JPH07112430B2 - Plasmid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Object of the Invention] <Industrial field of application> The plasmid shown in the present invention can host, as a host, industrially produced bacteria such as amino acids, nucleic acids, various enzymes, and antibiotics.
It can be used for breeding useful strains by the recombinant DNA method.

<Prior Art> Conventionally, as a vector that can be propagated using a bacterium of the genus Bacillus as a host, a large number of Staphylococcus-derived vectors such as pUB 110 are known, but these are all Bacillus subtilis strain M. Bacillus, which is known as an industrial producer,
There are few examples of vectors that can be propagated using the Subtilis K strain as a host. For example, pUB 110 〔Keggins, KM, Lovett, PSa
nd Duvall, EJ, Proc.Natl.Acad.Sci., 75 , 1423 (197
8)] did not transform the Bacillus subtilis K strain at all, and pSA 2100 could transform the K strain but could not maintain the genetic trait stably, so that neither was a practical vector.

<Problems to be Solved by the Present Invention> A problem to be solved by the present invention is a plasmid that can be propagated using Bacillus subtilis K strain as a host, and has a large number of cloning sites and stabilizes a cloned gene. The purpose is to create a practical vector that can be maintained.

[Structure of Invention]

<Means for Solving Problems> In constructing the target vector, the following three types of plasmids were used as materials. Plasmid pC 194 (Iordanescu,
S., Surdeanu, M., Latta, PDand Novick, R., Plasmid, 1 ,
468 (1978)] is a Staphylococcus aureus-derived plasmid having a molecular weight of 1.9 megadalton, which grows using Bacillus subtilis M strain as a host and expresses chloramphenicol resistance. Plasmid pUR 222 [Ruthe
r, U., Koenen, M., Otto, K.and Muller-Hill, B., Nucl.Aci
ds Res., 9 , 4087 (1981)] and plasmid pUC 13 [Vie
ira, J. and Messing, J., Gene, 19 , 259 (1982)] is a plasmid vector for E. coli and both express ampicillin resistance. Moreover, pUC 13 has a multi-cloning site derived from the M13mp11 phage. The inventors of the present invention, using the above-mentioned three types of plasmids as materials, and the method shown in Examples,
Using a bacterium belonging to the genus Bacillus as a host, we succeeded in constructing a plasmid vector pHR 1 having a restriction enzyme cleavage map shown in FIG. pHR 1 has a chloramphenicol resistance gene and has a large number of cloneable sites despite its small molecular weight of 1.6 megadalton.
The sites are the restriction enzymes EcoR I, HgiA I, Sst I and Sma.
I, Ava I, BamH I, Xba I, Hinc II, Sal I, Pst I, Hi
It is a site that is cleaved by nd III, Hae III, Pvu II, and Cla I. Further, the present plasmid vector is not only stably retained in the host itself, but also highly stable in retaining the incorporated foreign gene. Furthermore, this vector can be propagated not only as a bacterium of the genus Bacillus but also as a host in Escherichia coli, and is extremely superior as a vector.

Strains that can be used as the host bacterium of the present invention belong to Bacillus bacteria and Escherichia coli, and examples thereof include the following.

Bacillus subtilis ATCC 6051 (M Inc.) Bacillus subtilis IAM 1523 (K Corporation) bus Chirusu-Ami Lori Beautique tumefaciens ATCC 23350 Escherichia coli C600r ^- m ^- as a plasmid of the present invention, gene foreign or heterologous is inserted into pHR 1 It includes things that have been done. Even better results can be obtained by using a mutant strain having a reduced restriction enzyme activity as the host of the plasmid of the present invention.

Example (1) Preparation of material plasmid Plasmids pC 194, pUR 222 and pUC 13 were prepared as follows.

Bacto-Penassay Broth
(Manufactured by Difco) 1 at 37 ° C. Bacillus subtilis 1E17 (strain carrying plasmid pC 194), Escherichia
E. coli C222 (strain holding plasmid pUR222) and Escherichia coli C13 (strain holding plasmid pUC13) were cultured until the late logarithmic growth phase, respectively, and the bacterial cells were collected. After lysing the obtained bacterial cells with lysozyme and SDS, the bacterial cells were washed with 20,00
Centrifugation at 0 rpm for 30 minutes gave 90 ml of supernatant. The plasmid DNA in the supernatant was collected by adding 2 volumes of cold ethanol to the supernatant for precipitation. This precipitate was dissolved in 6 ml of TEN buffer.

To 3.5 ml of this sample, 1 ml of 0.25M EDTA, 0.5 ml of ethidium bromide solution (4.6 mg / ml), and 5 g of cesium chloride were mixed and dissolved, and the refractive index was adjusted to 1.390.
Equilibrium density gradient centrifugation was performed at 000 rpm for 20 hours.

After the centrifugation, D under UV irradiation of 3,650 Å
The fluorescent band of NA was detected, and the plasmid DNA fraction was fractionated, purified, and 250 μg of pure plasmid DNA was isolated.

(2) Construction of plasmid pAN 40, which was an intermediate for construction of pHR 1. 0.1 μl of restriction enzyme Sau 3 was added to 1 μg of plasmid pC 194.
Add A1 (Boehringer Mannheim) and add 60 at 37 ℃.
The reaction was carried out for a minute to partially decompose the DNA.

1 unit of restriction enzyme BamH per 1 μg of plasmid pUR 222
Add I (Boehringer Mannheim) and add 60 at 37 ° C.
The DNA was completely decomposed by a decomposition reaction.

The obtained DNA fragments were treated with phenol and chloroform, precipitated with ethanol, and then mixed. In the presence of ATP and dithiothreitol, 10 units of T4 were added at 10 ° C for 10 hours.
DNA ligase (Boehringer Mannheim) was allowed to act. T4 DNA ligase was inactivated by treatment at 65 ° C for 10 minutes, and precipitated with ethanol to recover DNA.

The composite plasmid thus obtained was used for transformation.

Transformation is performed by the method of Ehrlich, SD et al. [Dagert, M. and Ehr
Lich, SD, Gene, 6 , 23 (1979)], Escherichia coli C600r ⁻ m ⁻ was used as a host.

Transformants were 8μg / ml chloramphenicol and 30μ
The cells were selected by culturing at 37 ° C. for 24 hours in L medium containing g / ml of ampicillin.

Birnboim et al. (Birnboim, HCand Doly, J., Nucleic Acicl
s Res., 7 , 1513 (1979)], the plasmid of the transformant was extracted, and the plasmid pAN 40 was selected by examining the restriction enzyme cleavage pattern. pAN 40 has a molecular weight of 3.
It exhibits ampicillin resistance and chloramphenicol resistance at 7 megadalton, and has the restriction enzyme cleavage map shown in FIG.

(3) Construction of pHR 1 20 μg of pUC 13 was treated with 20 units of restriction enzyme EcoR1 and Hind II.
Double-decomposes with 20 units at 37 ℃ for 1 hour. The polycloning gel electrophoresis method separated and extracted the multi-cloning site of 45 base pairs to obtain 0.05 μg.

1 µg of pAN 40 is double digested with 1 unit each of restriction enzymes EcoR1 and Hind III at 37 ° C for 1 hour.

The obtained DNA fragment is treated with phenol and chloroform,
After precipitation with ethanol, mixing was performed, and 0.01 unit of T4 DNA ligase was allowed to act for 10 hours at 10 ° C in the presence of ATP and dithiothreitol. T4 DNA ligase was heated at 65 ° C. for 10 minutes to inactivate it, and precipitated with ethanol to recover DNA.

The DNA thus obtained was used for transformation. Bacillus subtilis RM 125 (AJ 11711) (arg -, leu -, r -,
m ^-) was used as a host for transformation.

Bacillus subtilis RM 125 (AJ 11711) to "Pennassa
y Btoth "(Difco) and shake-cultured at 30 ° C. overnight, and cultured in the first culture medium (glucose 0.5 g / dl, (NH ₄ ) ₂ SO _{4
0.2g / dl, KH 2 PO 4} 0.6g / dl, K 2 HPO 4 1.4g / dl, MgSO 4 · 7H
₂ O 0.02g / dl, sodium citrate 0.1g / dl, yeast extract
0.2g / dl, L-arginine 25mg / dl, L-leucine 5mg / dl
Culturing was carried out at 37 ° C. for 4 hours with shaking, and then the second culture medium (glucose 0.5 g / dl, (NH ₄ ) ₂ S was added.
O ₄ 0.2 g / dl, KH ₂ PO ₄ 0.6 g / dl, K ₂ HPO ₄ 1.4 g / dl, MgSO ₄
・ 7H ₂ O 0.12 g / dl, sodium citrate 0.1 g / dl, yeast extract 0.02 g / dl, L-arginine 5 mg / dl and L-leucine
0.5 mg / dl) was inoculated and shake-cultured at 37 ° C. for 1.5 hours to prepare so-called competent cells (having DNA uptake ability) (Reference: J. Bacteri).
ol., 81,741 (1961)). After adding the solution of the DNA obtained in (2) to this competent cell suspension and culturing at 37 ° C for 2 hours with shaking to complete the transformation reaction, the cell suspension was mixed with 5 µg / ml of a broth. A transformant was selected by plating on L medium containing rhamphenicol and culturing at 30 ° C. for 24 hours. Birn
The plasmid of the transformant was extracted by the method of Boin et al., and one having a plasmid having a molecular weight of 1.6 megadalton was selected. Among the obtained transformants, Bacillus subtilis AJ
12283 was selected as a representative, and the plasmid contained in this strain was named pHR 1. pHR 1 is a plasmid having a molecular weight of 1.6 megadalton and expressing chloramphenicol resistance. The restriction enzyme digestion map of this plasmid is shown in FIG. In addition, Bacillus subtilis AJ 12 who owns pHR 1
283 has been deposited as FERM P-8650.

(4) Transformation of various hosts with pHR 1 By the same method as in (1), pHR 1 was prepared from Bacillus subtilis AJ 12283 carrying the plasmid pHR 1.
50 μg was obtained. This plasmid using the Bacillus subtilis strain M (AJ 11711), Bacillus subtilis strain K (IAM 1523) and Escherichia coli C600r ^- m ^- was transformed. C600r ^- m ^- were transformed in the same manner as in (2) for.

For M and K strains, a method of transfecting plasmid after protoplastization [Chang, S. and Choen, SN, Molec. Ge
n.Genet., 168, 111 (1979)] and transformed with the plasmid. The results are shown in Table 1.

When the plasmid of each transformant was extracted by the method of Birnboin et al., The same plasmid as before introduction was confirmed in each case, and it was found that this vector was stably introduced into each host.

(5) Stability of genes cloned in pHR 1 Generally, when Bacillus subtilis was used as a host, remarkable loss of genes cloned on the plasmid vector was observed. This is one of the biggest factors that make DNA experiments difficult. Therefore, the practicality of this vector was examined by cloning the gene in pHR 1 and examining its stability.

pHR 1 0.5 μg 37 with 1 unit of restriction enzyme BamHI
Completely decomposes at ℃ for 1 hour. After treatment with phenol and chloroform, the DNA is recovered by ethanol precipitation.

3.4 kilobase restriction enzyme Bgl containing K strain his J gene
By mixing 0.1 µg of the II cleavage fragment with the above DNA, 0.01 unit of T4 was added for 10 hours at 10 ° C in the presence of ATP and dithiothreitol.
DNA ligase was applied. T4 DNA ligase at 65 ℃ for 10
It was inactivated by treatment for a minute, and then used for transformation.

Bacillus subtilis AJ 11732, FERM P-6245 FERM BP
^{-224 (arg -, leu -,} hisJ, r - m -) as a host,
Transformation was carried out in the same manner as in (3). The transformant was a minimal medium plate containing 5 μg / ml of chloramphenicol (glucose 0.5 g / dl, (NH ₄ ) ₂ SO ₄ 0.2 g / dl, KH ₂ PO ₄ 0.6 g / d.
l, K ₂ HPO ₄ 1.4 g / dl, MgSO ₄ .7H ₂ O 0.02 g / dl, sodium citrate 0.1 g / dl, L-arginine 10 mg / dl, L-leucine 10 mg / dl, agar 2 g / dl, pH 7 .2), and cultured at 37 ° C for selection.

A plasmid was extracted from the transformant by the method of Birnboim et al., And the one in which the 3.4 kilobase fragment was incorporated into pHR 1 was selected, and this plasmid was named pHRB 2.

PHRB 2 was prepared in the same manner as in (1) to obtain 160 μg. AJ 11732 was retransformed with pHRB 2 in the same manner as described above, and it was confirmed that pHRB 2 has the hisJ gene.

Using the method of Chang et al. Described in (4), the plasmid pHR
Bacillus subtilis IAM 1 at 1, pHRB 2 and pSA 2100
523 was transformed. Transformants were selected in the presence of 5 μg / ml of chloramphenicol, 20 strains were selected from each of the obtained transformants, and the plasmid was extracted by the method of Birnboim et al. Table 2 shows the proportion of those having an intact plasmid among the 20 strains examined.

From Table 2, it was shown that both pHR 1 and the plasmid in which the gene was cloned were stably retained in the host.

[Brief description of drawings]

Figure 1 is a restriction enzyme digestion map of pAN 40, and Figure 2 is
It is a restriction map of pHR 1.

Claims (3)

[Claims] 1. A plasmid having a molecular weight of 1.6 megadalton and having the following restriction enzyme cleavage map. 2. The plasmid according to claim 1, which has a foreign gene linked thereto. 3. A transformant belonging to the genus Bacillus obtained by introducing a plasmid having a molecular weight of 1.6 megadalton and having a restriction enzyme cleavage map shown below.