JPS62198391A - Plasmid - Google Patents

Abstract

PURPOSE:To multiply a plasmid using a Bacillus subtilis K strain as a host, by preparing a plasmid having 1.6 megadaltons molecular weight and a specific restriction enzyme cleavage map or a plasmid obtained by inserting an exogenote thereinto. CONSTITUTION:A plasmid pC194 is used as a material to prepare a plasmid vector pHR1 using a bacterium of the genus Bacillus as a host. The resultant vector pHR1 has chloramphenicol-resistant gene and enormous parts capable of cloning though it is small-sized such as 1.6 megadaltons molecular weight. The parts are parts cleaved by restriction enzymes EcoRI, HgiAI, SatI, SmaI, AvaI, BamHI, XbaI, HincII, SalI, PartI, HindIII, HaeIII, PvuII and ClaI. The plasmid vector is individually and stably held in the host.

Description

[Detailed description of the invention] [Objective of the invention] It can be used for breeding useful bacteria4 by DNA method.

<Prior art> Many vectors derived from Staphylococcus bacteria, including pUBllo, are known as vectors that can propagate using Bacillus bacteria as hosts.
Most of these vectors use Bacillus subtilis strain M and strains thereof as hosts, and there are few examples of vectors that can propagate using Bacillus subtilis strains, which are known as industrially produced bacteria, as hosts. For example, pUB 110 (Keggin
a, K, M,.

Lovett+ P, S, and Duvall,
E*J,, Proc, Natl.

Acad, Set, 75.1423 (1978
)] did not transform the Bacillus subtilis strain at all, and psA 2100 transformed the strain into Bacillus but could not stably maintain the genetic traits, and neither of them could be considered a practical vector.

Problems to be Solved by the Present Invention The problems to be solved by the present invention are that Grasmid, which can propagate Bacillus subtilis strains as hosts, has many cloning sites, and is capable of stably storing cloned genes. The objective is to create a practical befter that can be maintained.

[Structure of the invention]

Measures to Solve the Problems> The following three types of plasmids were used to construct the target vector. f2 sumido pc 194CIor
daneseu+ S,, 5urdeanu, M,
, Latta, P.D.

and Novick+ R, Plasmid, L
468 (1978)] is Staphylococcus spp.
B. aureus-derived grasmid with a molecular weight of 1.9 megadaltons is used to propagate Bacillus subtilis strain M as a host, and develops resistance to chloramphenicol. Plasmid pUR222 (Ruther, U., Ko@nsn
, M, + 0tto, K, and Mullor-
H4ll+ B,, Nucl, Ac1ds Res
, L 4087 (1981)') and Grasmid pUC13 [Vieirs, J.

and Messing, J., Gone, 19,
259 (1982):) is a grasmid vector for E. coli, and both express ampicillin resistance. Also, p
UC13 has multiple cloning sites derived from M13mpH7age.

The present inventors constructed a plasmid vector pHR1 having the restriction enzyme cleavage map shown in FIG. 2 using Bacillus bacteria as a host using the above-mentioned three types of grasmids and the method shown in the examples. Successful. pHR1 has a chloramphenicol resistance gene, and although it has a small molecular weight of 1.6 megadaltons, it has an extremely large number of cloning sites. The site is restriction enzyme EeoRI, Hg1A Is Sst I%Sma
I, Ava I, BamHl, Xbal.

Hlna m, Sal I, Pst i, Hlnd
[[, 1 (as ll, Pvu II, C1ml cleavage site). In addition, this grasmid vector not only maintains itself stably in the host, but also maintains the integrated ectopic gene. Stability is also high.Furthermore, this vector can propagate not only in the genus Bacteria and Rus but also in Escherichia coli as a host, making it extremely superior as a vector.

Bacterial strains that can be used as host bacteria of the present invention include those belonging to the genus Bacillus and Escherichia coli, such as the following.

Bacillus subtilis ATCC6051 (M stock) Bacillus subtilis IAM 1523 (K stock) Bacillus amyloliquefaciens ATCC23350
Grasmids of the present invention include those in which a foreign or heterologous gene is inserted into pHR1.

Even better results can be obtained by using a mutant strain with reduced restriction enzyme activity as a host for Grasmid of the present invention.

Example (1) Preparation of material cilasmid Grasmid pc 1.94, pUR222 and pU
C13 was prepared as follows.

Bacto-Penas
+5ayBroth) (Difco 1) II, 3
At 7°C, Bacillus subtilis IE17 (7' lasmid pc194 carrier strain), Escherichia coli C222 (
A strain carrying plasmid pUR222) and Escherichia coli C1'3 (a strain carrying f lasmid pUC13) were each cultured to the late logarithmic growth phase, and the bacterial cells were collected. After dilyzing the obtained bacterial cells with IJzozyme and SDS, the bacterial cells ? , centrifuged at 20,000 rpm for 30 minutes,
A supernatant liquid of m1 was obtained. Plasmid DNA1 in the supernatant,
The supernatant was precipitated by adding 2 volumes of cold ethanol and collected. This precipitate was dissolved in 6 rnl of TEN buffer.

0.25 MEDTA 1 m for this sample 3.5-
Ethidium bromide solution (4.6mp/m) 0.5
mJ, cesium chloride 5! i is mixed and dissolved, and the refractive index is set to 1.
After solidifying at 390°C, this solution was heated at 15°C and 40°C.
Equilibrium density gradient centrifugation was performed at ,000 rpm for 20 hours.

After centrifugation, the fluorescent band of the DNA was detected under 3,650 angstrom ultraviolet irradiation, and the Grasmid DNA fraction was fractionated and purified to produce 250 μy of pure Grasmid DNA.
Separated from A. - (2) Plasmid p that served as an intermediate for pHR1 construction
Preparation of AN 40 Grasmid pc1941μs and O51 unit of circumcision enzyme Sau 3A1 (Pellinger Mann Ir)
1) was added and reacted at 37°C for 60 minutes to partially decompose the DNA.

One unit of restriction enzyme BamHI (Berry/Ger Mannheim #) was added to 1 μg of plasmid pUR222, and the DNA was subjected to a digestion reaction at 37° C. for 60 hours to completely degrade the DNA.

The obtained DNA fragments were treated with phenol and chloroform, precipitated with ethanol, mixed, and ATP
and dithiothreitol for 10 hours at 10'C.
01 unit of T4 DNA ligase (Pellingo Mannheim *) was applied. The T4 DNA IJ gauze was inactivated by treatment at 65° C. for 10 minutes, and the DNA was recovered by precipitation with ethanol.

The composite grasmid thus obtained was used for transformation.

Transformation was performed according to the method of Ehrlich, S.D. et al.
agerteM, and Ehrlich, S.D.
, Genet 43123 (1979)] using Escherichia coli C600r-m- as the host.

The transformed strain was treated with 8μμ of chloramphenicol and 30μμ
In L medium containing l/rug ampicillin at 37°C, 2
The cells were cultured for 4 hours and selected.

Birnboim et al.
nd Doly, J.

Nucleic Ac1cls Res,, 'L 1
Plasmid PAN40 was selected by extracting the plasmid of the transformed strain by the method of 513 (1979) and examining the restriction enzyme cut WR pattern. pAN 40
has a molecular weight of 3.7 megadaltons, expresses ampicillin resistance and chloramphenicol resistance, and has the restriction enzyme cleavage map shown in FIG.

(3) Construction of pHR1 320 μg of pUc1 was added to the restriction enzyme EcoR120”=y
Double decomposition at 37°C for 1 hour at 20 units, followed by polyacrylamide gel electrophoresis,
Separate and extract the 45 base pair multi-cloning site,
0.05μ, pt-obtained.

1 μg of PAN 40 with restriction enzyme EcoR1)
ndII[, 1 unit each at 37°C for 1 hour in duplicate 4
Understand.

The obtained 7'j DNA fragment was treated with 7 enol and chloroform, precipitated with ethanol, mixed, and treated with 0.01 unit of T4 DNA ligase at 10°C for 10 hours in the presence of ATP and dithiothreitol.

T4 DNA f-ase was inactivated by heating at 65° C. for 10 minutes, precipitated with ethanol, and DNA i was recovered.

The DNA thus obtained was used for transformation.

Bacillus subtilis RM 125 (AJ 1171
1) (arg", 1eu-, r-, m-) was used as a host for transformation.

Bacillus subtilis RM 125 (AJ 1171
1) to r Pennassay Btoth J (
Difeo) was inoculated and cultured overnight at 30°C with shaking.
(mt4) 2so4o, 2 Ji'/bao, KW2P
2HPO41,41y false to O40,6ji/dt%, M
gSO4・7H200.02# Sodium citrate 0.19/di.

゛Yeast extract 0.29 liters, L-arginine 251 dl
.

L-loidino5 rne/dt) was inoculated to 37
After shaking culture for 4 hours at
040.21. y, IQ (2PO40,6gAIt%
2HPO41,411/di, MgSO4・7H
200, 12fi/dl % Sodium citrate A 0
．． 197 g of yeast extract, 0.02 g/di of yeast extract, 0.5 vlde of L-arginine, and 0.5 vlde of L-arginine) and cultured with shaking at 37°C for 1°5 hours to obtain so-called competent cells. cells (having DNA uptake ability) were prepared (References: J, Ba
cterfol, 8L 741 (1961)).

The DNA solution obtained in (2) was added to this competent cell suspension, and the transformation reaction was completed by further shaking culture at 37°C for 2 hours.
of chloramphenicol t-containing trL medium, and
Transformants were selected by culturing at ℃ for 24 hours. Birn
Extract the plasmid of the transformed strain by the method of Boln et al.
A plasmid with a molecular weight of -1.6 megadaltons was selected. Among the obtained transformed strains, Bacillus subtilis AJ 12283 was selected as a representative, and the plasmid possessed by this strain was named pHR1. pHR1 is a plasmid expressing chloramphenicol resistance with a molecular weight it 1.6 megadaltons. A restriction enzyme cleavage map of this Grasmid is shown in FIG. In addition, Bacillus subtilis AJ 12283, which has pHR1, is F
It has been deposited as ERM P-8650.

(4) Transformation of various hosts with pHR1 By the same method as in (1), pHRI of Bacillus subtilis AJ 12283 carrying f:5 smid pHRI was prepared and 150 μI was obtained. This plasmid was used to transform Bacillus subtilis strain M (AJ 11711), Bacillus subtilis strain (IAM 1523) and Escherichia coli C6C600r-. C6C
600r-'' was transformed in the same manner as in (2).

For M and Ni strains, the method of transferring grasmid after glotograstization [Chang, S., and Cho
en, S, N,, Mo1ac, Gono Gon
s', 1681111 (1979)) and transformed with Grasmid.

The results are shown in Section 16.

Table 1 Bacillus subtilis strain 8.7 x 10' Bacillus subtilis M strain 2.0 x 10' Escherichia coli C600r''m-5, 0 x 10 * Number of drug-resistant colonies appearing per 1 μg of Grasmid DNA.

When the grasmids of each transformed strain were extracted by the method of Blrnboin et al., the same grasmids as before introduction were confirmed in each case, indicating that this vector can be stably introduced into each host.

(5) Stability of genes cloned into pHR1 Generally, when Bacillus subtilis is used as a host, significant loss of genes cloned onto plasmid vectors is observed, and this problem is a problem that can be solved by recombination using Bacillus subtilis. This is one of the biggest factors that make DNA experiments difficult.

Therefore, we decided to clone the gene into plIRl and examine its stability, and examined the practicality of this vector.

pHR10.5 μg and 1 unit of restriction enzyme BamH
Completely decompose at 37°C for 1 hour. After treatment with phenol and chloroform, DNA is recovered by ethanol precipitation.

The above DNA was mixed with 0.1 μy of a 3.4 kilobase restriction enzyme Bgl I-cleaved fragment containing the his J gene of the K strain, and incubated at 10°C in the presence of ATP% dithiothreitol.
0.01 unit of T4 DNA ligase was allowed to act for 10 hours. T4 DNA IJ gauze at 65℃, 1
After inactivation by treatment for 0 minutes, it was used for transformation.

Bacillus subtilis AJ 11732. FERM
P-6245-(arg″″, 1eu-, hisJ,
r-m-'') was used as a host and transformed in the same manner as in (3).The transformed strain was treated with chloramphenicol 5μ
y4! Containing minimal medium grade (glue: I-su 0.5 j
i/dis(NH4)28040.29/dt.

KH2PO40.6g/dt, K2HPO41.4
97g, MgSO4・n120-o, 02g
/dt, sodium citrate 0.1 g/dl, L
-Argini 710 m9/dt %L -oIran 10
$t, agar 297di, pif 7.2).
The cells were cultured at 37°C and selected.

Cypwosmid was extracted from the transformed strain using the method of Birnboim et al., and the 3.4 kilobase fragment was incorporated into pHR1. Select this Grasmid to pHR
It was named B2.

Prepare pHRB 2 in the same manner as (1), and use 160μ
I got yt. AJ 1 with pHRB 2 in the same manner as above
1732 was transformed again, and pHRB2 was transformed into hisJ
It was confirmed that they had the gene.

Using the method of Chang et al. described in (4), '7''' lasmid pHR1, pHRB 2 and psA 210
Bacillus subtilis IAM 1523 was transformed with 0. The transformed strain was treated with chloramphenicol 5 μg Al
1, and 20 of the obtained transformants were randomly selected, and plasmids were extracted by the method of Birnboim et al. Among the 20 strains investigated, the percentage of those with intact plasmids is shown in the second column. 5 Table 2 Plasmid Selectable Marker - Stability ←) * psA
2100 Cmr, Sm' 10p
HRI CmrloopHRB 2
Crnr, hisJ+100 hiatus Cm': chloramphenicol resistance Smr: streptomycin resistance 2nd generation fi, pHR1 and Grasmid, a gene cloned therein, have both been shown to be stably retained in the host9.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme cleavage map of pAN40 and the
The figure is a restriction enzyme cleavage map at pH11.

Claims (1)

[Claims] 1. A plasmid with a molecular weight of 1.6 megadaltons and a restriction enzyme cleavage map shown in FIG. 2. A plasmid having a molecular weight of 1.6 megadaltons, having the restriction enzyme cleavage map shown in FIG. 2, and into which a foreign gene has been inserted. 3. A plasmid having a molecular weight of 1.6 megadaltons, having the restriction enzyme cleavage map shown in FIG. 2, into which a foreign gene has been inserted, and which has been incorporated into the host microorganism.