GB2145093A - Plasmid - Google Patents

Abstract

The plasmid is circular and is derived from a Bacillus subtilis (natto) strain deposited at the Fermentation Research Institute, Japan under the number Ferm-BP 315, and has a molecular weight of between 5.6 and 5.8 kb and a copy number of 15 to 20. Endonuclease cleavage sites for a plurality of enzymes are specified.

Description

SPECIFICATION A novel plasmid and methods for its use The present invention relates to a plasmid, more particularly to a plasmid obtained from Bacillus subtilis (natto) and method for its use. "Natto" produced by B. subtilis (natto) and steamed soybean, is one of the most traditional fermentation foods in Japan.

Plasmids, which are extrachromosomal genes, have been used widely in recently developed genetic enginnering techniques as a vector for use in vitro recombination of genes. The in vitro genetic recombination technique has a wide application, e.g., to breeding of useful microorganisms.

Now, Escherichia coli, B. subtilis, yeast, etc. are mainly used as hosts in genetic engineering.

However, when, cloning of genes of higher organisms origin is attempted with view to producing useful substances which higher organisms produce such as growth hormones, etc.

using microorganisms, yeast and B. subtilis may be used more advantageously than E. coli since the former excrete their products outside the cells. Further advantage of use of yeast and B.

subtilis is that they are not harmful to humans nor parasitic to various animals and plants, thus providing a genetic operation system having a high safety.

Extensive researches have been made in order to develop a new genetic operation system using B. subtilis and as a result thereof a new plasmid being present at a high copy number has been isolated from B. subtilis (natto) GC 18.

The present invention is based on the above finding and provides a circular plasmid, a molecular weight 5.7 + 1 kb, is present at a copy number of 1 5 to 20 and has endonuclease cleavage sites as follows: Eco R I 1 site Bam HI 2 sites Hind 111 4 sites This new plasmid is hereinafter referred to as "pGC 18".

Further, the present invention provides a starting material for producing a pGC 18 hybrid plasmid by cleaving a pGC 1 8 plasmid with a restriction endonuclease Eco RI and splicing thereto structural gene for producing a physiologically active substance.

Still further, the present invention provides a starting material for producing transformed bacteria by introducing a pGC 1 8 plasmid into a host cell.

The plasmid of the present invention has been isolated by screening from about 1 90 B.

subtilis (natto) strains on fermented soybeans available from various areas of Japan.

The strain which provides the plasmid of the present invention has the following bacteriological characteristics.

(a) Morphology (1) Shape rod (2) Spore + (3) Gram's staining Gram positive (b) Growth (1) Growth in 7% Saline water + (2) Growth at pH 6.0-8.0 + (c) Physiological Properties (1) Reduction of nitrate + (2) Hydrolysis of Starch + (3) Utilization of citric acid + (4) Growth range Optimal growth temperature 30'C Optimal pH pH 7.2 Heat resistance can grow at 45"C but not at 65"C (5) Behavior toward oxygen aerobic (6) Formation of Acid and Gas from Sugars Acid Formation 1) L-arabinose 2) D-axylose + 3) D-glucose + 4) D-mannitol + 5) maltose + (7) Hydrolysis of gelatin + (8) Hydrolysis of casein + With reference to Buchanan, R. E. s Gibbons, N.E. (eds.): Bergey's Manual of Determinative Bacteriology (8 th ed.) Williams 8 Williams Baltimore, 1 974, 529-550, the above strain reveals to be strain of B. subtilis.

This strain has been deposited at Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on July 6, 1 983 under deposition No. FERM-BP No. 315 (named B. subtilis(natto) GC 18.

Isolation of plasmids can be carried out by the method described in Tanaka, T. and T.

Koshikawa: J. Bacteriol. 131, 699-701(1977).

B. subtilis (natto) GC 1 8 strain was cultured in 500 ml of L broth (10 g of peptone, 5 g of yeast extract, 1 g of glucose and 5 g of NaCI, water to make 1 liter) at 30"C with shaking, and cells were harvested by centrifugation. The cells thus collected were suspended in 1 8 ml of 25% sucrose TES solution (TES solution: 30mM Tris HCI (pH 8.0), 50mM NaCI, and 5mM EDTA).

To the cell suspension were added 1 ml of 0.5 M EDTA (pH 8.0) and 1 ml of lysozyme (10 mg/ml) and the mixture was allowed to react at 37"C for 30 minutes.

To the reaction mixture were added 10% SDS in 2 ml of TES and 2 ml of 5 M Nacl and the mixture was allowed to stand overnight at 4"C and then subjected to centrifugation (30,000 rpm) at 4"C for 30 minutes.

Polyethylene glycol was added to the supernatant to a final concentration of 10% and the mixture was allowed to stand overnight at 4"C and subjected to centrifugation at 10,000 rpm for 10 minutes.

The residue was dissolved in TES and subjected to cesium chloride-ethidium bromide density gradient centrifugation at 60,000 rpm for 1 6 hours to obtain plasmid DNA.

Then, the plasmid DNA was washed with isoamyl-alcohol in a conventional manner to remove ethidium bromide and dialyzed against 1,000 volumes of TES at 4"C.

The plasmid pGC 1 8 thus obtained have the following characteristics.

(1) Molecular Weight pGC 1 8 is a cyclic plasmid having a molecular weight of 5.7 + 1 kb.

(2) Cleavage Sites pGC 1 8 has the following restriction nuclease cleavage sites.

Number of Enzyme Cleavate Sites Eco R 1 Bam HI 2 Hind lil 4 Bgl II 0 Pst I 3 Sall O Kpn I 0 Pvull 1 Hpa I 0 Hae l l l 2 Hpall 4 Xba 1 0 Bcil 2 Sau 961 3 CIa I 0 Avall 3 Taql 4 Sam I 0 Ball O (3) Confirmation Test pGC 1 8 was tested whether it was actually a plasmid as follows.

(A) Electron Microscopic Analysis It has been confirmed by electron microscopic analysis that pGC 18 is a circular plasmid.

(B) Agarose Gel Electrophoresis On agarose gel electrophoresis DNA having a uniform molecular weight of 5.7 kb have been detected.

For the determination of the molecular weight of pGC 18, DNA fragment having a known molecular weight which was prepared by decomposing ADNA (DNA of A phage of E. coli) with Hind Ill was used as an internal marker. Particulars of the agarose gel electrophoresis device and conditions under which DNA was detached are as follows.

Electrophoresis device: Disc electrophoresis device (standard type) produced by Mitsumi Electric Co., Ltd. was used.

In a glass tube having an inner diameter of 0.5 cm and a length of 9 cm was placed 0.7 or 1.5% agarose gel and the tube was positioned between the upper and lower buffer solution troughs. DNA samples were placed on the upper end of the agarose gel in the tube.

Electrophoresis: Electrophoresis was carried out in a buffer solution (0.04 M Tris, 0.02 M sodium acetate and 1 mM EDTA, adjusted to pH 8.3 with acetic acid) containing 0.5 yg/ml of ethidium bromide at 50 V for 3 to 4 hours.

After conclusion of electrophoresis, the gel was taken out of the glass tube and irradiated with UV light (350 nm) to detect the position to which DNA migrated, and the position was photographed using a Polaroid film 105 through R1 filter.

(4) Decomposition with Restriction Enzyme pGC 1 8 plasmid isolated and purified as described above was reacted with a restriction enzyme in a known buffer solution at 37"C for 1 to 2 hours. The molecular weight of DNA fragments was determined by agarose gel electrophoresis. As an internal marker, DNA fragment prepared by decomposing ADNA with Hind Ill was used. The DNA fragments obtained have the following molecular weights (Table 1).

Table 1 pGC 18 DNA Fragments No. of Swn of (aeLermi- frag. sizes Enzyme nations Fragment size (kb) (kb) Eco RI 1 Bam HI 2 4.1 1.7 5.8 Hind III 4 3.8 0.9 0.5 0.4 5.6 Bam HI +Hind IIT h 1.8 1.7 0.9 Q.5 0.4 a Bam HI +Eco RI 3 3.6 1.7 0.48 5.78 Eco RI +Bam HI 7 1.8 1.7 O ().48 0.4 A "" +Hind ITT The number of sites at which plasmid DNA is cleaved with a restriction nuclease is determined by completely digesting plasmid pGC 18 with an excess amount of a restriction nuclease, subjecting the digested products to 0.8% agarose gel electrophoresis and counting the number of separable fragments.

The molecular weight of the plasmid is determined by calculating the molecular weight of each DNA fragment of digested pGC18 according to standard curve prepared by plotting the distance at which DNA fragments having known molecular weights obtained by digesting ADNA with Hind Ill (J. Mol. Biol. 98, 551-564(1975)) migrate on the same agarose gel upon electrophoresis. When a plurality of fragments are formed the molecular weights of the fragments are summed.

(5) Copy Number of pGC 18 The copy number of pGC was determined according to conventional method (Clowes, R. C., Bacteriol. Rev., 36, 361(1972), Helsinki, D. R., Clewell, D. B., Ann. Rev., Biochem. 40, 899 (1971), etc.). As a result, the copy number of pGC 18 was 15 to 20 per-chromosome.

Utility of pGC 18 Since pGC 1 8 is a plasmid derived from B. subtilis (natto), which produces exogenous enzymes, and is present at a high copy number, the plasmid is very advantageous.

The plasmid of the present invention can be used for providing a means for the extracellular production of useful substances on a large scale by cleaving pGC18 with a suitable restriction nuclease, for example Eco RI, in a conventional method and inserting a structural gene for producing physiologically active substances such as interferon (cf. e.g., Japanese Patent Application (OPI) No. 56-131598), HB vaccine (cf. e.g., Japanese Patent Application (OPI) No.

56-63995), urokinase, albumin, etc.

Further efficient manifestation of characteristic, i.e., efficient production of desired physiologically active substances, can be expected by introducing a suitable promotor gene such as, for example, a-amylase gene to the plasmid according to a conventional method.

It can easily be understood by analogy that the function enabling autonomous replication of pGC 18 is borne by a part thereof as in known plasmids and there is no reason why one skilled in the art could not expect that pGC18 derivatives such as fragment of pGC 18 or compound pGC18 comprising pGC18 (or a part thereof) and other DNA inserted thereto have a similar function provided that these derivatives contain the part bearing the function of autonomous replication. Therefore, not only pGC18 as a complete integrity but also its derivatives or modified DNA are useful as far as such modified DNA have an autoreplicability.

Application of pGC 18 At present, it is unclear what markers pGC18 isolated from B. subtilis (natto) has, therefore, a chimera plasmid was prepared using pGC 18 and pUB 110, a plasmid derived from Staphylococcus aureus. More particularly, after cleaving pUB 110 and pGB18 with Eco RI, these plasmids were bound to each other by conventional method as described in Birnboim, H.

C., and Doly: J. Nucleic Acids Res. 7, 1513 (1979), Scheer-Abramowitz, J. Gryczan, T. J., and Dubnau, D.: Plasmid, 6, 67 (1981), etc.

A plasmid pBR 322 can further be bound to the above chimera plasmid to obtain a shuttle vector of E. coli-B. subtilis.

Claims (3)

1. A circular plasmid comprising an extrachromosonal DNA of-Bacillus subtilis (natto) which has: (1) a copy number of 15 to 20, (ii) a molecular weight of between 5.6 and 5.8 kb, and (iii) endonuclease cleavage sites associated with respective enzymes as follows: Eco RI 1 site Pvu II 1 site Bam HI 2 sites Hae Ill 2 sites Bcl I 2 sites Hind Ill 4 sites Hpa II 4 sites

2. The plasmid as claimed in claim 1, which is derived from Bacillus subtilis (natto) GC18 (Ferm - BP No 315).

3. The plasmid as claimed in claim 1 or claim 2 which further comprises an amylase promoter gene.