JPS5936700A - Plasmid - Google Patents

Abstract

PURPOSE:Plasmid expected to be useful as a vector for preparing an active component of isoprenoid derivative, carotenoid derivative, etc., obtained from highly halophilic bacterium of Halobacterium halobium. CONSTITUTION:Cyclic plasmid having 27 megadalton molecular weight, obtained by subjecting a mold of variant derived from a highly halophilic bacterium of Halobacterium halobium R1(FERM-P 6677) to bacteriolysis in a low hypotonic solution, treating it with an alkali to give a plasmid fragment, removing protein from it with phenol, purifying it, which is decomposed with restriced enzyme BamHI into 6 fragments, with Hind III into 2 fragments, with BalI into 6 fragments, with PvuII into 7 fragments, with KpnI into 4 fragments, with HpaI into 2 fragments, and with SatI into 8 fragments, respectively. The plasmid has 61 GC(%) base composition and 92.5 deg.C melting temperature(Tm).

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to plasmids. Specifically, the present invention relates to a novel plasmid obtained from the highly halophilic bacterium Halobacterium halobium.

Grasmid, an extrachromosomal gene, has been developed in vitro.
vizro) vector in genetic recombination (v
ector) is widely used in genetic engineering.

The present inventors have demonstrated that high salt concentrations cause deoxyribonucleic acid (DNA
), we were searching for plasmids from Halobacterium halobium in order to investigate the effect on higher-order 41t instars, and we succeeded in isolating a new glasmid from Halobacterium halobium, thus achieving the present invention. That is, one gist of the present invention is obtained from the highly agglomerative bacterium Halobacterium halobium.

Limit r! #Molecular weight with the following decomposition characteristics for cumulative, 2
The 2-megadalton circular plasmid Ba-H1 was used to generate 1 fragments, Ba11 was used to generate 2 fragments, and pvu H was used to generate 7 fragments (r(, Kpnl). * It is decomposed into two fragments at Hpa 1 and into ? fragments at 8θtl.

exists in

To explain in detail the present invention, the test lock in the present invention is as follows:
Highly halophilic bacteria] 10 Bacterium norobilum (Halo
It is a mutant strain derived from Bacterium halobium) R. Harobakuburi Rhamno 10 Bium R0 is a strain (Feikoken Bacteria No. 77).

This bacterium belongs to the so-called archaebacteria (Archebacteria), has the only plasmid among archaea, normally grows only in the presence of 2j% concentration of salt, and has a low concentration of salt inside the cell. Therefore, it has a mechanism to sandwich the cell membrane and actively exclude Na ions. It has bacteriorhodopsin, which is similar to rhodopsin, and has a mechanism that moves electrons in response to light stimulation. Furthermore, ATP can be produced anaerobically using arginine as the only energy source, and useful components such as isoprenoid derivatives and carotenoid derivatives have been discovered as its constituent components.

The plasmid of the present invention is a glasmid fraction obtained by lysing the bacteria of the above-mentioned 710 Bacterium halobium R1 strain with a hypotonic solution and treating with Ashikari according to the method specifically described in the Examples below. It can be separated by deproteinizing with 7 enol and purifying it.

The molecular weight of the plasmid of the present invention can be determined from the results of measurements using agarose gel electrophoresis and transmission electron microscopy.
, 27 megadal tons (,yz,s kilobases). In addition, the base composition is aC (Tsu = Otsu/) and the % melting temperature ('
I1m) is an octopus, 5°0.

Furthermore, the molecular weight of the restriction enzyme digested fragment of the plasmid of the present invention is as follows.

Molecular weight (unit: megadacyton) BamH7: '? 0.3, 3.9. ．． f, 3. ．． 2
．． 9j,,2. ri, 2゜7H1ni Ill: ,
22,91. i, IBad Engineering: /, J'(,12
Gag, ! , / , , 2.7 , . 2. Z, /, 3
Pvu [1: //, 2.3.9. ,? ,0. ．． 2
,7. ．． 2. u, 2-, /, VKpn l:
//j, 10,1.3 fertilizer, 7.2Hpa l:
/,? ,? , /3. θSsz l: 9. and 1.0.2,2. ♂、2j、／、７、／、
! , /, j , 0°7 As stated above, the plasmid of the present invention.

stomach. Furthermore, due to the properties of the above-mentioned halophilic bacterium Robacterium no. 13- Robium R1 strain, the plasmid of the present invention is expected to be used as a vector for producing useful components such as isoprenoid derivatives and carotenoid derivatives.

The present invention will be described below with reference to Examples.

Example (1) Cultivation of Bacteria Halobacterium halobium R1 (2) was inoculated onto an agar medium having the following composition and cultured in an apparatus at 37°C for about 1 day.

Composition of agar medium/l (pH 4, j) Yeast extract rg Batatotryptone! 9 Na (12,1011 Mg80.・ riH, O, tli Oa O1t (7, -y agar
/jll The obtained coco cr=- was transferred to a liquid medium with the following composition (main culture 7
Inoculate the cells into a 770-fold culture medium and culture with shaking at 37°C for about 3 days to perform preculture.

4- Subplant this into a single culture liquid medium and grow it for about 7 hours at 3°C.
Perform shaking culture for 1 day.

Composition of liquid medium/l (pHt, J) Casamino acids 7. j 5 yeast extract 1017NaO1 toy Mg80.・7H,0209 /-'s/;t, 9t%F6804@riH*O(W,/
'V) (2) Preparation of plasmid Collect bacterial cells from the culture solution obtained in (1) by centrifugation. This was suspended in an antibacterial buffer solution with the following composition, and
rpm, centrifuge for 10 minutes.

Discard the supernatant.

Composition of antibacterial buffer 100- (pH 4j) Mail
, 2! 1Mg80.・7H, 0,
2F Sodium citrate 0.3g
K course, 70mM 0yDTA (cyclohexanediaminetetraacetic acid), J-tmn Tris-HCl pH 0]/-, and then add 9 mg of the same solution and dissolve uniformly. Next, add solution ■ [0°, 2M MaOH
, 7% (W/V) B D 8 (sodium dodecyl sulfate)), 2 (7-) and stirred gently at 0°C for 70 minutes.
CHaCOOH] / j Add me, O゛0 to 2
Leave for 0 minutes to remove denatured protein, 8D13 denatured DNA, etc.
Precipitate. This was centrifuged at /200 θ rpm for 75 minutes, the supernatant was taken, and twice the amount of isoglopanol was added, stirred, and left overnight at -θ°C.

Then, centrifugation was performed for 1.5'0θOrpm, /θ minutes, the supernatant was discarded, and the precipitate was diluted with 30mM Tris-HCl (psi, 0)-o.
, /M OH,00ONa buffer, add 1 equivalent of water-saturated phenol, shake with one hand, and collect the aqueous layer by centrifugation.

Furthermore, an equal amount of chloroform-isoamyl alcohol (2 g/v/v) was added, and the mixture was shaken and inverted.

This is centrifuged to collect the aqueous layer, and chloroform-isoamyl alcohol is added again to remove protein.

5.2 times the amount of ethanol is added to the aqueous layer thus obtained, stirred and left at 11.theta.C for 2 to 3 hours. The DNA was then precipitated by centrifugation for 0 min/0 min and treated with 20 mM Na
j]l! Dissolve in 1DTA buffer (pHr, 0), precipitate again with double the amount of ethanol to remove OH,0OONa, remove as much alcohol as possible, and then add 10ml of
jomM) Lis-HCl-, 2j mM Na! 1EDT
Dissolve in buffer A and adjust the solution to 9 with the same buffer.

Next, add 73,217 cesium chloride.

! ■/ rnlHρ ethidium promitoko. Add the goo and mix gently. After putting this into a polyaromer tube and attaching an aluminum cap, liquid paraffin was layered through the hole at the top.

3JOOOrpm between gθ and erH, /! Far in °C - Do your heart.

When you shine the U and V lamps on it, you will see two sharp bands, so collect the bottom one.

Cesium chloride-ethidium bromide solution (,
tOmM) Lis-HCl (pHr, 0) -2/mM N
a11!1DTA J', 01/, Seshikumuku.

Ride rJ77, /, 2! vrg/l, lH, O engineering'f
Add jimp CIYid/, 4d) and re-fj(
Centrifuge at 3j, 000 rpm 4tQ~gr hours, i, t°C. When you shine a UV lamp on it, you will see one band, so take it out and add n-butanol to it! Extract twice to remove ethidium bromide.

Add 10mM Tris-HCl (pHJ') to a dialysis tube.
, θ) −/mM Na,F! against DTA buffer.

Dialyze at g °C for several hours or more. The obtained glasmid is
Store at 7°C.

(3) Measurement of molecular weight (The molecular weight of Grasmid obtained in step 21 was measured by agarose gel electrophoresis and electron microscopy, and the molecular weight was found to be 27 megadaltons.The agarose gel electrophoresis used The conditions for electrophoresis and DNA detection are as follows.

Electrophoresis device: Submarine type electrophoresis device (/7X/j
crn) was used.

Electrophoresis: 0.09M) Lis, θ, θri M Hocic acid, J
, j mM Na, 7% (W/
Dissolve the remaining agarose, place the DNA sample on top of it, and perform electrophoresis at 100V for 4 hours.

After electrophoresis, the gel is stained by soaking it in 00-2 μg/− of ethidium bromide solution and photographed with Polaroid film under TJV, 2/4 tnm irradiation.

(4) React the grasmid obtained in step (2) with restriction enzymes in the buffer shown in Table 7 at 37°C for 7 hours with the various restriction enzymes shown in Table. Add r OmM Wag E! to the reaction solution. D.T.A.

! %5DS1.26% Glycerif, 0.0jt% (W/
Y) A mixed solution of bromophenol blue in an amount of //j times was added to stop the reaction, and the molecular weight of the DNA fragment was measured by agarose gel electrophoresis. As an internal marker.

λDNA was digested with Hlndl and BamH7
NA fragment and coIE/(plasmid) to Ha
The one decomposed by el was used.

Table 2 shows the molecular weights of fragments obtained by digestion with various restriction enzymes.

Table / Applicant Mitsubishi Chemical Industries, Ltd. Procedural Official Report (spontaneous) Commissioner of the Patent Office Kazuo Wakasugi 1 Case Description Patent Application No. 147035, filed in 1982
No. 2 Name of the invention Plasmid 3 Relationship with the person making the amendment Applicant (596) Mitsubishi Chemical Industries, Ltd. 4 Agents 2-5-2 Marunouchi, Chiyoda-ku, Tokyo (1 idiot) 5 Specification subject to amendment Column 6 of “Detailed Description of the Invention” Contents of amendment (1) rHpn in the second row from the bottom of Table 1 on page 11 of the specification
IJ is corrected to rHpa IJ.

(2) On page 12 of Akira Am, in group 2, line 5 says “PVa...
” it says! Correct as fvul[J.

that's all

Claims (1)

[Claims] (1) A cyclic grasmid with a molecular weight of 27 megadaltons, which is obtained from the halophilic bacterium Halobacterium novum and has the following decomposition characteristics with respect to restriction enzymes. BamHI to 2 fragments, Bindl to - 2 fragments, Bal to 6 fragments, Pvul to 7 fragments.
kpn I to 5 fragments* apa
It is decomposed into 2 fragments by I, and into r fragments by sat 1.