JPH0367590A - Plasmidp ni10 and gene recombinant method using same plasmid as vector - Google Patents

Abstract

NEW MATERIAL:A plasmid pNI 10 having about 3.7Kb molecular weight and enzyme cut map expressed by the formula and derived from the genus Pseudomonas which is not cloven by restriction enzymes BamHI, SacI and Sal I. USE:A vector for gene recombinant. PREPARATION:For example, Pseudomonas flavide IF-4 (FERM P-10865) strain is inoculated into a cultue medium and subjected to shake culture one night at 30 deg.C and the bacteria are collected by cooling and centrifuging the cultured liquid and suspended in a buffer and lysozyme, pronase and RNAase are added thereto and allowed to stand on ice and cooled and centrifuged to separate supernatant and isopropanol is added to supernatant and the resultant precipitate is separated to give crude DNA specimen and ethidium bromide and cesium chloride are added to the crude DNA specimen and the mixture is subjected to super centrifugation to separate a plasmid DNA, which is then subjected to electrophoresis and part having same moving degree as straight-chain DNA of 2.3Kb is dissolved and plasmid DNA is absorbed into a filter by sucking and filtering the solution with a filter to provide the pNI 10 plasmid.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a novel plasmid useful as a vector for genetic recombination using Pseudomonas bacteria as a host, and a genetic recombination method using the same as a vector. More specifically, the molecular weight is about 3.7 Kb, and the first
The present invention relates to a novel plasmid pNI10 characterized by the restriction enzyme cleavage map shown in the figure and a gene recombination method using the same as a vector.

In the present invention, gene DNA encoding a target protein is ligated to any restriction enzyme site of pNI10, and this is introduced into Durham-negative bacteria such as Pseudomonas bacteria and Escherichia coli. Targeted proteins such as peptide hormones and various enzymes can be mass-produced inside or outside the bacterial cell.

[Prior art]

Conventionally, genetic recombination experiments have been widely conducted mainly using Escherichia coli as a host system, and insulin.

Great results have been achieved, including the mass production of interferon, human growth hormone, and various enzymes using Escherichia coli. In addition to E. coli, host-vector systems have been developed using yeast, Bacillus subtilis, actinomycetes, etc., and ways to apply them are being considered.

Bacteria of the genus Pseudomonas have a wide habitat range, can utilize a variety of organic compounds as a single carbon source, and can utilize chemically synthesized compounds. The development of microorganisms has become a more effective means for breeding industrially useful microorganisms.

[Problems to be solved]

The existence of several plasmids has been reported so far from Pseudomonas bacteria, but all of these have molecular weights of 30 Kb or more, and because they involve various operational difficulties, it has been difficult to create straw niblasmids using various restriction enzymes. Attempts have been made, but nothing satisfactory has been achieved yet.

In addition, genetic recombination experiments with Pseudomonas bacteria include
Examples of using a broad host range plasmid such as RSFIOIO that can be used as a multi-copy plasmid in Durham-negative bacteria are known, but this plasmid also has an 8.9Kb plasmid.
It is relatively large, and the transformation efficiency is not very high.

Therefore, there has been a strong desire to develop a small vector that can insert a foreign gene of any length, transform Pseudomonas bacteria with high efficiency, and easily detect it.

[Method for solving problems]

Against this background, the present inventors conducted intensive studies to develop hosts and vectors suitable for Pseudomonas bacteria, and finally developed a new bacterium of the Pseudomonas genus, Pseudomonas flavida (IF).
From the -4 strain, they discovered a plasmid pNI10 suitable for use as a vector and completed the present invention.

Since the plasmid pNI10 of the present invention can be maintained in E. coli, it can be used as an E. coli-Pseudomonas shuttle vector.

Also, pNllo is clearer from Figure 1, Pst
l, BcoRl, old nc If, Pvu I
It has cleavage sites by restriction enzymes such as f, 5eal, and Smal at specific and limited positions. This is advantageous in that when pNllo is used as a vector, the introduction site of the desired gene to be inserted can be significantly selected.

In order for plasmid DNA to be used as a vector, it is essential that the plasmid has the ability to autonomously reproduce within the host and a selection marker (a marker indicating that the plasmid is present within the host). Generally, Pseudomonas bacteria are kanamycin (hereinafter abbreviated as Km).
and streptomycin (hereinafter abbreviated as Sm), and if such drug resistance is imparted, the presence of the vector plasmid can be easily identified. We inserted pUc-4K (Vieria, J. and Me.
ssing+J, Gene+vol. 19, p. 259 (19
82) ) derived Km resistance gene and RSFIOIO (
Scherzinger, I! , et al., Proc, Na
tl, Acad.

Sci, USA, vol. 81, p. 654 (1984)
)-derived Sm resistance gene was introduced, and a pNIIO-derived plasmid vector having a selection marker was completed.

The plasmid pNI10 of the present invention has a large number of copies within cells, and therefore a large amount of gene amplification can be expected when recombining with a foreign gene. It is also possible to insert foreign genes of 20 Kb or more.

Furthermore, by using the plasmid pNI10 of the present invention as a vector, breeding of useful Pseudomonas bacteria that assimilates a large number of compounds can be significantly improved.

Plasmid pNI10 is a strain newly isolated by the present inventors from soil! The mycological properties of the 0 strain obtained from F-4 are as follows.

■, cell shape JILI ~ short rods with two polar flagella 0°7-1.
I There is a glow.

(2) Standard agar slant culture Straight, smooth surface, buttery, iridescent, medium growth.

(3) Standard liquid culture Slight powdery precipitate, no film formation.

(4) Standard gelatin puncture culture liquefy.

(5) Ridmus milk alkalinity ■、Physiological properties Catalase: positive Cytochrome oxidase: negative CC content 263% Pigment live TC: yellow (trypto soy agar medium).

water-insoluble dye).

Yellow (KingB medium, fluorescent aqueous solution) sex pigment) 0-F test: oxidized type Acid production: glucose xylose mannit milk sugar + + white sugar maltose Salicin Fructose 10 development: 5℃ + 41℃ Mac Conkey + SS ten Nac 10 C, V, T, + 6χNaC1+ DL-α-HB Polybeta hydroxybutyrate accumulation nitrate reduction gas from nitrites Egg yolk reaction 1 Arginine dihydrolase + Lysine decarboxylase ornithine carboxylase acylamidase Tween80 decomposition gelatin decomposition Casein degradation Starch decomposition DNA degradation Estarine degradation Urea decomposition cellulose decomposition decomposition of chitin Agar decomposition + VP, MR reaction Indole, hydrogen sulfide reaction Production of dioxyacetone Use of citric acid T, S, I.

0, N, P, G.

Mucoid production - + Alkalinized lidmus milk Phenylalanine reaction Growth temperature (°C) 5-37 Growth pH 5.0-9.2 Based on the above mycological properties, the Berger manual (Berg.
ey's Manual of Determi
Native Bacteri-ology) 8th Edition, Cowan Steel Manual (S, T, Co
Wan et al.: 1974 Manual
for the identification of
Medical Bacteria), CDC Manual (R,EJever and D,G,Ho
1lis: 1983 Gran-Negative
Organis+++s: An approach
t.

Identification Centers for
r Disease Control 1), this strain was found to be Pseudomonas (Pseudomonas).
as) was recognized.

However, as for the species, it is a new strain that is not described in each manual, and because it carries a new plasmid pNI10, the 0 strain, which was determined not to be any of the conventional species, is a particularly shiny yellow bacteria. For some reason, Pseudosonas flavi
da) The strain named IF-4 strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FERM P-10865.

Lost ship group (1) Pseudomonas flavida IP-4 (Pf!RM
pNllo DNA from cultured cells of P-10865)
Isolation of PY medium (5 g of peptone, 5 g of yeast extract, 1 part of water)
ffi, pH 7,0) was inoculated with Pseudomonas flavida IF-4 (FERM P-10865) strain, and -
At night, culture with shaking at 30°C and add 1 liter of culture solution to the same fresh medium 10011! and cultured at 30°C for 16 hours.

This culture solution was cooled and centrifuged to collect bacteria, and SET buffer (20
% Sucrose, 505M [EDT^, 50mM)
Suspend in lithium hydrochloric acid (p)I 8.0) 20d.

To this solution, 2.25 d of lysozyme (10 mg/II & Boehringer Mannheim product) and 2.25 d of pronase (10 s
rg/I11. Kaken Kagakusha product) 250μm, RN
Add 250 IIN (5 tig/ld, Boehringer Mannheim product), stir gently, and leave on ice for 5 minutes. Next, add 45 m of alkaline SDS solution (1% S
D S , 0.2N NaOH) and stir by swirling up and down in a centrifuge tube. After cooling on ice for 10 minutes, 321
Add the 3M sodium acetate solution (pH 4,8) from step d, mix, and let stand in ice water for 1 hour. After cold centrifugation at 19,000 rpm for 15 minutes, the supernatant was separated. Furthermore, the same volume of isopropanol as the supernatant was added, and after cooling on ice for 2 hours, centrifugation was performed at 8000 rpm for 10 minutes. After centrifugation, the precipitate was washed with a 70% ethanol solution. After drying the precipitate under reduced pressure in a desiccator using a vacuum pump, 8jd
TE buffer (10s+M) Lis-HCl, 1mM EDT
A (pH 8.0) and used as a crude DNA sample.・To this, add 0.56 m of ethidium bromide of 10 μ/d and 8.
Add 48g of cesium chloride and 1 at 50,000 rpm+i.
Plasmid DNA was isolated by ultracentrifugation at 16°C for 7 hours. The separated plasmid DNA was treated with isopropanol saturated with saturated cesium chloride water to remove ethidium bromide, and then dialyzed against TE buffer to obtain plasmid DNA. Next, the DNA was transferred to 0.7% agarose. Gel, TBE buffer (5 hM) Lis-borate, 2 gM
Electrophoresis was performed using IEDTA, pH 8,0). When the former ndll1 digest of λ phage DNA was used as a DNA molecular weight marker, the mobility of the circular plasmid pNllo was the same as that of a linear DNA fragment of about 2.3 Kb. This part is cut out from the agarose gel, the gel is dissolved in 8H sodium perchlorate solution, and this solution is suction-filtered through a Whatman 10FC filter to adsorb the plasmid DNA onto the filter. After washing the filter with 6M perchloric acid solution and 100% ethanol, the filter was immersed in TE buffer to collect plasmid DNA. By the above operation, high purity pNI
Ten plasmids were prepared.

(2) Measurement of molecular weight of pNI10 plus pNllo
The molecular weight of pNllo was determined by 0.7% agarose gel electrophoresis of a fragment obtained by cleaving pNllo with the restriction enzyme Pstl. As a molecular weight marker in this case, λphage D
A Hindl1-digested product of NA and a BstB-digested product were used. From this, the molecular weight of pNIIO was calculated to be 3.7 Kb.

(3) Creation of restriction enzyme map of pNI10 Purified pNI
10 plus ξ DNA was cut with various restriction enzymes. After the cleavage, the molecular weight of the restriction enzyme-cleaved fragment of this plasmid DNA was determined by 1% agarose gel electrophoresis in the same manner as in section (2).

Table 1 shows the number of cleavages for various restriction enzymes.

A restriction enzyme cleavage map as shown in FIG. 1 was created from the restriction enzyme cleavage patterns shown in Table 2.

(4) Measurement of copy number of pNllo Pseudomonas flavida IF-4 (Fil!RM P
-10865) strain was cultured using the method described in (2) above, and 0 bacterial cells were obtained using the known method of Saito and Miura [Biochis + i
ca et Bio-physica Acta),
72, pp. 619-629 (1963)), the total DNA was extracted and purified.
μg was completely digested with the restriction enzyme EcoRI, and a portion thereof was subjected to agarose gel electrophoresis using the method described in section (2) above.

The separation pattern by agarose gel electrophoresis was photographed, and the ratio of the peak of the band derived from pNI10 to the peak of other DNA portions was measured using a densitometer. The total amount of DNA per cell of Pseudomonas bacteria is 4X10
``Assuming Dalton, the copy number of pNllo per cell was calculated to be 30-40/cell.

(5) Plus straw pNI105. pNllo6. p
Nllo7. Construction of pNllll a〉Plus ρNll0 restriction enzyme old ncII cleavage site was inserted into the known plasmid pUc-4K (Vieria,
J, and Messing+J, Gene, 1
9, p. 259, (1982)) with the restriction enzyme old nc [
The approximately 1400 bp DNA fragment containing the tar gene produced by the treatment was transformed into T.

Pseudomonas flavida IP-42 (FBRM-P
10865) strain by the method described in the next section, transform K11
A transformed strain was selected using PY medium containing 25uglx&Km'. From this transformed strain, plasmid pNI105 having a Km' marker was obtained.

b) Using the same method as in section a), add 90C-4K to the restriction enzyme Sma I cleavage site of pNIIO.
The approximately 1400 bp K-gene-containing DNA fragment generated by the Il treatment was ligated and transformed into Pseudomonas flavida strain IF-42, thereby obtaining plasmid pNI106 having a Ks' selection marker.

c) By changing the restriction enzyme old nclI cleavage site of pNllo6 to the restriction enzyme Bas + old cleavage site,
pNllo7 was obtained.

The construction procedure of pNllo7 is as follows.

First, pNllo6 is cut with restriction enzyme C1all.

Add a commercially available phosphorylated Ba5HI linker to this DNA fragment.
(manufactured by Takara Shuzo ■, having the CGGATCCG sequence) was ligated using T and DNA ligase. Then Ram
Apply T4 DNA to make both ends sticky and add T4 DNA again.
A circular DNA was prepared using ligase. Using this, Shademonas flavida strain IF-4 (FORM-P 10
865), thereby obtaining pNIIO7.

d) I) N1107 is partially digested with restriction enzyme C1al, and the DNA fragments cut at the ly position are separated and recovered by agarose gel electrophoresis. The obtained positive DNA
After making the ends blunt using DNA polymerase, RS
Approximately 1.9 Kb DNA containing the Sm resistance gene excised from FIOIO with the restriction enzyme old nc II -Pvun
The A fragment was ligated using T4 DNA ligase. Using this plus ξ DNA, Pseudomonas putida (Ps.
eudoaonas putida) IF5 strain was transformed, and an Sm-resistant strain was isolated. When we prepared a plasmid from the Sm-resistant transformant and examined its structure, we found that the second
As shown in the figure, Cla in the Km resistance gene of pNIIO7
Plasmid pNI with Sm resistance gene inserted at l site
111 was obtained. FIG. 2 shows an outline of each vector.

(6) Transformation of Pseudomonas bacteria and other Durham-negative bacteria Pseudomonas bacteria can be transformed using known methods (M, B
Agdasarian et al., Gene.
16, p. 237 (1981)) with some modifications.

Inoculate the PY medium with a stock seed of Pseudomonas bacteria,
Shaking culture was performed at 30°C for 15 hours. Collect 1 ale of this culture solution, then add 2.5 m of transformation buffer 1 (10 mW MOPS, 10+sM Rb
C1, 100mM MgC1z, pH 7.0) was added to suspend the bacteria. After standing at 0°C for 30 minutes, the cells were collected by centrifugation, and 0.3-buffer I was added thereto to resuspend them to obtain competent cells. Add 25 μl of DNA sample to competent cell solution and let stand at 0°C for 45 minutes. Next, heat treat at 40°C for 5 minutes, add 1.7 ml of PY medium, and culture with shaking at 30°C for 3 hours. 0.15 d of the culture solution was applied to the surface of any selective medium to select the desired transformed strain.

(7) Transformation efficiency According to the method of (6), the transformation efficiency of several Pseudomonas bacteria was measured. The efficiency of transformation is lp
The number of transformed strains (Km-resistant strains) obtained per g of pNI105 plasmid DNA was expressed as a standard. Table 2 shows the results.

Also, pNI106. pNI107. pNllll
When each of the vectors (Sm resistant) was used, the same results as in Table 2 were obtained.

Table 2 (8) Search for host bacteria of pNI105 It was investigated whether plasmid pNI105 could be replicated and expressed using Escherichia coli as a Gram-negative bacterium other than Pseudomonas bacteria as a host. The transformation method is a known method (Ku
shner, S.R., Genetic Engine
ering 17 pages, B15evier (1978)
) Competent cells were prepared according to the following method.

As a result, Escherichia coli (f!5 cherichia col
i) It was found that the pNI105 vector was stably maintained in the C-600 strain. especially[! , coli C
The transformation efficiency in 600 strains is 2 XIO' cel
1g/#g DNA, very high rate, pNI105
could be satisfactorily used as a shuttle vector between Shademonas bacteria and Escherichia coli.

Also pNI106. pNI10? , pNI111 and pNI111 gave similar results to pNI105.

As described above, pNI10 plus ξd is useful as a shuttle vector between Pseudomonas bacteria and E. coli.

(9) Length of DNA that can be inserted An experiment was conducted using the following method to determine how much foreign DNA could be inserted into pNI107.

First, foreign DNA was prepared from Pseudomonas putida according to the method described in the previous section (4).The DNA was then partially digested with the restriction enzyme EcoRI to obtain a foreign DNA preparation.

On the other hand, pNI107 DNA was cut with EcoRI, ligated with this vector DNA using the foreign DNA @ Ta ligase, and Pseudomonas
Putida IF3 strain was transformed. 5 Km-resistant strains arbitrarily
0 strain was selected and plasmid DNA was extracted from the obtained transformed strain.
was isolated. Plasmid DNA obtained from each strain
Foreign DNA inserted by cutting A with restriction enzyme BcoRI
I cut out A. The length of this DNA was determined by agarose gel electrophoresis according to the method described in the previous section (2). As a result, it was revealed that DNAs of various lengths were inserted into this vector, and that the largest DNA was 20 Kb or more.

〔Effect of the invention〕

Plasmid pNI obtained according to the invention as described above
Reference numeral 10 is a small vector, into which a gene DNA encoding a protein of interest can be ligated to any restriction enzyme site. Therefore, by introducing this into various bacteria, it is possible to mass-produce target proteins such as peptide hormones and various enzymes inside or outside of these bacteria.

[Brief explanation of drawings]

FIG. 1 shows the restriction enzyme map of the plus turtle pNI10 of the present invention. FIG. 2 shows the restriction enzyme map of each plasmid derived from the plasmid pNI10 of the present invention, and a) in the figure shows plasmids pNI105, b) is plasmid pN
I106, C) is plasmid DNI10? , d) show the restriction enzyme map of plasmid pNI111, respectively. In the figures, - indicates DNA derived from pNIIO, "K" indicates DNA derived from pUc-4, and "1" indicates DNA derived from RSFIOIO.

Claims (1)

[Scope of Claims] 1) A plasmid pNI10 derived from a Pseudomonas bacterium that has a molecular weight of approximately 3.7 Kb, has the enzyme cleavage map shown in FIG. 1, and is not cleaved by restriction enzymes BamHI, SacI, and SalI. 2) A plasmid in which a selection marker for kanamycin resistance or streptomycin resistance is added to plasmid pNI10. 3) Gene recombination method using pNI10 or a plasmid derived from pNI10 as a vector.