2mM BDTA, pH 8,1). The gel was then washed with 1 μg of ethidium bromide.
/mA' solution for staining. This gel was irradiated with UV light [-1] to determine the number of generated fragments, and from the migration distance of each fragment,
The molecular weight of each was calculated. Molecular tFi, Hind of lambda 7age DNA run simultaneously on the same agarose.
It was calculated based on a standard line created from the migration distance of each fragment of known molecular weight produced by u-cleavage.

Example 2 Isolation and purification of plasmid from Gluconobacter souffleleutelas IAM1842 was performed using G medium (
2% gluconic acid, 0.5 Tiglucose, 0.5 Tiglycerol, 0.2 Ti yeast extract, 0.2 Tivorivept/,
piis, 5), and the volume of new medium was 20 [1 mJ
Purified cilasmid pMv201i was obtained in the same manner as in Example 1, except that the culture time 'fr was changed to 24 hours. Specific cleavage with restriction enzymes and molecular weight measurement were also carried out according to Example 1.

Agent Patent Attorney 1) Parent Male

Claims (1)

[Scope of Claims] (1) It is derived from acetic acid bacteria, has only one recognition site for at least one restriction enzyme, and has a molecular weight of 0.9 to 6.
6 megadalton plasmids pMV x group and pMvy group (21gcoRI or FiHin dl recognition site) plasmid pMV x group according to claim 1. (31pMV106, Plasmid pM according to claim 1 consisting of pMV201 and pMV107
Vx liver. (4) Plasmid pMvy according to claim 1, which has only one A c c I recognition site
IR,. (51pMV108, pMV202, pMV203.p
MV204, pMV205, pMVID9, pMVl
10, pMVI 11, PMV115, pMVl 14
The plasmid pMVy group according to claim 1, consisting of , and pMVl 15.