JPH025393B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [] BACKGROUND OF THE INVENTION Technical Field The present invention relates to the transformation of Bacillus subtilis. More specifically, the present invention relates to the transformation of Bacillus subtilis, which is characterized by the type of plasmid used and the transformation expression operation.

With the recent development of molecular biology, technology for endowing bacteria with the ability to produce useful substances by incorporating them into bacteria, that is, so-called genetic recombination or transformation technology, is becoming a reality. be. In this case, the bacterium to be transformed, that is, the host bacterium, is mainly used as Sunbacterium, and many studies have been conducted on vectors to introduce foreign genes into this host, and various hosts and vectors have been used. system is being developed.

By the way, Bacillus solarium is a Gram-negative bacterium, but Gram-positive bacteria also exist, so it would be beneficial to develop a suitable host-vector system for Gram-positive bacteria, especially Bacillus subtilis. is big. This is not only due to an increase in the number of host types, but also because it is expected that it will be possible to express traits that are difficult to express in Gram-negative bacteria.In addition, microorganisms belonging to Gram-positive bacteria such as Bacillus subtilis are expected to be able to express traits that are difficult to express in Gram-negative bacteria. Bacillus subtilis has been used for the mass production of flavor components, antibiotics, and various enzymes, and a long period of experience and technology has been accumulated in its industrial mass cultivation, and Bacillus subtilis is not pathogenic to animals or plants. Moreover, it is a microorganism that can be safely handled because it is not parasitic in the intestinal tract of animals such as humans.

[] Summary of the Invention The present invention aims to solve the above-mentioned problems, and attempts to achieve this purpose by providing a hybrid plasmid useful as a vector for Bacillus subtilis.

Therefore, the hybrid plasmid according to the present invention contains a DNA fragment homologous to the hisA ^- gene portion of the chromosome of Bacillus subtilis, which is to be a host, and a DNA fragment derived from a plasmid that can be propagated in Bacillus subtilis,
The above-mentioned first DNA is characterized in that it forms a non-histidine auxotrophic gene through complementarity through recombination with a homologous portion of the host chromosome, and is capable of propagating in the host Bacillus subtilis. It is.

In addition, the Bacillus subtilis DNA recombination method according to the present invention using this hybrid plasmid is as follows:
It contains a DNA fragment homologous to the hisA ^- gene part of the chromosome of Bacillus subtilis, which is to be the host, and a DNA fragment derived from a plasmid that can be propagated in Bacillus subtilis, and the first DNA mentioned above is a homologous part of the host chromosome. The hybrid plasmid, which forms a non-histidine auxotrophic gene through complementation through recombination with the host Bacillus subtilis and can be propagated in the host Bacillus subtilis, is transformed into the host Bacillus subtilis which has recombination ability. The method is characterized in that the hybrid plasmid is introduced into the host Bacillus subtilis to produce a chromosomally integrated transformant in which the hybrid plasmid is integrated multiple times into the chromosome of the host Bacillus subtilis, and the chromosomally integrated transformant is selected.

Here, the term "chromosomally integrated transformant" means a transformant in which a hybrid plasmid has been integrated into the host chromosome.

Effects According to the present invention, a novel host-vector system for Bacillus subtilis that is safe to handle is provided, but the hybrid plasmid of the present invention allows Bacillus subtilis to be transformed with high frequency, and Therefore, more than 40 copies of this hybrid plasmid are integrated onto the host chromosome in the same direction.

Traditionally, Bacillus subtilis was used under competent conditions.
DNA recombination is said to be due to double crossover between the donor single-stranded DNA and the recipient bacterial chromosome. However, the recombination in the present invention occurs during the logarithmic growth phase of the host bacteria,
Plasmids are continuously integrated into the host chromosome multiple times in the same direction by so-called Campbell-type recombination. This fact was not predicted from conventional knowledge.

As described above, in the present invention, the hybrid plasmid to be a vector is integrated multiple times into the chromosome of the host Bacillus subtilis, so when a useful gene, such as a bioactive polypeptide producing gene, is integrated into this vector, this useful gene is integrated into the host Bacillus subtilis chromosome. It will exist in an amplified state on the chromosome, and therefore it can be expected that the expression of useful genes will be carried out efficiently. In particular, since pUB HA31 (details described below), which is a representative example of the hybrid plasmid of the present invention, has a small molecular weight of 4.1 megadaltons (Md), multiple integration of useful genes will be carried out effectively. Also,
Since this plasmid is integrated into the chromosome, it is stable and less likely to fall off during storage of the transformant strain or due to the external environment, which is advantageous in handling the strain.

[] Detailed Description of the Invention 1 Hybrid Plasmid (1) Definition (1) Structure The hybrid plasmid according to the present invention has the chromosome of Bacillus subtilis to be the host.
hisA ^- fragment of DNA homologous to the gene part,
derived from plasmids that can be propagated in Bacillus subtilis
It contains a fragment of DNA. Here, the first DNA, that is, the hisA ^- gene part of Bacillus subtilis that is to be the host, is
DNA is a gene that forms a non-histidine auxotrophic gene through phase correction through recombination with a homologous portion of the host chromosome, that is, it allows the resulting chromosomal integration transformant to express a histidine-nonauxotrophic trait useful for selection. It is something that can be done. This hybrid plasmid must be capable of propagation in the host Bacillus subtilis.

The hybrid plasmid according to the present invention contains two types of DNA fragments.
"Containing" a fragment means that it contains not only these two types of fragments, but also other DNA fragments.
It also includes fragments, especially useful substance producing gene DNA.

(2) Homologous DNA fragment The DNA fragment homologous to the hisA ^- gene portion of the chromosome of Bacillus subtilis, which is to be the host for this hybrid plasmid, consists of the DNA strand of the histidine auxotrophic (hisA ^- ) gene.

As mentioned above, this DNA fragment must be able to cause the resulting chromosomal integration transformant to express traits useful for selection through complementation through recombination with a homologous portion of the host chromosome. . One of the conditions for meeting such requirements is that the auxotrophic (hisA ^- ) portion of the host Bacillus subtilis chromosome that is homologous to the DNA fragment of the hybrid plasmid is transferred from a non-auxotrophic gene to a portion of its DNA. This mutation is caused by a mutation occurring in the plasmid DNA and the host chromosome.
It occurs in a different part from DNA,
It is. Specifically, for example, regarding a histidine auxotrophic gene that is a homologous portion between plasmid DNA and the host chromosome, the gene on the plasmid side has a deletion of some of the nucleotides at one end of the gene, resulting in a histidine The gene on the host chromosome side is auxotrophic, whereas the gene on the host chromosome is
Due to a point mutation in one nucleotide within the DNA (but only in both DNAs)
When parts are juxtaposed to cause recombination,
This point mutation has a histidine requirement (it must be located at a different site from the deleted plasmid DNA). In this case, Campbell-type recombination allows the plasmid to be located on one side of each attachment site.
With complementary contributions from the non-deleted portion of the DNA and the non-mutated portion of the host chromosomal DNA,
The original gene without DNA mutation, ie, the non-histidine-requiring gene, is reproduced, and this trait, ie, the non-requiring for histidine, makes it possible to select the desired transformant.

(3) Plasmid-derived DNA fragments Various plasmids that can be propagated in Bacillus subtilis may be used to provide plasmid-derived DNA fragments. in particular,
For example, pUB110, pLS11, pLS13,
pLS14, pC194, pE194, pSA2100,
There are pT84, pTP5, and others. Preferred among these are pUB110, pC194,
pE194, especially pUB110. pUB110
is derived from Staphylococcus aureus and is a multicopy plasmid with a molecular weight of approximately 3 Md and is kanamycin resistant (km ^R ). Markers such as km ^R can be used for primary selection of transformants.

(2) Construction (1) Principle Construction of the hybrid plasmid of the present invention basically consists of ligation of two essential DNA fragments. However, homologous to the host chromosome
As for the DNA fragment, it is necessary to satisfy the above-mentioned requirements with the corresponding homologous part of the host chromosome. In other words, for example, if this homologous part has a histidine auxotrophy due to a mutation in the DNA of a normal gene related to histidine auxotrophy, This hybrid plasmid must be constructed using two types of plasmids.
This can be considered from two aspects: ligation of DNA fragments and fulfillment of this requirement.

(2) Preferred method A preferred method for constructing the hybrid plasmid of the present invention is to use a plasmid such as
Phage derivatives incorporating pUB110 and host chromosomal DNA fragments, such as ρ ₁₁ phisA ⁺ ,
is digested with an appropriate restriction enzyme such as Eco RI, and the resulting plasmid fragment and the host chromosomal DNA fragment excised from the phage are combined.
Ligate with DNA ligase e.g. T4 ligase and connect this to competent B. subtilis e.g.
For example, Spizizen in Bacillus subtilis61242
method [J.Bacteriol. 81 , 741-746 (1961)]
This method consists of recovering a cloned target hybrid plasmid, such as pUB HA31, from the resulting transformant. In this method, the raw conjugate obtained by ligation with ligase is
Since there is no deletion in the hisA ⁺ DNA, this plasmid is still hisA ⁺ , and by subjecting it to transformation of B. subtilis,
The desired genetic trait hisA ^- results. Note that instead of this method, an artificial DNA mutation or deletion induction method may be used (details will be described later).

Phage derivatives incorporating host chromosomal DNA fragments used in the former preferred construction method can be prepared from various temperate phages by Kawamura's method [Gene, 5 (1979), 87-
91) (Elsevier), “Proteins, Nucleic Acids, and Enzymes”
Vol. 26, pp. 469-475 (1981)].

Temperate phages that can be used include ρ ₁₁ (molecular weight = approximately 78 megadaltons (Md)), φ105, φ3T, and others. Among these, ρ ₁₁ and φ105 are preferred, especially ρ ₁₁ .

target chromosomal DNA fragment from host Bacillus subtilis,
For example, to create a phage (e.g., ρ ₁₁ ) derivative incorporating hisA ⁺ , Bacillus subtilis chromosomal DNA (e.g., hisA ⁺ ) and ρ ₁₁ DNA are both cut with Eco RI, and the resulting fragments are ligated with DNA ligase and All you have to do is clone it. Bacillus subtilis
As a derivative of the phage ρ ₁₁ containing the hisA ⁺ gene, ρ ₁₁ p hisA ⁺ is already known and is one of the preferred ones for use in the present invention.

On the other hand, various plasmids that can be propagated in Bacillus subtilis can be used.
Some of the specific examples are as described above. Plasmids with high copy numbers are preferred. Preferred is pUB110 from Staphylococcus aureus.

In order to ligate the above-mentioned phage derivative and the above-mentioned plasmid, they may be cleaved with the same restriction enzyme and the resulting fragments may be ligated with ligase, as described above. Restriction enzymes that can be used are capable of cleaving both the phage derivative and the plasmid, while also cutting the necessary DNA parts (e.g., the drive unit for autonomous propagation of the plasmid, the hisA ⁺ part, and the necessary drug resistance). can be any that does not cleave the involved gene). To give a specific example,
There are Eco RI, Bam HI, Hin d, and others. Among these, Eco RI is preferred.

In order to convert the resulting ligation product into the hybrid plasmid of the present invention, Bacillus subtilis is transformed with this ligation product, but the Bacillus subtilis used here is the same as the Bacillus subtilis used for multiple integration into the chromosome described below. It can be anything that is defined. Specifically, for example, B. subtilis strain 61242 [Agency of Industrial Science and Technology Research Institute of Microbiology, accession number
No. 6028”. ^hisAI- , ^trpC2- , ^cysB3- ],
Bacillus subtilis PS9-16 [Feikoken Bacteria Collection No. 6029]. ^hisAI- , ^metB5- , ^leuA8- ,
trpC2 ^- , nonAI ^- , nonBI ^- ], UOT278, and UOT378 (UOT means a strain kept by the Saito Laboratory at the Institute of Applied Microbiology, University of Tokyo, and is freely available). All of these bacteria are mutant strains of B. subtilis marburg, and their main genotypes are as shown above.

Transformation was performed using the method of Spizizen [J.
Bacteriol. 81 741 (1961)]. Selection of transformants is
Km ^R (if the plasmid is pUB110)
The plasmid can be further assayed by performing a primary selection using the plasmid and a secondary selection using the desired His ⁺ . For details, please refer to the above-mentioned literature and the experimental examples described below.

(3) Product In this way, for example, after ligating the ρ ₁₁ phisA ⁺ fragment to pUB110, B.
By transforming the subtilis 61242 strain, the hybrid plasmid pUB of the present invention can be obtained.
HA31 is obtained. pUB HA31 is a plasmid with a molecular weight of 4.1 Md as determined by agarose gel electrophoresis.

pUB HA31 is another plasmid pUB
Separated at the same time as HA81. Eco for both
As a result of examining the cleavage pattern (agarose electropherogram) (see Figure 1) by RI,
pUB HA81 is a 3.3Md Eco RI fragment cloned into ρ ₁₁ hisA ^+, cloned in the same size (therefore,
His ⁺ ) is a plasmid that is expected to be isolated from transformants when genetic DNA is introduced into host cells by the transformation method. On the other hand, pUB HA31 of the present invention unexpectedly has this 3.3Md Eco
This is a plasmid in which a portion of the RI fragment has been deleted (therefore, hisA ^- ), that is, a 1.1Md fragment has been cloned.

(4) Other construction methods In the above preferred construction method, ρ ₁₁ phisA ⁺ is used as a phage derivative.
If ρ ₁₁ phisA ⁻ is used instead, it is possible to directly obtain a hisA ⁻ hybrid plasmid.

In addition, by deleting a part of the above-mentioned his ⁺ hybrid plasmid pUB HA81, for example, his ⁺ can be transformed into his ^-
can be converted to . For example, a restriction enzyme that cuts a fragment containing a normal histidine gene at two or more sites (in the above case, Hin
By treating pUB HA81 DNA in step d), ligating with _T4 ligase, and then transforming Bacillus subtilis, a plasmid in which part of the histidine gene has been deleted can be obtained.

2. Construction of chromosomal integration transformants Using the hybrid plasmid of the present invention obtained as described above, such as pUB HA31, for example, the method of S. Cohen [Molec.gen.
Genet. 168 , 111-115 (1979)].

(1) Hybrid plasmid The hybrid plasmid of the present invention constructed as described above can be used as is for transformation, but if necessary,
pUB110, pC194, pE194, pSA2100,
It may be re-cloned into a plasmid vector such as pTP4 or pTP5 for use. In addition, genes that produce useful substances, such as mammalian active peptides (hormones such as insulin, interferon, growth hormone, ACTH, somatostatin, etc.), vitamins,
Genes involved in in vivo production of oxygen, antibiotics, amino acids, and other substances can also be incorporated before transformation. Note that such useful substance-producing genes can also be incorporated at a stage prior to this process, for example, at the stage of phage derivatives.

(2) Host Bacillus subtilis is recombinant (Rec ⁺ ) and has a hisA ^- gene portion homologous to the hybrid plasmid DNA, and the corresponding non-auxotrophic trait can be achieved by partial deletion or mutation of this gene. The ^site of this mutation is
Any Bacillus subtilis can be used (different in the corresponding DNA portion of the hybrid plasmid).

Specific examples of such Bacillus subtilis include, for example,
B. subtilis 61242 strain, B. subtilis PS9−16,
Such as UOT278 and UOT378.

(3) Transformation For details of S. Cohen's method, please refer to the above-mentioned document.

For details on applying this method to the present invention, please refer to the experimental examples described later.

(4) Selection of transformants Selection of chromosomal integration transformants from the generated transformants is based on the traits (for example, His ⁺ ) expressed by complementation through recombination as described above.
However, it is preferable to first select all transformants, including transformants other than chromosomal integration transformants, from non-transformants using plasmid-derived traits, such as drug resistance. The obtained transformants are propagated to increase their quantity, and then the trait in question, i.e., expressed by the above-mentioned complementation, is
Preferably, secondary selection is by His ⁺ .

In addition to such genetic means, selection of transformants with chromosomal integration may also be carried out by utilizing the improvement in activity due to multiple integration of hybrid plasmids.

(5) The resulting chromosomal integration transformant B. subtilis strain 61242 (B. subtilis UOT477) obtained by transformation with the hybrid plasmid pUB HA31 was
No. 6030" and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.

3 Experimental example Creation of ρ ₁₁ phisA ⁺ Kawamura's method below [Proteins, Nucleic Acids, Oxygen]
26, No. 4 (1981), pp. 469-475], ρ ₁₁ phisA ⁺ was constructed.

That is, His ⁺ Bacillus subtilis DNA (4 μg) and ρ ₁₁ DNA (10 μg) are cut with Eco RI and ligated with T4 ligase. Histidine auxotrophs that have lysogenized Phage _ρ11 as recipient bacteria, such as B. subtilis.
(his A1, leu A8, ρ ₁₁ ) is used. The ligated DNA is used to transform competent cells (1 ml) of this strain. Usually about 100~ per 0.1ml
1000 transformants are obtained.

Next, 50–100 His ⁺ transformants were collected to select and enrich for _ρ11 phages carrying hisA ⁺ genes.
Divide into portions and suspend in 5 ml of L-broth. OD ₆₆₀ =
Dilute to 0.05 and culture with shaking at 37℃. _OD660 =0.2
~0.3, mitomycin C (0.5μg/
ml) treatment to induce phages, and the lysate is treated with a Millipore filter to remove viable bacteria. Plaque forming ability and HiS ⁺ in these lysates
Test transduction potential. The ability to transduce His ⁺ is examined using non-lysogenic bacteria that require histidine during logarithmic growth.

The thus obtained HiS ⁺ transductants are subjected to single colony isolation on a selection plate, a lysate is prepared again, and the plaque forming ability and His ⁺ transducing ability are examined.

Construction of hybrid plasmid (1) Ligation of heterologous DNA and detection of hybrid plasmid 1 μg each of pUB110 and ρ ₁₁ phisA DNA
, S buffer (20mM Tris-HCl PH7.2,
It was treated with Eco RI in 10mM MgCl ₂ , 0.1M NaCl) at 37°C for 1 hour, further heated at 65°C for 10 minutes, and then rapidly cooled. Add this to buffer solution (20mM Tris-HCl PH7.6, 10mM dithiothreitol, 2mM
ATP), T4 ligase (1 unit) was added, and the mixture was reacted at 10°C for 24 hours.

In order to transform B. subtilis strain 61242 (Rec ⁺ hisAI, trpC2, cysB3) with this DNA mixture, we first transformed B. subtilis strain 61242 (Rec + hisAI, trpC2, cysB3) according to the method of Spizizen.
subtilis61242 strain in CI medium (10x Salts 10ml, distilled water 90ml, 50% glucose 1ml, 1M
MgSO ₄ 0.4ml, 2mg/ml tryptophan 2.5
ml, 5% Casamino Acid (manufactured by Difco) 0.4ml), and further cultured in C medium (10x Salts 10ml, distilled water 90ml).
ml, 10% glucose, 1 ml, 1M _MgSO4 0.4
ml, tryptophan (2mg/ml) 0.25ml, 5%
After culturing in casamino acids (0.2 ml, manufactured by Difco) for 60 to 90 minutes, this DNA mixture was added to the culture solution.
Thereafter, the cells were incubated for 60 minutes.

The transformed strain was first incubated with Trypton Blood Base Agar (Difco
A kanamycin-resistant strain was selected using a medium produced by the company, and this resistant strain was further replicated onto a histidine-free minimal medium (containing trp and cys), and a histidine-nonrequiring strain was selected.

Bacillus subtilis (Km ^R ,
His ⁺ strain), the size of the plasmid it possessed was investigated using a simple detection method. That is, the colony was added to 30 μl of LRS solution (50 mM Tris-HCl).
PH7.5, 10mM EDTA, 0.1M NaCl, 10mg/ml
After suspending in lysozyme, 40 μg/ml RNaseA, 25% sucrose) and treating at 37°C for 200 minutes, add 3 μl of 10% SDS (sodium dodecyl sulfate) and heat. This DNA solution was subjected to agarose gel electrophoresis to assay the size of the plasmid. As a result, two types of plasmids with larger molecular weight than pUB110 were confirmed.

(2) Isolation and purification of hybrid plasmid and its identification The separation and purification of the plasmid from Bacillus subtilis (Km ^R , His ⁺ strain) carrying the above two types of plasmids was carried out using the method of Gryzan et al. [J. Bacteriol. 134 , 318
(1978)] was available and the method was followed.

That is, Bacillus subtilis (transformant) was mixed with 10 g of L medium (Bacto-trypton (manufactured by Difco)), yeast
extract (manufactured by Difco), 5 g of NaCl, 2 g of glucose, 10 mg/ml of kanamycin, adjusted to neutrality with NaOH) and cultured until the late logarithmic growth phase.The culture solution was centrifuged to obtain bacterial cells. Lysozyme treatment followed by 5M NaCl and 10% SDS
added. This was left at 0°C overnight, centrifuged, ethanol was added to the supernatant, further centrifuged, the precipitate was treated with RNase and protease, and then phenol, and the ethanol precipitate was extracted from the aqueous phase. DNA was obtained as follows.
This DNA was subjected to Low Melting Point agarose gel electrophoresis, and after electrophoresis, it was stained with ethidium bromide and the plasmid was excised. After destaining, TE buffer was added, the DNA was dissolved by heating at 68°C for 10 minutes, and treated with phenol. After ethanol precipitation, this aqueous phase was dissolved in TE buffer and dialyzed.
A purified plasmid was obtained. Using this method, more than 50 μg of cccDNA was obtained from 1 liter of culture solution.

(3) Preparation of restriction enzyme cleavage map and others The properties of the isolated hybrid plasmid are examined by preparing a cleavage map with restriction enzymes, measuring molecular weight by agarose electrophoresis, and measuring transformation activity.

For example, a hybrid plasmid (pUB HA31) obtained by ligating the ρ ₁₁ hisA fragment to pUB110 and then transforming the B. subtilis 61242 strain has the following properties.

This plasmid is isolated at the same time as another type of plasmid (pUB HA81), but both
The cleavage pattern (agarose electropherogram) with Eco RI as shown in Figure 1 was obtained.

Furthermore, in order to investigate whether the hisA ⁺ gene was cloned into pUB HA31, the presence or absence of the activity of transforming the recipient bacillus subtilis strain UOT378 (hisAI ^- ) into His ⁺ was determined. The results are shown in Figure 2, and in the Eco RI degradation product, His ⁺ transforming activity was observed in the fraction corresponding to the 1.1Md fragment. Although this is His ⁺ from a plasmid-carrying bacterium, the host chromosome DNA also has transforming activity. In addition, pUB HA31 was used to generate Bacillus subtilis recombination-deficient mutant strain GSY908.
(recE4hisA1), the resulting kanamycin strain was histidine auxotrophic. Therefore, although pUB HA31 has the vicinity of the hisA1 ⁺ point and has deleted part of the hisA gene, it has homology with the host chromosome regarding hisA, so it is not possible to create a recombinant by introducing it. can.

Next, in order to obtain a breakpoint map of pUB HA31, the number of cuts with nine types of restriction enzymes was investigated, and the cleavage site was determined by combining with double cuts. The restriction enzymes used were BamH, Bgl, Bgl,
Hind, Kpn, Sma, Pat, and Sac
The cutting point map is shown in Figure 3. This result suggests that the deletion was probably continuous in the Sac-Kpn region of the 3.3Md fragment.
This 1.1Md fragment is one of the enzymes mentioned above, BamH
and Bcl cleavage sites are only shown.

(4) In vitro construction of a mutant plasmid with partial deletion of the hisA gene from pUB HA81 0.3 μg of pUB HA81 DNA was completely digested with a restriction enzyme Hind that cuts at two sites, and then ligated with T4 ligase according to a conventional method. Bacillus subtilis strain UOT278 was transformed into Km-resistant using this ligated DNA. When the plasmid possessed by the obtained transformant was examined using a simple detection method, it was found that
A plasmid smaller than pUB HA81 was detected. This plasmid had a molecular weight of approximately 5.6 Md, was obtained by deleting the Hind fragment of pUB HA81 by approximately 0.7 Md, and also contained a portion of the hisA gene.

Transformation with hybrid plasmid (pUB HA31) (1) Preparation of protoplasts B. subtilis strain 61242 (Rec ⁺ , hisA) at 30°C.
The cells were precultured in TBAB medium overnight. This pre-cultured bacteria was added to 50 ml of Pennassay medium (manufactured by Difco).
inoculated to OD ₆₆₀ = approximately 0.05 and heated at 37℃.
The cells were cultured with shaking until OD ₆₆₀ = 0.4. After centrifugation, suspend the bacterial cells in 5 ml of SMMP solution,
Transferred to a sterile triangular kolben. Add lysozyme to this so that the final concentration is 1 mg/ml,
The cells were cultured at 37°C with gentle shaking. 3
After an hour, centrifuge with a horizontal rotor to collect bacteria, wash the bacteria with 5 ml of SMMP solution, centrifuge again,
The bacterial cells were suspended in 5 ml of SMMP solution to obtain brotoplasts.

(2) Transformation and selection of kanamycin-resistant strains 0.5 B. subtilis protoplasts obtained above
0.1 μg of pUB HA31DNA per ml (DNA solution and 2
An equal volume mixture of double strength SMMP) was added and immediately 1.5 ml of 40% polyethylene glycol (average molecular weight 6000) was added. After 2 minutes, 5ml
It was diluted with SMMP solution and centrifuged horizontally. The bacterial cells were suspended in 1 ml of SMMP solution and cultured with shaking at 30°C for 90 minutes. The obtained bacterial cells were 150μg/ml
inoculated into DM3 regeneration medium containing kanamycin,
Kanamycin-resistant strains were selected by culturing at 37°C for 48 hours. Kanamycin resistant strain is 10 ⁶ /μg
It showed an appearance more than DNA.

(3) Selection of multiple integrants The kanamycin-resistant strain obtained above was
L medium containing g/ml kanamycin [Bacto-
trypton (manufactured by Difco) 10g, yeast extract (manufactured by Difco)
) 5g, NaCl 5g, glucose 2g,
Neutralize with NaOH], dilute the culture 100-fold at 37°C until the late logarithmic phase, and then
Inoculate 0.02-0.1 ml onto histidine-free minimal agar medium and culture at 37°C for 48 hours to obtain His ⁺
Strains, ie multiple integrants, were selected (the frequency was between 1×10 ⁻³ and 10 ⁻² based on the number of viable bacteria).

(4) Confirmation of multiple integration (1) Proof by Southern analysis
The host chromosome was isolated and purified from the Rec ⁺ His ⁺ recombinant by a conventional method, and 1 μg of this DNA was subjected to 0.8% agarose electrophoresis, denatured, and transferred to a nitrocellulose filter.
[α ³² P] Nick-translated with dATP
This filter was hybridized with pUB110DNA as a probe, and an autoradiogram was taken. Rec ⁺ His ⁺ recombinant is
Hybridization activity was clearly seen in the host chromosome region, indicating integration of the plasmid into the host chromosome (Figure 4).

(2) Quantification of multiplicity by densitometry The above His ⁺ recombinant host chromosome was
or cut with Bgl, the plasmid
Compared to the band that occurs when DNA itself is cut with the above enzyme, it appears much darker in the cut pattern photograph.

This indicates that the plasmids were integrated in multiple ways in the same direction.
1 μg DNA of the His ⁺ recombinant host chromosome was completely cleaved with 2 units of the above-mentioned restriction enzymes according to a conventional method, subjected to 0.8% agarose electrophoresis, and the negative film was traced at 600 nm. The area ratio of the peaks showed that about 40 plasmids were integrated (5th
figure).

(3) Quantification of multiplicity by Hind cleavage pUB HA31 is not cleaved by Hind. Therefore, after approximately 40 plasmids have been integrated, this part of the host chromosome becomes a large DNA fragment that is completely uncleaved by Hind. Therefore, His ⁺ recombinant host chromosome
1 μg of DNA was completely cleaved with 2 units of Hind according to a conventional method and subjected to 0.8% agarose electrophoresis. Bacillus subtilis virulent phage φ1 DNA (105Md) was used as a DNA with a known molecular weight as a molecular weight marker, but larger DNA fragments were detected (Figure 6). The molecular weight of pUB HA31 is
Since it was 4.1 Md, it indicates that at least 26 plasmids were integrated into the chromosome in a connected manner.

[Brief explanation of drawings]

Figure 1 shows the hybrid plasmid of the present invention.
FIG. 3 is an agarose electrophoretogram showing the cleavage pattern of pUB HA31 and others by EcoR. 1...uncut plasmid pUB110, 2...uncut plasmid pUB HA31, 3...uncut plasmid pUB HA81, 4...EcoR cut plasmid
pUB110, 5...EcoR cut plasmid pUB
HA31,6...EcoR cleavage plasmid pUB
HA81, 7...3.3Md hisA fragment (φ105d hisA ⁺
prepared from ), 8...molecular weight marker (φ105EcoR
cleavage products). Figure 2 shows the transformation activity of pUB HA31, where A is an agarose electropherogram, and B and C are migration distances on this electropherogram and His ⁺ transformants and Met ⁺ His ⁺ transformants. It is a graph showing the appearance frequency of bodies. FIG. 3 shows various plasmid cleavage maps. Figure 4 shows Southern analysis of His ⁺ recombinant host chromosomes. A...Agarose electrophoresis pattern of each DNA,
B... pUB110 that was nick-translated with ^32P
Autoradiogram of A as a probe, 1
...pUB HA31DNA, 2...168Ti chromosome
DNA, 3...BD366 (pUB110) chromosomal DNA,
4...61242pUB HA31His ⁺ recombinant chromosome (Rec ⁺ ), 5...UOT279pUB HA31His ⁺ recombinant chromosome (recE4). FIG. 5 shows the results of densitometry of the cleavage pattern of His ⁺ recombinant chromosomal DNA. In the figure, and indicate the negative electrode and anode, respectively, in electrophoresis. Figure 5 is Bg1
is related to digest, and is related to EcoRI digest. FIG. 6 is an agarose electropherogram showing the Hind cleavage pattern of His ⁺ recombinant host chromosomal DNA. C2...His ⁺ recombinant chromosome Hind cleavage, φ...
...φ1DNA (105Md).

Claims (1)

[Claims] 1. A DNA fragment homologous to the hisA ^- gene portion of the chromosome of Bacillus subtilis, which is to be a host, and a DNA fragment derived from a plasmid that can be propagated in Bacillus subtilis,
The above-mentioned first DNA forms a non-histidine auxotrophic gene by complementation through recombination with a homologous portion of the host chromosome, and is capable of propagating in the host Bacillus subtilis. Plasmid. 2. The hisA ^- gene part of the chromosome of Bacillus subtilis, which is to be the host, and the hybrid plasmid homologous to this.
2. The hybrid plasmid according to claim 1, wherein both DNA genes are derived from a normal gene related to histidine requirement and have mutations at mutually different sites. 3 pUB with a molecular weight of 4.1 megadaltons
The hybrid plasmid according to claim 1, which is HA31 DNA. 4 Contains a DNA fragment homologous to the hisA ^- gene portion of the chromosome of Bacillus subtilis, which is to be the host, and a plasmid-derived DNA fragment that can be propagated in Bacillus subtilis,
The above-mentioned first DNA forms a non-histidine auxotrophic gene by complementation through recombination with the homologous portion of the host chromosome, and the hybrid plasmid, which can be propagated in the host Bacillus subtilis, is The hybrid plasmid is introduced into a competent host Bacillus subtilis by a transformation method to generate a chromosomally integrated transformant in which the hybrid plasmid is multiplex integrated into the chromosome of the host Bacillus subtilis, and this chromosomally integrated transformant is selected. Bacillus subtilis, which is characterized by
DNA recombination method. 5. Selection of chromosomal integration transformants is carried out by combining the first DNA of the hybrid plasmid and the host chromosome.
5. The DNA recombination method of Bacillus subtilis according to claim 4, which is carried out by a trait expressed in a chromosomally integrated transformant through complementation through recombination with a hisA ^- gene portion. 6. The hisA ^- gene part of the chromosome of Bacillus subtilis, which is to be the host, and the hybrid plasmid homologous to this.
Both genes of DNA are derived from normal genes related to histidine auxotrophy by having mutations in mutually different sites, and the trait that should be used to select chromosomal integration transformants is non-nutrient auxotrophic. The Bacillus subtilis DNA recombination method according to claim 5, wherein the Bacillus subtilis DNA recombination method is 7. The plasmid-derived DNA fragment has a gene that expresses a trait different from the trait that can be used to select chromosomal integration transformants,
The method according to any one of claims 4 to 6, wherein the transformant is subjected to a primary selection in which the latter trait is used as a lever to select the transformant from non-transformants prior to the selection of the transformant with chromosomal integration. DNA recombination method for Bacillus subtilis. 8. The Bacillus subtilis DNA recombination method according to claim 7, which comprises multiplying the transformants after the primary selection, and then selecting the transformants based on the trait expressed in the chromosomally integrated transformants. .