JP2983602B2 - Circular plasmid - Google Patents

Description

The present invention relates to a novel plasmid, and more particularly to Rh
The present invention relates to a novel circular plasmid derived from a microorganism belonging to the genus odococcus.

[Conventional technology and problems]

Microorganisms belonging to the genus Rhodococcus are known as microbial catalysts for hydrating nitriles to produce the corresponding amides, and that microorganisms belonging to the species Rhodococcus rhodochrous have extremely high nitrile hydration activity It has been known. Under these circumstances, Rhodococcus
The development of a genus host-vector system has long been expected. However, for strains belonging to the genus Rhodococcus, development of vectors suitable for using these microorganisms as hosts has been delayed, and strains in which plasmids have been found in the genus Rhodococcus are Rhodococcus sp.H13-A strains (J. Bacter
iol. 170, 683-645 (1988)]. Therefore, there is a strong demand for the development of new vectors for breeding and improving industrially usable microorganisms from strains belonging to the genus Rhodococcus.

[Means for solving the problem]

The present inventors have conducted intensive studies to develop an industrially useful host-vector system using a strain belonging to the genus Rhodococcus. The present inventors have found a novel circular plasmid which can be used as the present invention, and have completed the present invention.

That is, the present invention has a size of about 2.6 kb and the number of restriction enzyme cleavage sites is Sac I: 2, BamH I: 1, Pvu II: 1, Sca
R, characterized by I: 1, Sph I: 1 and Xho I: 1
It is a circular plasmid derived from a microorganism belonging to the genus hodococcus.

The cyclic plasmid of the present invention is specifically, for example, Rhod
ococcus rhodochrous ATCC4276, ATCC14349 and ATCC1
4348, each of which is approximately 2.6 kb in size
And a novel circular plasmid having the characteristic of degrading restriction enzymes shown in Table 1 below. Hereinafter, these plasmids are referred to as pRC001, pRC002, and pRC003, respectively.

Next, examples of the present invention will be described.

Example 1 (1) Separation and Purification of Plasmid Rhodococcus rhodochrous ATCC4276, ATCC14349 and ATCC14348 were each added to 400 ml of MY medium (polypeptone).
0.5%, Bacto yeast extract 0.3%, Malt's extract 0.3
%, Glucose 1%). OD660 = 0.1
At around 5 to 0.2, add 0.5 U / ml of penicillin G. OD660 = 1.
After culturing to 0, the cells are collected by centrifugation. 40 ml of cells
TES (10 mM Tris-HCl (pH 8), 10 mM NaCl, 1 mM EDTA)
After washing with buffer, 11 ml of 50 mM Tris-HCl (pH 8) /12.5%
Suspend in sucrose 100 mM NaCl / 1 mg / ml lysozyme and shake at 37 ° C. for 3 hours. 0.6ml of 0.5M EDT
A, 2.4 ml of 5 M NaCl and 4.4 ml of 4% SDS-0.7 M NaCl are sequentially added, mixed gently, and left on ice for 18 hours. At 4 ° C
After centrifugation at 65,000 × g for 1 hour, a supernatant is obtained, and 4.6 ml of 50% polyethylene glycol (MW = 6,000) is added thereto. 3 on ice
Let stand for a while and centrifuge at 1,000 xg for 5 minutes. Precipitate 5 ml TB
After dissolving in S buffer, 7.5 g of CsCl and 2 ml of 1.5 mg / ml ethidium bromide-TES buffer were added and mixed. 42 hours for this solution
The cells were subjected to density gradient centrifugation at 130,000 × g.

After fractionating the plasmid fraction detected by ultraviolet irradiation, it was treated with n-butanol to remove ethidium bromide. After dialysis against a TE buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA), a purified plasmid fraction was obtained by ethanol precipitation. This was subjected to 0.7% agarose gel electrophoresis, and the presence of the plasmid was confirmed by staining the gel with ethidium bromide.

(2) Measurement of Molecular Weight of Plasmid A part of the plasmid prepared as described above was subjected to 0.7% agarose gel electrophoresis. At this time, E. coli plasmids pUC18, pUC118, pBR322 (each
2.69kb, 3.16kb, 4.36kb) were run simultaneously. Rhodococ
cus rhodochrous ATCC4276, ATCC14349 and ATCC14348
Were named pRC001, pRC002 and pRC003, respectively, and the sizes determined by agarose gel electrophoresis were all about 2.6 kb.

(3) Specificity of cleavage by various restriction enzymes A part of the plasmid prepared as described above is reacted with various restriction enzymes, and after completion of the reaction, the reaction solution is analyzed by 0.7% agarose gel electrophoresis and 5% acrylamide gel electrophoresis. did. Lambda phage as size marker
Using the Hind III digest and the Pst I digest of DNA, the size of each restriction enzyme fragment of the plasmid was calculated. pRC001, pR
C002 and pRC003 showed the same restriction enzyme cleavage properties as shown in Table 2.

[Brief description of the drawings]

FIG. 1 shows the plasmids pRC001, pRC002 and pR of the present invention.
It is a restriction map of C003.

Claims (2)

(57) [Claims] 1. The size is about 2.6 kb and the number of restriction enzyme cleavage sites is Sac I: 2, BamH I: 1, Pvu II: 1, Sca I: 1, Sph I:
1 and Xho I: 1, a circular plasmid derived from a microorganism belonging to the genus Rhodococcus. 2. The microorganism is Rhodococcus rhodochrous ATCC42.
76. The circular plasmid of claim 2 which is ATCC14349 and ATCC14348.