JPS6087789A - Composite plasmid - Google Patents

Abstract

PURPOSE:To provide a vector having the domains of the tetracycline-resistant and ampicillin-resistant genes, recognizing the cleavage site with a specific restriction enzyme between both domains, and having the characteristics of a shuttle vector shuttling between Escherichia coli and Bacillus subtilis. CONSTITUTION:The plasmid pAMalpha1 of Streptococcus faecalis DS5 (ATCC 14508) and the vector of Escherichia coli pACYC177 are incised enzymatically in a manner to contain the domain of the tetracycline-resistant gene (Tc) originated from pAMalpha1, the domain of the ampicillin-resistant gene (Amp) originated from pACYC177, the replication origin domain (OripAMalpha1) originated from pAMalpha1, and the replication origin domain (Ori177) originated from pACYC177. The objective composite plasmid is produced by connecting the above domains. The plasmid is characterized by the sequence of the restriction enzyme recognition cleavage sites of the figure.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the treatment of Streptococcus faecalis, a Durham-positive bacterium.
The present invention relates to a novel composite plasmid useful as a cloning vector for Escherichia coli and Bacillus subtilis, which is synthesized from a plasmid of Escherichia coli (Escherichia coli) and a Gram-negative bacterium, Escherichia coli, or Escherichia coli.

More specifically, the present invention combines the tetracycline resistance gene region (1+C) derived from the plasmid pAMα1 of Streptococcus-7 echaris and the vector pA (
, 'Ampicillin resistance gene region derived from YC177 (A
+np), and the replication initiation region derived from the plasmid pAMα1 (Or! Menya α1) and the pAcYc1
The tetracycline resistance gene region has a replication initiation region derived from Jri 77 (Jri177), and the recognition cleavage site of the entire domain 'Jri177' for the restriction enzymes I and Jri11 is located in the tetracycline resistance gene region, and the ampicillin resistance gene region In the genetic region,
This invention relates to a novel complex plasmid characterized in that only one recognition cleavage site is located in the entire region for each of restriction enzymes II, Pvu I, and psti'.

In general, in vitro genetic manipulation requires the use of vectors compatible with host cells in order to transfer desired foreign DNA into host cells and express the genetic information.

Regarding vectors for genetic manipulation, the host-vector system of Escherichia coli, which has been extensively studied, is the most commonly used system (which has been elucidated); Research is being actively conducted to develop host-vector systems, including the actinomycetes that produce substances, and the instruments that are widely used in the brewing field.

By the way, it is known that the essential conditions for a vector are the presence of a gene sequence necessary for self-replication and a restriction enzyme recognition cleavage site for inserting foreign DNA.
For practical use, for example, the types of restriction enzymes and their recognized cleavage sites to enable and facilitate genetic manipulation, the efficiency of expression, the presence of marker genes necessary for detecting transformants, etc. , compatibility with host cells, host range, stability within host cells, and adaptability to biological containment aimed at reducing the risk of hypothetical biological disasters. Because the characteristics are required,
Currently, there are not many examples of the development of practically useful vectors regarding the host-vector systems of E. coli and Bacillus subtilis.

In view of the current situation, the present inventors have so far established a host-vector system for Escherichia coli and Bacillus subtilis, which is industrially useful as a producer of amylase, etc. (and developed a useful plasmid vector). As a result, a composite plasmid synthesized from Streptococcus faecalis plasmid pAMα1 and E. coli vector pAc YC177 possesses various characteristics suitable as a vector for genetic manipulation of E. coli or Bacillus subtilis. However, in order to improve the ratio of vectors, we also aim to measure their stability within the host and increase their tolerance to foreign DNA, assuming the industrial practical use of these new vectors. As a result, they succeeded in downsizing the vector and completed the present invention.

The composition of the complex plasmid of the present invention is Strep):'
t'jJ l SuDS 5 (AT
Ce14508) 0. ) The plasmid pAMα1 and the E. coli vector pAcYe177 were combined to create the above pAMα1.
The tetracycline resistance gene region (Tc) derived from the above pACYC-177, the ampicillin resistance gene region (A+np) derived from the above pACYC-177, and the replication initiation region of the above pAMα1 Yamaki (Ori pAM(!1) and the above pAcY
It is a composite plasmid created by enzymatically cleaving and ligating pHY340 so that it contains the replication initiation region (Ori 177) of pHY340.
It is a complex plasmid named .

The above-mentioned complex plasmid according to the present invention has a resistance gene region for tetracycline and ampicillin on its DNA, and therefore, upon transformation, it has the property of imparting resistance to both drugs to E. coli host cells. There is. This characteristic serves as a selection marker for detection and selection when obtaining a recombinant plasmid-carrying strain, ie, a transformant, which has integrated the desired foreign DNA. In addition, in the tetracycline resistance gene region of the composite plasmid according to the present invention, the only recognition cleavage site among the entire region for the restriction enzyme Ba1 (and Hpa I) is located, and the ampicillin resistance gene region contains three types of Since the only recognition cleavage site in the entire region for each of the restriction enzymes Eli, Pvui, and IstI is located, these restriction enzymes can reduce the 1) NA of the complex plasmid according to the present invention.
As long as 1) NA is specifically cleaved, there will be no possibility that a large number of undesired cleavage sites will be generated in NA and it will become fragmented.

Therefore, any one of the only cleavage sites in the above-mentioned entire region can be used as the insertion site of a desired foreign rice foreign species 1) NA.

In particular, the recognition cleavage site of the restriction enzymes I and I of the tetracycline gene region is D.
The so-called flush end, in which the center of the 6 bases of NA is cleaved, is completely formed and then cleaved, so DNA created by cleavage with the same type of restriction enzyme
Not only fragments but also I) NA fragments cut with any other flash-end restriction enzymes can be used with linker-DNA.
It is possible to perform direct ligation without the use of other methods, and furthermore, it has the feature that it is possible to clone artificially flush-ended DNA fragments.

The complex plasmid according to the present invention can stably proliferate in Bacillus subtilis as well as E. coli by the action of the replication initiation region (Or i pAMα1) derived from plasmid pAIVα1.
Since the tetracycline resistance gene is expressed in both of the above bacterial species, it has the characteristics of a shuttle vector l-(5huttlevector) that can shuttle between E. coli and Bacillus subtilis.

I would like to add that DNA is currently the most advanced in research and development.
The cloning system uses Gram-negative bacteria.
(Nizierisia Aikoli K) and its vector system (EK system)
In this system, genes derived from Gram-negative bacteria and certain genes derived from Gram-negative bacteria can be expressed.

However, on the other hand, there are still many problems to be solved regarding the Gram-negative bacterial system, and a great deal of attention is being paid today.In particular, among the Gram-negative bacteria, Bacillus subtilis 5ubtilis), although it is a useful microorganism with moderate practical advantages,
Since its biological properties are completely different from those of Escherichia coli, its genetic analysis and further development of gene cloning systems have become of great interest in recent years.

Under these circumstances, it is an urgent industrial need to establish a host-vector system for Bacillus subtilis. The development of a shuttle vector that can shuttle between Bacillus subtilis, a representative species of Gram-negative bacteria, is significant as it overcomes one step toward establishing a DNA cloning system for Gram-negative bacteria. The complex plasmid according to the present invention is industrially useful.

In addition, other Gram-negative bacteria such as Lactobacillus 7%, Bihydrobacterium spp.
It is industrially promising in that it has the potential to be used extensively in the field of genetic analysis and molecular breeding of microorganisms belonging to the erium).

Next, the complex plasmid pHY-340 according to this invention
The synthetic route and various intermediates of vector pACMC177 (Figure 1 (A)) will be explained based on Figures 1 to 5.
and plasmid pAMα1 (Fig. 1 (B)) as starting materials, intermediate composite plasmids pHY780 (Intermediate 1) (Fig. 1 (C), Fig. 2), pi-IY600
(Intermediate 2) (Figure 1 1Fl), Figure 3), pHY4
60 (Intermediate 3) (Figure 1 (IC), Figure 4) and p
It is synthesized via HY385 (intermediate 4) (FIG. 1F1, FIG. 5).

Next, the structure of the composite plasmid according to the present invention and the composite plasmid as the various intermediates described above will be explained in detail as follows.

Construction of composite plasmid pf4Y 780 (intermediate 1) (11p HY 780 is a plasmid pAM containing the tetracycline resistance gene region (TC) of Streptococcus faecalis D85 (ATCC1450B)
D obtained by cutting α1 with restriction enzyme 11ind ■
N A 1iji fragment and E. coli ampicillin resistance gene region (Amp) and kanamycin resistance gene region (K
This is a composite plasmid with a molecular weight of approximately 7.8 megadaltons, which is synthesized by ligating the plasmid vector pAcye177 containing the above vector pAcye177 with the DNA fragment obtained by cleaving the vector pAcye177 with restriction enzymes Hind and II.

Plasmid pAMα1 and vector pAeYc177 used as raw materials for such synthesis are both known (pAMα1: proc.

Natl, Acad, Sci,, USA, 72, 172
0-1724 (1975), pAcYc177:
J, Bacteriol, 134.1141-11
56 (1978)).

In addition, when determining the molecular weight of the synthesized complex plasmid, 1-1 indll of E. coli λ phage I) NA
Molecular fragments obtained by digestion with Mo1 (J, Mo1
．． Biol, 98.551-564 (197
5)) was drawn on a 0.8% agarose gel (by comparison with the standard line of migration distance, the molecular weight of each fragment of 1Y780 (plasmid p) digested with various restriction enzymes was measured, and their total sum was calculated. .

+21 p) IY780 has 9 unique recognition cleavage sites for each of the restriction enzymes yl (and Psti) in its ampicillin resistance gene region (Amp), and furthermore, its tetracycline resistance gene region ( TC),
Since it has all the recognition cleavage sites for the restriction enzyme Bali, these recognition cleavage sites can be used as insertion sites for foreign DNA.

The cleavage sensitivity (number of recognized cleavage sites) of pMY780 to various restriction enzymes is as follows.

Number of restriction enzyme cleavage sites ■1 January 2 Bgl l I U! Jcol (i 2 Hae l [4 Hi Direct 1 dl [3 Hpa I 2 1 (pHI 2 normal + t ■ 1 heart 17 2 support ■ l 5 al I I Xba I 1 stop ■ 1 The above number of cleavage sites is pl-IY780 It is determined according to the number of distinguishable bands when the digested product obtained by complete digestion in the presence of an excess of various restriction enzymes is subjected to electrophoresis on an agarose gel.

Furthermore, a detailed restriction enzyme cleavage map is shown in FIG.

(3) pHY780 is not only able to self-reproduce within the ecosystem of Escherichia coli, a Gram-negative bacterium, but also has ampicillin (A+np) and tetracycline (IllC) resistance present in a specific region on its self-replicated DNA. Because it imparts resistance traits derived from genes to its host bacteria,
This can be used as a selection marker for transformants.

ill p) LY600 was synthesized by cleaving the above p) LY780 with restriction enzyme unHI to delete a DNA fragment of about 1.8 megataltons, and then ligating the obtained DNA fragments with 14 ligase. Molecular weight approximately 6.0
It is a megadalton complex plasmid.

(21pl (Y2O2 is the above p) Similar to LY780, the ampicillin resistance gene region (A+np) contains the entire region 9 for each of the restriction enzymes Bgl and Psti.
In addition, the tetracycline resistance gene region (Tc) has 9 unique recognition cleavage sites for restriction enzyme L (al), so these recognition cleavage sites can be used to transfer foreign DNA. can be the insertion site.

1, and the cleavage sensitivity (number of recognition cleavage sites) of pi-IY600 to various restriction enzymes is as follows.

Restriction enzyme cleavage site number Nagi 11 □Old 1 Restriction enzyme cleavage site number Bg'l 1 1 Stop mowing Rl 2 River re II 2 , Pressure Dull I Hpa　I　2 Crab 11 True 2 Fallen CI [1 Dangenko 11 Responsibility>aI 1 N111 Furthermore, a detailed restriction enzyme cleavage map is shown in FIG.

(3) pHY600 is similar to pHY780,
It is capable of self-replication within the ecosystem of Escherichia coli, a Gram-negative bacterium, and imparts resistance to tetracycline and ampicillin to the host bacterium, so it can be used as a cloning vector for at least Escherichia coli.

+1) pHY460 is obtained by cleaving the above pHY600 with restriction enzymes, io■ and 3al■.
This is a composite plasmid with a molecular weight of approximately 4.6 megadaltons, which was synthesized by deleting a 4 megadalton DNA fragment and ligating the obtained DNA fragments using +1t4 ligase.

(2) pHY460 contains restriction enzymes 1×, 1×, and P in its ampicillin resistance gene region (A+np).
The entire region has 9 unique recognition cleavage sites for each of the
c) has 9 recognition cleavage sites in the entire region for each of the restriction enzymes Dan1 and Seimi, so these recognition cleavage sites can be used as insertion sites for foreign rice DNA.

The cleavage sensitivity (number of recognized cleavage sites) of pHY460 to various restriction enzymes is as follows.

Restriction enzyme cleavage site number Dansue 11 holder l Hg1i I EcoRl 2 Rib chewing ■ 2 raw>a ■1 Si±11 Haue■1 Suction pressure [I K rib-11 Furthermore, a detailed restriction enzyme cleavage map is shown in FIG.

(31pHY460 is capable of self-replication in the ecosystem of not only Escherichia coli, which is a representative of Gram-positive bacteria, but also Bacillus subtilis, which is a representative of Durham-positive bacteria. Since it imparts traits, it can be established as a cloning vector (shuttle vector) compatible with E. coli and Bacillus subtilis.

(1) PX conjugate plasmid pHY385 has a molecular weight of approximately 3
．． 85 megadalton cyclic deoxyribonucleic acid (L) NA
).

(21p) lY385 is obtained by subculturing Bacillus subtilis transformed with the above pHY460 at 37°C for about 50 generations.
pl (+31 pi, a complex plasmid obtained by deleting a portion of Y460 (equivalent to about 0.75 megadaltons)
-IY385 has its ampicillin resistance gene region (A
11ll)) has 9 recognition cleavage sites in the entire region for each of the restriction enzymes, Ygl (, Katarie I, and Pvul), and furthermore, the tetracycline resistance gene region (Tc) has restriction enzymes, Since there are 9 recognition cleavage sites in the entire region for each of Sn and Hpai, these recognition cleavage sites can be used as insertion sites for foreign DNA.

The cleavage sensitivity (number of recognized cleavage sites) of pHY 385 to each restriction enzyme is as follows.

Restriction enzyme cleavage site number Le-shaped II Bgl I I EcoRI 2 Tsuji Satoru-■　2 Hpai1 Yamiyu I I PvuI, 1 Responsibility 1 Furthermore, a detailed restriction enzyme cleavage map is shown in FIG.

(4) Like p)lY460, pHY385 is capable of self-replication in the ecosystem of Escherichia coli, a Gram-negative bacterium, and Bacillus subtilis, a Durham-positive bacterium, and also imparts tetracytalline resistance to these host bacteria. It can be used as a cloning vector for E. coli and Bacillus subtilis.

Composition of complex plasmid pi (Y340) (11 complex plasmid pHY340 has a molecular weight of approximately 3.4
It is a megadalton cyclic deoxyribonucleic acid (1) NA).

(21pHY340 is obtained by cutting the above pi-1Y385 with a restriction enzyme dish
) A composite plasmid with a molecular weight of approximately 3.4 megadaltons was synthesized by cutting out the NA fragments and ligating them using T4 +1 gauze.

+3) 1) l-IY340 contains restriction enzymes Bgl I and P in its ampicillin resistance gene region (A+np).
It has 9 recognition cleavage sites in the entire region for each of stI and Pvu I, and further has 9 recognition cleavage sites in the entire region for each of the restriction enzymes Bali and Nagi I in its tetracycline resistance gene region (Tc). Therefore, these recognition cleavage sites can be used as insertion sites for 1) NA.

So. The cleavage sensitivity (number of recognized cleavage sites) of pi-IY340 to various restriction enzymes is as follows.

Restriction enzyme number of cleavage sites T al (1 horse II EcoRI2 sub [1 f(pa)' I Pst) I Pvu ■1 Restriction enzyme number of cleavage sites Sac II 1 In addition, a detailed restriction enzyme cleavage map is claimed as a claim. Second
Described in section.

+31 pHY340 is capable of self-replication in the ecosystem of not only Escherichia coli, which is a representative Gram-negative bacterium, but also Bacillus subtilis, which is a representative Durham-positive bacterium, and it also imparts resistance traits derived from tetracycline resistance genes to these host bacteria. , it is established as a cloning vector (shuttle vector) compatible with E. coli and Bacillus subtilis.

The composite plasmid pHY340 according to the present invention, configured as described above, completely satisfies the necessary conditions as a vector when cloning any gene by DNA recombination technology using Escherichia coli and Bacillus subtilis as host bacteria. Therefore, according to the complex plasmid of the present invention, specific genes related to biosynthesis of useful substances or their regulation can be cloned from pond microorganisms into host bacteria such as Escherichia coli and Bacillus subtilis. , by causing the production of useful substances within the ecosystem of the host bacterium, and further by strengthening the biosynthetic system through the amplification of genetic information related to the above-mentioned specific genes, increasing the productivity of useful substances. It is possible to provide an effective means for

Since various genes derived from other microorganisms can be expressed within the ecosystem of Gram-positive bacteria such as Bacillus subtilis, it is an effective means for gene analysis and molecular breeding of Gram-positive bacteria. can also be provided.

Next, embodiments and reference examples of the present invention will be described as follows.

tl) Preparation of pAcYc 177 12 WA802r+n+ (pACYc177
) (held by Saito Laboratory, Institute of Applied Microbiology, University of Tokyo), L
-Pros (Bacto-1ry)
pton) 1%, yeast extract XO, 5%, NaC
l0.5%, glucose 0.1% in N-NaOH
After culturing overnight in PI (adjusted to 7.0), the bacteria were collected by centrifugation, and then transferred to 100 mj' (7.0
J20+nM Tris-IQ+nM nDTA (1)
Washed twice with H8,O). After washing, the bacterial cells were washed with 9mr.
100+nM Nael-2Q+nM Tris-IQ
+nM EDTA. Next, lysozyme and Na5e were added at 100 μg/+n/ and 50 μg/+n/, respectively.
μg/ml! After reacting at 0°C for 10 minutes, 1+nl of 2% SDS was added and the bacteria after the reaction was further lysed at 37°C for 5 minutes. After leaving this solution at 0°C for 10 minutes, 36.000 r p+n
Centrifuge the separated supernatant for 30 minutes at 1) N
A sample was collected as a crude specimen. Add an equal weight of cesium chloride to this, and get 5+ng/+nl! 0.71 n/ethidium bromide was added. To this, 36.00Orpm,
After 40 hours of centrifugation, 1) the NA fraction was collected, and further centrifuged at 36,000 rl) for 40 hours. EtBr was removed by washing the separated I) NA fraction with isopropanol saturated with saturated cesium chloride water. Then, 1 mM
Tris-Q, 1mM EDTA (pH 7,4)
(hereinafter referred to as buffer A), and further, 1) N
An equal amount of phenol (saturated with buffer A) was added and shaken. Then, the aqueous layer obtained by the centrifugation process was dialyzed against buffer A to prepare pA, cYc 177 DNA shown in FIG. 1 (cut map of N).

[21 Preparation of pAMα1 Streptococcus faecalis DS5 (ATCC1
4s013) in 500+nl (7J O Gosa medium (
Rogo-sa + nediu + n 211 medium Glucose 20g,) Lipticase-Peptone 10g 1 Yeast Nikis 5g, Trypticase 3g,
K2HPO43g.

KHzP (J43 g, ammonium citrate 2 g.

Sodium acetate Ig, Tween 801g, IVIg8
(J, ・R2 (J O, 515g% L-cystine hydrochloride 0.5g, Mn5U4 @ 2Hz (J O, 12
g, l″esO4@7H2U 84Ing
: Cultured overnight at pH 6, 8).

The DNA extraction process described below is performed using the pAeYC-
This was carried out in the same manner as in the case of the adjustment of No. 177.

0.5 ml of the extracted DNA was then transferred to 121 nl! in a nitrocellulose centrifuge tube. 5% ~ 3096
dli [placed on the gradient end. As a buffer solution,
50InMTTris-5Q+nMnDTA-50InM
Nacl (pi-18,0) was administered. 20.000
rp+n, after centrifugation for 16 hours at 10°C.
A hole was made in the bottom of the nitrocellulose centrifuge tube and 10 drops were fractionated. 0.896 μl of DNA in each fraction
The molecular weight of the DNA was confirmed by electrophoresis on an agarose gel. Eight fractions with a molecular weight of 6 megadaltons were then dialyzed against buffer A. After the above processing, Figure 1+1
pAMα1, represented by a cleavage map of 11, was isolated from a mixture of β1, rl.

(10 μl of cleavage and ligation of 31pkCYC177 and pAMα1 obtained by the treatment described in (1) and (2) above)
For each DNA (0.1-0.2μg DNA amount), 1μ
E buffer (10 InM Tris-HCI)
(pH 7,6), 7InMMgCI2.7 +nM β
-Mercaptoethanol, 50mM NaCl) was added, and then 1 µl of 1-1indll of '' was added, and this was reacted at 37°C for 600 minutes. And 6
After stopping the reaction at 0°C for 1o min, a small amount of 5M
NaCl was added, and twice the total amount of cold ethanol at 20°C was added, followed by cooling at -20°C for 30 minutes. Furthermore, this is 15.000 rp+n, O'C15
minutes, centrifugation minutes! After the treatment, the supernatant was discarded and the precipitate was washed again with ethanol at -20°C. This is 15,
000rp+n.

After centrifugation at 0°C for 2 minutes, the supernatant ethanol was discarded and the ethanol was completely evaporated.

1) The NA fragment thus obtained was dissolved in 20 μl of sterile water, and 31 tl of 10 InM ATP was added.

100 +nM dithiothreitol 3ttl, 66
0 +nMTris-HCI (pH7,6) -66
Add 3 μl of InM MgCl2, and add 400 μl of InM MgCl2.
/ltl (0.5 μl of 7JT4 ligase was added. 1
After reacting overnight at 5° C., 120 μl of sterile water was added to bring the total volume to 150 μl.

(4) Transformation of Escherichia coli 12 C600r In (Applied Microorganisms of the University of Tokyo (held by Saito Laboratory, Research Institute)) was cultured overnight in L-broth. −
In addition to the broth, shaking culture was performed at 37°C for 2 hours and 10 minutes. After collecting bacteria by centrifugation, the obtained bacteria are
°C Q, l R4CaC1□Wash with water, 0,25
Competent cells were generated by dissolving in 0.1 M CaCl2 water at 0°C. And 0.1
0.11 nlO,) per ml competent cells,
DNA obtained by the treatment described in (3) above (1 μg ia,
After adding Anri and leaving it for 5 minutes at 0℃, 0.8+n
l! of L-broth was added and cultured at 37°C for 1 hour.

Next, this culture solution was added to L-broth with 1.596 agar,
20gg/ml of tetracycline and 30gg
The cells were spread on the surface of a selective medium supplemented with ampicillin/l and cultured overnight at 37°C. At that time, the size of the plasmid possessed by the 10 transformants that formed colonies on this selective medium was examined.

5) Plasmid J) Extraction of NA and measurement of its molecular weight Culture solution 5+ obtained by culturing the transformant overnight in L-gloss
n/ was collected by separation treatment. Then, the isolated bacteria were treated with 0.5 Inl of 100mM NaCl-50InM.
It was dissolved in Tris-10+nM EDTA (pH 7,4).

To this solution was added 0.21n7 of 2+ng/+nl lysozyme-Q, 5 mg/+nI! 1 (INase was added and reacted at 37°C for 5 minutes. 0.2 In l
Add a 2% SD8 solution of
Centrifugation was performed at 0°C for 10 minutes at 00 rpm+n. Separated supernatant 0.5+nI! 0.5 In l of phenol saturated with buffer A was added to the mixture and mixed well. Furthermore,
This was centrifuged at 15,000 rp+n for 3 minutes at room temperature, the aqueous layer was collected, 50 μl of each was subjected to 0.8% agarose gel electrophoresis, and its molecular weight was measured.

As a result of the above treatment, the size of the plasmid of 10 transformants was examined, and the molecular weight was approximately 7.8 megadaltons.

In this way, among the complex plasmids synthesized from pAcYc177, I) AMαl, which is represented by Hitoshi's cleavage map in Figure 1, f(,') in Figure 1, more specifically, The one represented on the map was named pl(Y780), and the other one was named pi-iy 11.

As a result, it was confirmed that at least pHY780, which is represented by the tc+ cleavage map in FIG. 1, grows stably in the E. coli ecosystem and expresses its characteristics.

In addition, when linking pHY780, L te f; t , pA
CYC-177(7) DNA fragment and l) Another complex plasmid p of the same molecular weight with a different direction of connection with that of AMα1
HY11 is also produced simultaneously, but both complex plasmids p
HY780 and pi-iy 11 can be separated into two groups with different fragment sizes by cutting using addition H1. And, Figure 1 (C
Based on the cut map of 1, two replication start regions (Or
i pAMα1 ), (Ori177) The number of cutting sites should be increased (
, from the viewpoint of the presence or absence of a restriction enzyme that can be used to cut and remove a portion in the entire region containing the same cleavage site as the restriction enzyme recognition cleavage site in both gene regions (Tc) (A+np).
Both complex plasmids pHY780, p of pHyH
HY 780 was selected as a promising candidate for subsequent downscaling.

<Reference Example 2> Composite plasmid p) lY600
Synthesis of (intermediate 2) (1) pl-IY780 (7J cleavage and ligation reference example 1
Escherichia coli C600r-m, a transformant prepared with
-(pHY780) Kara, Reference Example 1 (Plasmid 1) NA was extracted by the same method as described in Section 51. 1) 1 μg/20 μl of extracted NA and 2 μl of buffer (100+nM Tri 5-HCI (pH
-7,6), 70+nM MgCl2.70mM β
-Mercaptoethanol, 500mM NacI) and 4u/μl restriction enzyme Ba + 1μl of nHI.
1 was added, and the reaction was carried out at 37°C for 60 minutes. 60℃, 10
After stopping the reaction by heat treatment for 1 min, 80 μE of water was added, 2 pl of 5M Nael was added, and further washed for -2
Add 200μl of 0℃ ethanol and incubate at -20℃ for 30 minutes.
10. Leave for 5 minutes at 0°C. QQQ
DNA was collected by r p +n centrifugation. After washing this with 200 μl of ethanol at 20°C,
Dissolve in 2oμl of water and 'I'4! The ligation treatment was carried out using I gauze in the same manner as described in Reference Example 1 (3).

(2) Transformation of E. coli Next, using this DNA, the large intestine It<12C60Q
Transformation into rm- was performed. The transformants are 2
0ttg/m! Tetracytalline and 30 pg/ml ampicillin showed copper properties. Among these transformants, 10
Regarding the strain, the size of the plasmid was determined by the same method as described in Reference Example 1 (5), and the molecular weight of all plasmids was approximately 6 megadaltons.

In this way, the complex plasmid synthesized from pHY780, represented by the cleavage map in Figure 1 (C1), and more specifically represented by the cleavage map in Figure 3, was
It was named HY600. Through the above treatment, it was confirmed that this pHY600 can also be used at least as a cloning vector for E. coli.

p)lY600 produced in Reference Example 2 was treated with restriction enzyme λh.
Cleavage was performed using enzyme treatment similar to that described in Reference Example 1 (3) using oI and Sal I, and further, 1) N
The A fragments were ligated by enzymatic treatment using T4 ligase, and then transformed into Escherichia coli using the same method described in Reference Example 1 (4) to obtain a plasmid (puy -460) was produced. As a result, pHY-460, which is represented by the cleavage map in FIG. 1 (hil, more specifically, FIG. 4), was produced from pHY600, which is represented by the cleavage map in FIG. 1(D).

Reference Example 4 Transformation of Bacillus subtilis using intermediate 3 (1) Preparation of competent cells
5ubtilis) IVlarburg 16B
(Owned by Saito Laboratory, Institute of Applied Microbiology, University of Tokyo) as a medium■
(Spizizen mineral medium (5pizizen +
ni-neral +nedium): K2H
PO41.4%, KH2PU40.6%, (NH4)+
5 (740.2%, sodium citrate 0.196,
IVgS (J4・7) 1zU O,0291, casein hydrolyzate 0.02% for glucose 0.596
, L-) with 50 μg/Inl of liptophan) for I x 108/+nI! Inoculated to some extent.

This was cultured with shaking at 37°C, and after about 4 hours, when it entered the stationary phase, medium (L-tryptophan in medium I was adjusted to 5 μg/mJ, and 5 InM Mg5 (J
4) was diluted 10 times and culture was continued. Bacteria in the culture solution become competent (comp) after 90 minutes.
state has been reached. This competent cell 0.
9+nl! pi-IY460 produced in Reference Example 3
Complex plasmid DNA/solution 0.1+nt! was added and cultured with shaking at 37°C for 90 minutes to perform transformation.

(2) Detection of transformants Transformants incorporating DNA (p14Y460) were transformed into L containing 20 μg/+nl of tetracycline.
- It was spread on an agar plate and cultured at 37°C for 24 hours.

The presence of plasmid was confirmed in the obtained colonies by the same method as in Reference Example 1. replicated plasmid p
HY460 has a molecular weight of about 4. %, 6 Q It was Gadalton's.

Colon lK12 C600r-+n- (7J transformation was also confirmed by the same method as described in Reference Example 1 (5). As a result, complex plasmid pi-IY
460 is a host for at least both Escherichia coli and Bacillus subtilis.
It turned out to be a cloning vector in the vector system, ie a shuttle vector.

In addition, as a transformant, large 1] W@ ni 12 C600
r-+n- (pHY460) and Bacillus subtilis 168 (p
)lY460) have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession numbers No. 6873 and No. 6874, respectively.

Reference Example 5: Complex plasmid pHY385
Synthesis of (middle drug 4) Using the method described in Reference Example 4, pi-IY460 was used (1
By transforming Bacillus subtilis, the obtained transformant was subcultured for about 50 generations at 37°C. The culture at that time was carried out in the same manner as in the method described in Reference Example 4 (2).
On L-agar plates containing 20 μg/+nl of tetracycline (Bacto-trypto
n) l%, yeast extract 0.5%, Na1l'
0.5%, g/l/: I-x O, 1% IN-
The transformants were plated on a plate (including one whose pH was adjusted to 7.0 with NaUH) at 37°C.

Regarding the colonies obtained in the above culture, Reference Example 1 (5)
The presence of the plasmid was confirmed by a method similar to that described in section 2.

As a result, 4 out of 12 transformants had a molecular weight of 3.
．． It was confirmed that the virus contained a plasmid of 85 megadaltons. When we analyzed the structure of this plasmid by cutting it with restriction enzymes, we found that this plasmid was
From pHY460, a portion thereof, more specifically, Xba
It was found that this is a novel plasmid produced by deletion of a region with a molecular weight of approximately 0.75 megadaltons containing recognition sites for I and Ba+nH1.

In this way, Figure 1! The composite plasmid synthesized from pHY460 shown in the cleavage map of FIG. 1 (l-), more specifically shown in the cleavage map of FIG. 5, synthesized from pHY460 shown in the cleavage map of E) was named pHY385.

Then, according to the same method as described in Reference Example 1 f41F51, 12C was added to E. coli using this pHY385.
Since transformation of 600r-+n- was confirmed, this p
HY385 is useful as a cloning vector in E. coli and B. subtilis host-vector systems.

In addition, 12 CC600r was added to E. coli as a transformant.
-(pHY385) and Bacillus subtilis 168 (1) l-L
Y385) has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under accession numbers 7113 and 7111, respectively.

(Example)...Composite plasmid p) IY340
Synthesis of pHY385 prepared in Reference Example 5 was cleaved using the restriction enzyme Hae by enzymatic treatment similar to that described in Reference Example 1 (3), and then using T41J gauze, the obtained 1 ) NA fragments were ligated.

Next, according to a method similar to that described in Reference Example 1 (4), the DNA obtained in the above treatment (pHY-34
0) to E. coli by 12C,600r-+n (7)
Transformation was allowed to occur, thereby obtaining transformants exhibiting resistance to tetracycline and ampicillin. When the size of the plasmid possessed by these transformants was examined in the same manner as described in Reference Example 1 (5), all of the transformants had a molecular weight of approximately 3.4 megadaltons. It turns out that there is something. - A transformant of Bacillus subtilis Marburg 16B could also be obtained by a method similar to that described in Reference Example 4.

In this way, the complex plasmid synthesized from pHY385 represented by the cleavage map of tFl in FIG. 1, fGl in FIG. did. By obtaining the above-mentioned transformant, it was confirmed that this p)LY340 is useful as a cloning vector for E. coli and Bacillus subtilis.

In addition, 12 C600r- was added to E. coli as a transformant.
+n- (pI-IY340) and Bacillus subtilis Marbur
g168(+)HY340) have been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology under accession numbers 7196 and 7197, respectively.

[Brief explanation of the drawing]

Figure 1 shows the composite plasmid pHY34 according to the present invention.
This shows the synthetic route of 0. In the figure, CAI and (Hitoshi) represent the restriction enzyme cleavage maps of vector p as starting materials and plasmid pAMα1, and (C1, +Di, tg+, and (Fl) represent the cleavage maps of synthetic intermediates. And (Gl each shows the cleavage map of the complex plasmid pHY340 according to the present invention. Figures 2 to 5 show the cleavage map of the above synthetic intermediate in detail. Patent application People Yakult Honsha Co., Ltd. Figure 2 Figure 3 Figure 4 Figure 5

Claims (1)

[Claims] +11 Streptococcus-7 echaris (Strep
a tetracycline resistance gene region derived from plasmid pAMα1 of S. to-coccus faecalis;
Schericbia coli (1)
Vector 9A (') has an ampicillin resistance gene region derived from YC177, a replication initiation region derived from the above plasmid pAMα1, and a replication initiation region derived from the above vector pAcY0177, and the tetracycline resistance gene region contains restriction enzymes Ba1i and The only recognition cleavage site for kI in the entire region is located, and furthermore, the ampicillin resistance gene region contains restriction enzymes 1I, Pvu) and p
A complex plasmid characterized in that a unique recognition cleavage site in the entire region for each of sil is located. (2) The composite plasmid plff 340 according to claim 1, which is characterized by a molecular weight of about 3.4 megadaltons and the following sequence of restriction enzyme recognition and cleavage sites.
,.