JP2003250563A - Dna cloning method using bacterial genome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective insertion method of DNA (LPS) which becomes a foothold for transducing DNA having a large size into a genome vector of a bacterium of the genus Bacillus or an extreme thermophilic bacterium. <P>SOLUTION: When inserting an LPS to a host genome by homologous recombination, the insertion of a homologous sequence used for the recombination can simply be carried out by using a vector inserted with only one fragment of the LPS into a vector used for the recombination by using a sequence of a marker gene which is not LPS. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a DNA cloning technique. More specifically, a method for inserting a homologous region into the genome in a DNA cloning method characterized by performing homologous recombination using the genome of a Bacillus bacterium or an extreme thermophile, and for use in insertion Related to vector etc.

[0002]

2. Description of the Related Art Use of a genomic DNA library is sometimes essential when analyzing the structure and function of a mammalian gene (eg, human or rat). Genome DN
There are several ways to construct an A library. One of the currently widely used methods for constructing a DNA library is to use a plasmid vector, a phage vector, or the like. Genomic DNA extracted from a target biological sample (tissue, cell, etc.) is a suitable restriction enzyme. It is a method of constructing a genomic DNA library by digesting with the above method to obtain a fragment of an appropriate length, ligating it into a vector having the same restriction enzyme site, and transforming this into a host. As a host, a number of systems have been developed which use plasmids such as Escherichia coli as a host or phages as vectors.

Although a plasmid vector or a phage vector using Escherichia coli as a host has the advantage of being relatively easy to handle, there is a limit to the size of DNA that can be cloned, and a large size DN of 100 kb or more is used.
It is difficult to clone A. Generally, the size of a gene is about 1 kb per gene in bacteria, but it may be 100 kb or more in higher eukaryotes because the gene is divided by introns. Therefore,
There is a need for a technique capable of cloning a DNA having a size of 100 kb or more. As a vector for cloning such a huge DNA, BAC (Shizuya, H.,
Others, Proc.Natl.Acad.Sci.USA, 87, 8242-8247 (1990))
And YAC (Burke, DT, etc., Science, 236, 806-811 (198
7)) has been developed, but the basic operation of cloning is the same as that of a plasmid vector, and new problems have occurred in terms of physical DNA damage during cloning operation, cloning efficiency, stability, and the like.

There are some humans, mice and plants whose nucleotide sequences have already been determined. In the future, a new cloning technique will be conducted on the premise that the nucleotide sequences are known, that is, only the target region will be extracted. However, the technology that enables subsequent DNA manipulation becomes important. PC is a technique for amplifying only the DNA in the target region whose nucleotide sequence is known.
The R method is known. However, there is a limit to the size of DNA that can be amplified even by the PCR method, and DNA of 50 kb or more is used.
Amplification of is not possible at this time.

As a novel cloning technique for solving the above problems, the present inventors directly cloned the target DNA into the genome of Bacillus subtilis, and cloned D
A method for converting the entire NA into a plasmid and recovering it was developed (Japanese Patent Laid-Open No. 2000-00093). Bacillus subtilis genome vector that uses Bacillus subtilis genome as a vector
It is an excellent vector that stably holds A and can be manipulated freely (Mitsuyasu Itaya "Naturally learned Bacillus subtilis genome vector" Bioscience and Industry, vo
l.57, No.8, 540-543 (1999)). Using this Bacillus subtilis genome vector, a huge DN derived from bacteria has been used so far.
A, bacteriophage λ DNA, or extreme thermophile pTT2
7DNA cloning has been reported and it has been demonstrated that large size DNA can be cloned (It
aya, M. Biosci. Biotech. Biochem., 56, 685-686 (199
2); Itaya, M. Biosci. Biotech. Biochem., 63, 602-6
04 (1999); and Itaya, M. Mol. Gen. Genet., 248, 9-1.
6 (1995)).

The outline of the cloning method using the Bacillus subtilis vector previously developed by the present inventors is shown in FIGS. 2A and 2B. First, both outside DNAs that sandwich the DNA region to be cloned are obtained. This outer DNA
Is essential for cloning into the Bacillus subtilis genome,
It should serve as a scaffold (base) for inserting the DNA to be cloned into the Bacillus subtilis genome. Both of the above-mentioned outer (scaffolding) DNAs (hereinafter referred to as "LP
S]) may be referred to as S] in the Bacillus subtilis genome. The method of integration is by genetically inserting a DNA having a common sequence with both LPS into an appropriate vector (FIG. 2A: pE [n / n + 1]) and transforming the Bacillus subtilis genome with the vector. This homologous recombination is due to one side of LPS (portion shown as [nth] in FIG. 2).
And a DNA having a common sequence as a homologous region (portion indicated by a vertical line in FIG. 2A). Here, the cloning site is between the two integrated LPSs. Next, when a DNA solution containing a DNA fragment containing the DNA to be cloned and DNA outside both of them is added to Bacillus subtilis whose cloning site is thus integrated in the genome, Bacillus subtilis removes the DNA fragment from the DNA solution. Incorporate spontaneously. The DNA incorporated into the cells undergoes homologous recombination at the cloning site (LPS portion) previously incorporated into the genome (portions shown as [nth] and [n + 1th] in FIG. 2B). As a result, the DN you want to clone
A is inserted into the Bacillus subtilis genome (hereinafter, this may be referred to as the "Shakutori method").

[0007] It is possible to increase the size of the DNA cloned in the Bacillus subtilis genome by repeating the cloning into the Bacillus subtilis genome while overlapping the target region to be cloned with the homologous region and shifting them little by little. Is. However, in the cloning method using the Bacillus subtilis vector as described above, it is necessary to always insert two LPS DNA fragments at both ends of the insertion sequence into the vector for inserting LPS into the Bacillus subtilis genome ( FIG. 2A: pE [n / n + 1]), which has a problem of complicated operation and long time.

[0008]

SUMMARY OF THE INVENTION The present invention has been made to solve the problems of the prior art described above. That is, the problem to be solved by the present invention is Bacillus
It is an object of the present invention to provide an efficient method for inserting LPS to recombine large-sized DNA into a genomic vector of a genus bacterium or an extreme thermophile.

[0009]

As a result of intensive studies to solve the above problems, the present inventors have found that when inserting LPS into the host genome by homologous recombination, the homologous sequence used for recombination is used as a scaffold. It has been found that by using a marker gene sequence that is not the following sequence, only one fragment of LPS needs to be inserted into the vector used for recombination. The present invention has been accomplished based on these findings.

That is, according to the present invention, (1) a host genome comprising transforming a host in which sequences at both ends of an insert sequence to be introduced are previously inserted into the genome with DNA having the insert sequence. In the method of inserting DNA into DNA, a method of sequentially inserting the sequences at one end of the inserted sequence into the genome is
(I) Partial sequence of the marker gene previously introduced into the host genome, one end sequence (n + 1) of the insertion sequence, a marker gene different from that introduced into the host genome, common sequence in this order, where n and n + 1 sequences are DNA having an insertion sequence
And a homologous recombination between the vector and a host genome into which the marker gene has been previously introduced adjacent to one end sequence (n) of the insertion sequence. And (iii) selecting a strain in which the expression of the marker gene previously introduced into the host genome is lost, (iv) transforming the obtained host with the DNA having the insertion sequence, and further (i) To (iv) are repeated, (2) the host is Bacillus
The method according to (1) above, which is a genus bacterium or an extreme thermophile, (3) the method according to (1) or (2) above, wherein the marker gene has drug resistance activity, (4) above. For use in the method according to any one of (1) to (3), a partial sequence of a marker gene previously introduced into the host genome, one end sequence (n + 1) of the insertion sequence, and one introduced into the host genome A different marker gene, in the order of the consensus sequence, except that the n and n + 1 sequences are ligated in the same direction as the DNA having the inserted sequence.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method and an embodiment of the present invention will be described. The method of the present invention comprises a method of inserting a DNA into a host genome, which comprises transforming a host in which sequences at both ends of the insert sequence to be introduced are previously inserted into the genome with a DNA having the inserted sequence ( Hereinafter, this may be referred to as "a method for cloning a DNA into a bacterial genome"), in which the sequence of one end of the inserted sequence is inserted into the genome in order (1) a marker gene previously introduced into the host genome. A vector containing a part of the sequence, one end sequence (n + 1) of the inserted sequence, a marker gene different from the one introduced into the host genome, the common sequence, and the DNA ligated in the same reading frame (Fig. 1A: pE
[/ N + 1]), (2) Inducing homologous recombination between the vector and the host genome (FIG. 1A: Genome) into which the marker gene has been introduced in advance adjacent to one end sequence (n) of the insertion sequence, 3) A strain in which the expression of the marker gene previously introduced into the host genome is lost (FIG. 1B: strain having the genome shown in Genome) is selected, and (4) the obtained host is used with DNA having an insertion sequence. The method is characterized by transforming and further repeating (1) to (4).

(I) Insertion Sequence and LPS The insertion sequence to be introduced in the present invention is a sequence of a foreign DNA to be cloned into a bacterial genome as a host, and its kind and size are not particularly limited. The types of DNA include genomic DNA fragments, DNA fragments that have already been cloned, DNA fragments amplified by PCR, and cDNA.
Although it may be any of fragments, it is not particularly limited, but genomic DNA is preferably used in the present invention because it has a particular effect on cloning of large-sized DNA. The size of the insertion sequence is 10 kb or more, preferably 10 kb to 1
A huge DNA of about Mb, more preferably about 10 kb to 500 kb is preferable. Also, the insert sequence is genomic DNA of an animal or plant or a useful microorganism. The type of animal is also not particularly limited, and preferable examples thereof include mammals such as humans and rodents such as mice.

The terminal sequence of the insertion sequence to be LPS is a sequence at the end of the insertion sequence, and the size thereof is preferably 5 to 15% of the insertion sequence. Specifically, when the insertion sequence is 50 kb, LPS is 5'and 3'of the insertion sequence.
The sequence is about 5 kb at the end. Insertion DN into host genome
To introduce A, it is necessary to previously introduce sequences at both ends of the insertion sequence as LPS into the genome. This LPS becomes a homologous region for homologous recombination when the inserted sequence is introduced into the genome. Here, homologous recombination is 1
It is recombination that occurs in a portion (homologous region) having a homologous base sequence of a pair of double-stranded DNAs. In addition, it is not necessary that the homologous sequence or the homologous region in the present specification has 100% sequence identity as long as the desired homologous recombination can be induced. That is, the two homologous sequences may be substantially homologous, for example, 60% or more,
The homology is preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more, and most preferably 95% or more. The length of the homologous sequence is not particularly limited, but generally 500 bp or more is used.

The cloning method of the present invention is carried out by repeating the introduction of such an insertion sequence into the host genome.
It is used to extend a foreign sequence cloned in the genome. Therefore, the method for cloning DNA into a bacterial genome of the present invention means that LPS is inserted into the genome in order on one side of the inserted sequence along the direction of extension, and the inserted sequence is introduced into this genome. It also refers to a method in which the insertion of one side into the genome is repeated.

The host used in the present invention isBacill
usBacteria of the genus or extreme thermophiles are preferably used.Baci
llusThe type of genus bacteria is not particularly limited, for example,B. subti
lis(Bacillus subtilis),B. megaterium(Giant fungus),B. anthrac
is(Splenoid bacillus),B. cereus,B. stearothermophilus
(Moderate thermophile) and the like. Preferably DNA
Excellent in uptake and recombination abilityB. subtilis(Dead grass
Fungus). Extreme thermophiles are thin thermophiles that can grow only at high temperatures.
It is a generic term for bacteria.

(II) Construction of scaffold insertion vector and insertion of scaffold into host genome The method for inserting this LPS into the host genome will be described below. LPS is inserted into the host genome using homologous recombination. Here, as the homologous region, a partial sequence (marked with a shaded box in FIG. 1A) of the marker gene previously introduced into the host genome and an appropriate common sequence (marked with a vertical stripe in FIG. 1A) are used. As a marker gene,
There is no particular limitation as long as its expression can be confirmed in the host, but an antibiotic resistance gene is preferred. Specific examples include a tetracycline resistance gene, a chloramphenicol resistance gene, and an erythromycin resistance gene.

The partial sequence of the marker gene is not particularly limited as long as this sequence has a function as a scaffold for homologous recombination, but preferably, a partial sequence in which the marker gene does not have the function. Is. The reason why some sequences in which the marker gene does not have that function is preferable is that
In the host genome recombined with this as a homologous region, the full length of the marker gene was contained before recombination, but since it becomes a part of the sequence that lost function when recombination occurs, it becomes an indicator of recombination. This is because it is used that the function of the marker gene is lost. The common sequence is not particularly limited as long as it has the function of the above homologous region, but preferably a vector sequence for inserting LPS is used.

As a vector used for recombination for inserting LPS into a host genome, a commonly used cloning vector is used. In this cloning vector, a partial terminal sequence (n + 1: box indicated by [n + 1st] in FIG. 1A) of the insertion sequence to be LPS was added to a part of the above-mentioned marker gene sequence (box indicated by hatching in FIG. 1A). A marker gene different from the one inserted in the host genome so as to be adjacent to it and introduced into the host genome so as to be adjacent thereto (boxes indicated by horizontal stripes in FIG. 1A), and the consensus sequence (indicated by vertical stripes in FIG. Insert the box shown) (FIG. 1A: pE [/ n +
1]; hereinafter, this may be referred to as "scaffold-integrating vector"). When ligating these DNA sequences,
The respective sequences are ligated so that the direction of the DNA is the same as that of the inserted sequence, and in the extension process of the DNA to be inserted into the host genome, a part of the above-mentioned marker gene is located at the end.

The scaffold-incorporating vector thus prepared was used to prepare one end sequence of the insertion sequence (see FIG. 1A).
(Box indicated by [nth] in Fig. 1) is transformed into a host genome into which a marker gene and a common sequence have been introduced to induce homologous recombination. Transformation and homologous recombination can be carried out by a conventional method, and specifically, a solution containing a scaffold-integrating vector may be added to a culture solution of a bacterium having a genome which is cultured under normal conditions. For details of transformation and homologous recombination, see Mitsuo Ogura, Bioscience and Industry, vol. 57, No. 8,
532-535 (1999), Mitsuyasu Itaya, Journal of the Japanese Society of Agricultural Chemistry, vol. 67, No. 4, 703-706 (19
93), and Mitsuyasu Itaya, Journal of the Japanese Society of Agricultural Chemistry, vol.
68, No. 11, 1545-1550 (1994) and the like.

(III) Selection of Transformant The transformed bacterium is cultivated by an appropriate method, and then an individual in which homologous recombination has been correctly performed is selected. The selection is not particularly limited as long as it can confirm that homologous recombination into the correct fragment has been performed in the correct place,
For example, in the host bacterium, confirmation of the deletion of the function of the marker gene that was partially inserted into the scaffold integration vector, and the function of the marker gene whose full length was inserted into the scaffold integration vector And the like. As a specific selection method, for example, a case where a partial sequence of the chloramphenicol resistance gene, a scaffolding sequence, or a full-length erythromycin resistance gene sequence is inserted from the 3 ′ downstream side as an example of a scaffold-incorporating vector explain. After transforming a host bacterium with the vector, the transformant was applied to an agarose gel plate medium and cultured, and the emerged colonies were added to erythromycin and chloramphenicol in the same plate medium (hereinafter, respectively, (Cm medium "and" Em medium "may be referred to) with an address such as a toothpick, and the cells are inoculated and cultured. After culturing, it grows in Em medium, but Cm
By selecting a transformant that does not appear in the medium, L
A transformant having a PS-inserted genome (hereinafter, this may be referred to as “scaffold-introduced body”) can be selected.

When an erythromycin resistance gene or a chloramphenicol resistance gene is used as a selection marker, it grows if the number of inoculated cells is small even if the genome has the antibiotic resistance gene. Since it may not be possible, insert an antibiotic resistance gene that is easy to obtain resistance into the scaffold integration vector, first check the resistance with this antibiotic and then select with a selection marker such as erythromycin or chloramphenicol The method is preferably used. Examples of the antibiotic resistance gene that easily obtains resistance include a spectinomycin resistance gene and the like.

(IV) Introduction of Inserted DNA into Host Genome The inserted DNA is introduced into the host genome by transforming the host having the genome into which the sequence to be the scaffold thus obtained is inserted, with the DNA having the inserted DNA. can do. For details of transformation and homologous recombination, see Mitsuo Ogura, Bioscience and Industry, vol.
57, No. 8, 532-535 (1999), Mitsuyasu Itaya, Journal of the Japanese Society of Agricultural Chemistry, vol. 67, No. 4, 70
3-706 (1993), and Mitsuyasu Itaya, Journal of the Japanese Society of Agricultural Chemistry, vol. 68, No. 11, 1545-155
0 (1994) and the like.

The transformant into which the inserted DNA has been introduced (hereinafter, this may be referred to as "DNA extender") is
This can be selected with an appropriate marker gene. Here, the marker gene is preferably one introduced into the host genome so as to be sandwiched between LPS. Such a marker gene can be used as a scaffold in the scaffold-incorporating vector described in (II) above (see FIG. 1).
A box) represented by [n + 1] of A, is inserted between the part of the sequence of the marker gene (box indicated by oblique lines in FIG. 1A) (box indicated by c I in FIG. 1A). The marker gene used here is different from the other marker genes inserted in the vector, and any marker gene can be used as long as the deletion of the marker gene can be confirmed in the host. Specifically, for example, c I
A repressor or the like is preferably used. When the c I repressor is used as the marker gene, the inserted DNA is obtained by using the phenotype in which neomycin resistance is imparted to the obtained transformant by deletion of the c I repressor as an index.
An introduction body can be selected (Japanese Patent Laid-Open No. 2001-321).
No. 170).

[0024] Actually, most of the scaffold-incorporating vectors into which the c I repressor is inserted lack the DNA near the c I repressor, and therefore the inserted DNA is not actually introduced. Despite this, transformants that become neomycin resistant may appear. To prevent this phenomenon, for example, in scaffolding insertion vector further different marker gene adjacent to the c I Ripuresssa (hereinafter, sometimes referred to as c I confirmation marker ") by inserting, this c I It is preferable to select the scaffold-introduced body based on the confirmation marker gene. Here, as the marker for confirming c I, the above (II
In I), it is preferable to use a marker gene inserted in a scaffold-incorporating vector as an antibiotic resistance gene that easily obtains resistance (box indicated by SpR in FIG. 1A).

(V) Construction of a scaffold-integrating vector and insertion of the scaffold into the host genome (IV)
The inserted DNA, its terminal sequence LPS (the box indicated by [n + 1] in FIG. 1C), and the marker gene adjacent to the 5'upstream side (for convenience, the marker B
)) (The box indicated by EmR in Figure 1C). When the inserted DNA is further extended using this transductant as a host, it can be performed by further inserting LPS into the transductant and then transforming it with a DNA containing the next inserted DNA.

Here, as the scaffold-incorporating vector, a partial sequence of the marker B gene inserted in the genome, one terminal sequence (n + 2) of the inserted DNA, a marker different from the one introduced into the host genome. It contains a gene and a common sequence, and can be constructed in the same manner as (II) above. Here, assuming that a marker gene different from that introduced into the host genome is a marker gene having a part thereof inserted in the vector described in (II), use of two different marker genes in the vector Then, LPS can be extended.

[0027]

The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples. Example 1 Extension of cyanobacterial genomic DNA incorporated into Bacillus subtilis genomic vector using scaffold insertion vector

(1) A plasmid p containing a marker gene
Construction of CISP334 and pCISP335 Vector pCIS used in the conventional shakutake method
P310B and pCISP311B (Itaya, M .;
et al. J. Biochem. , 128,869
-875 (2000); FIG. 3), using pCISP3
34 and pCISP335 were made by the following procedure.

First, in order to obtain a DNA fragment on the amino terminal side of the chloramphenicol resistance gene, pCISP3
Using 10B as a template and the primers shown in SEQ ID NOS: 1 and 2, a fragment of about 0.6 kb was amplified by polymerase chain reaction (PCR) to obtain pCR-XL-
It was cloned into the TOPO vector (Invitrogen). After confirming by sequencing that the obtained fragment was correct, the obtained plasmid was cleaved with the restriction enzyme Bgl II to obtain about 0.6 k
The DNA fragment of b, and cloned into the Bam HI site of PCISP311B, resulting in plasmid PCISP333.

The restriction enzyme I- Ppo I was used as a restriction enzyme for pCISP333.
And partially digested with Xmn I and E. coli plasmid pBR32
Approximately 2 kb fragment derived from 2 (I- Ppo I site in bold type of pCISP311B in FIG. 3 and a part indicated by capital X)
After blunting the remaining fragment after removal of the plasmid, plasmid pCISP335 was obtained by self-closing. Also, pCISP
For construction of a plasmid paired with 335, the DNA fragment at the amino terminus of the erythromycin resistance gene was cloned into pC
A fragment of about 0.7 kb was amplified by PCR using ISP311B as a template and the primers shown in SEQ ID NOS: 3 and 4, and the pCR-XL-TOPO vector (Invitr
(Ogen). After confirming by sequencing that the obtained fragment was correct, the obtained plasmid was cleaved with the restriction enzyme Bgl II, and the obtained DNA fragment of about 0.7 kb was pCISP3.
It was cloned into the Bam HI site of 10B to obtain the plasmid pCISP332. pCISP332 was partially digested with restriction enzymes I- Ppo I and XmnI to remove the approximately 2 kb fragment derived from Escherichia coli plasmid pBR322 (the part indicated by the bold I- Ppo I site in FIG. 1), and the remaining fragment was blunted . After that, by self-closing, the plasmid pCIS
P334 was obtained.

(2) Integration of the first cyanobacterial genome into Bacillus subtilis genome using pCISP310B As a vector for Bacillus subtilis integration and extension, RM125 strain, which is a derivative strain of Bacillus subtilis 168 strain ( Uoz
umi, T .; et al. , Mol. Gen. Gene
t. , 152, 65-69 (1977)), the BEST7003 strain in which the sequence of pBR322 was integrated into the NotI site of the proB gene in the genome (Itay) was used.
a, M. et al. J. Biochem. 128,
869-875 (2000)).

A pair of LPS for introducing the target DNA into the genome of Bacillus subtilis BEST7003 strain and a drug resistance gene sandwiched therebetween were introduced using pCISP310B by the following procedure. The target DNA is cyanobacteria (Sy
nechocystis sp. PCC6803) genomic DNA ([CyanoBase] http: // w
ww. kazusa. or. jp / cyano /) was used.

The above Cy of the cyanobacterial genomic DNA
Base number 1,759,556 in ananoBase
The 1,765,540 DNA fragment was prepared by amplification by PCR using the cyanobacterial genome as a template and the primers having the nucleotide sequences of SEQ ID NOs: 5 and 6. The obtained DNA fragment of about 5 kb (LPS [175
9-1765]) was cloned into the pCR-XL-TOPO vector (Invitrogen). The resulting plasmid was designated as pCR1759-1765.

Next, the above LPS [1759-1777].
From the cyanobacterial genomic DNA at about 7 kb, base number 1,77 in the above CyanoBase.
2,146-1,777,171 DNA fragment (LP
S [1772-1777]) was produced by amplification by PCR using cyanobacterial genomic DNA as a template and primers having the nucleotide sequences of nucleotide numbers 7 and 8. The obtained DNA fragment of about 5 kb (LPS [177
2-1777]) was cloned into the pCR-XL-TOPO vector (Invtrogen). The resulting plasmid was designated as pCR1772-1777.

Plasmid pCISP310B (Itay
a, M. et al. J. Biochem. 128,
869-875 (2000)) have c I cassette DNA spectinomycin resistance gene and the c I repressor gene are linked, and the chloramphenicol resistance gene. The c I repressor gene is the above BES
When it is integrated into the T7003 strain, it acts on the neomycin resistance gene in the genome and becomes sensitive to neomycin.
A Bam HI site and EcoRI site present on both sides of the c I cassette of this PCISP310B,
[1759-1765] excised with Bam HI from the above pCR1759-1765, and the above pCR1.
It was excised from 772-1777 by Eco RI [1.
772-1777] was inserted into the plasmid pC [175
9/1772] was constructed.

BEST7003 made into a competent cell
The strain was transformed with the above-mentioned pC [1759/1772] to give tetracycline sensitivity, neomycin sensitivity,
Select spectinomycin resistance as an index and select BEST
8375 shares were acquired. The selection operation by the combination of the c I repressor gene and the neomachine resistance gene is
aya, M .; et al. J. Biochem. , 1
28,869-875 (2000).

Cyanobacterial genomic DNA (LPS
[1759-1765] to LPS [1772-177]
7]) was incorporated into the genome of Bacillus subtilis by transforming cyanobacterial genomic DNA into competent cells of the Bacillus subtilis BEST8375 strain. In this transformation, LPS [175-9765] and LPS [1772-1777] and cyanobacterial genomic DNA undergo homologous recombination, resulting in [1759-1765] and [1772-177] in the Bacillus subtilis genome.
A Bacillus subtilis BEST8382 strain (FIG. 4) having a DNA of about 7 kb sandwiched between 7] was obtained. Examples of the selection marker, the neomycin resistance due to loss of c I cassette is due spectinomycin sensitive.

(4) Preparation of Bacillus subtilis BEST8386 strain into which a sequence serving as a scaffold for extension and a selection marker are introduced DN further 3 ′ downstream of the cyanobacterial genomic DNA introduced into the BEST8382 strain obtained in (3) above
L as an extending LPS for introducing A adjacent to the 3'downstream of the introduced genomic DNA
Scaffold insertion vectors pCISP334 and pCISP335 containing PS [1807-1812] were constructed.

LPS [1807-1812] is a sequence of SEQ ID NOs: 9 and 1 using cyanobacterial genomic DNA as a template.
Was prepared using the primer having the nucleotide sequence shown in 0, and the obtained fragment of about 5 kb was pCR-XL-TOPO.
It was cloned into a vector (manufactured by Invitrogen) and cut out from it with Eco RI, and this was cut into pC
It was cloned into the Eco RI site of ISP334.
This was designated as pC [/ 1807].

Competent BEST8382 strain was transformed with the above-mentioned pC [/ 1807] plasmid,
Neomycin sensitivity, chloramphenicol sensitivity, erythromycin resistance, and spectinomycin resistance were selected as indicators, and Bacillus subtilis BEST8386 strain was obtained (FIG. 4).

(5) Cyanobacterial genomic DNA
([1772-1777] to [1807-1812])
B. subtilis BEST8386 strain transformed into competent cells was transformed with cyanobacterial genomic DNA.
In this transformation, LPS [1772-177
7], and LPS [1807-1812] and cyanobacterial genomic DNA undergo homologous recombination, resulting in [1772-1777] and [1807] in the Bacillus subtilis genome.
-1812], a Bacillus subtilis BEST8387 strain incorporating a DNA of about 30 kb was obtained. in this way,
By inserting a pair of scaffold sequences into the Bacillus subtilis genome and transforming it with genomic DNA, the genomic DNA sandwiched between the scaffold sequences can be inserted into the Bacillus subtilis genome.

(6) Extension of cyanobacterial genome inserted into Bacillus subtilis genome By repeating the method of (4) and (5) above, the plasmid vector used is alternately repeated with pCISP334 and pCISP335 to obtain 600 kb in the Bacillus subtilis genome. B. subtilis BEST with cyanobacterial genomic DNA inserted
8566 strains were acquired (Fig. 4). Table 1 shows the sequence numbers of the nucleotide sequences of the LPS used and the primer for producing it.

[0043]

[Table 1]

[0044]

INDUSTRIAL APPLICABILITY According to the present invention, in a method for cloning DNA into a bacterial genome, it becomes efficient to prepare an integrating vector for inserting a DNA fragment serving as a scaffold into the Bacillus subtilis genome, and the operability of the above cloning method is improved. It has improved dramatically.

[0045]

[Sequence list] SEQUENCE LISTING <110> Mitsubishi Chemical Corporation <120> A method for DNA cloning <130> J08245 <160> 42 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 1 agatctttcgt tgactctaga ggatc 25 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 2 agatctggtg tttgggaaa caatttcccc g <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 3 agatcttcgt tggtcgagat ccg 23 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 4 agatcttgga accatactta atatagaaa tatc 34 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 5 ggatccagac ttcaacctgg ac 22 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 ggatccggtc gcctttgtgg ag 22 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 ggatccccgg catcttgg 18 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 ggatccctcc ctgatgaacc ag 22 <210> 9 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 ggccagtcag ccccg 15 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 10 ctggtctcca ttagtttctg gcatg 25 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 11 cacttgctag ggagcatttc tgg 23 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 12 ctgtggtggc tgtgacctg 19 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 13 cttcacccgg taatagtgtg cc 22 <210> 14 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 14 cccgccccct aactcg 16 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 15 gcctggaccc catccac 17 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 16 ccttgtttgt cccccagcc 19 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 17 cgaatcccgc agataggcc 19 <210> 18 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 18 ggcctatttg gcaggacgg 19 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 19 ccattgtccc tgcccagg 18 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 20 cccccaaact gggatcgag 19 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 21 cctgctcctc gcaaaccg 18 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 22 ctgagggagt aggggtcag 19 <210> 23 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 23 gccggagcct aaacgcc 17 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 24 caagccctaa tcatcatgac tggc 24 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 25 gtcacctcag tcagcctgc 19 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 26 cctttaggaa gactggcctg c 21 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 27 cagccacagg ggtggatc 18 <210> 28 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 28 cgctcgcaac tgggcc 16 <210> 29 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 29 ctgtccgagg gagcgc 16 <210> 30 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 30 ggctgtgcgt tgcaccc 17 <210> 31 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 31 cccagtacga tcctgccc 18 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 32 ggtgaggtac gcactgcc 18 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 33 ccaatctaag acccactgcc ttg 23 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 34 gctctgcttg gctgatagcg 20 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 35 ctaagtcaag ctcgctcctg g 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 36 catctcctgg gctggttgat c 21 <210> 37 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 37 ggggacgctc taccaagg 18 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 38 ccgcaaaacg aaacacctgc c 21 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 39 cccgaatcta cctacactcc tc 22 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 40 gccccctgag tcgttacg 18 <210> 41 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 41 gccgtcacca ctgtcacc 18 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 42 ggaggagtcc gtgttaggta ttg 23

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a method for cloning DNA into a bacterial genome of the present invention.

FIG. 2 is a schematic diagram showing a conventional method for cloning DNA into a bacterial genome.

FIG. 3 is a schematic diagram showing the constitution of a vector for inserting a scaffold into the bacterial genome of the present invention.

FIG. 4 is a schematic diagram showing an experiment for cloning into a Bacillus subtilis genome by the method of the present invention.

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4B024 AA20 CA03 CA04 DA07 EA04                       FA10 GA11                 4B065 AA01Y AA19X AA99Y AB01                       AC10 AC20 BA02 CA46

Claims (4)

[Claims] 1. A host having a sequence in which both ends of an insert sequence to be introduced are previously inserted into the genome, and the DNA having the insert sequence.
To the host genome including transformation with
In the insertion method, the method of sequentially inserting one end sequence of the insertion sequence into the genome is (1) a partial sequence of a marker gene previously introduced into the host genome, one end sequence of the insertion sequence (n + 1), the host A vector containing a marker gene different from the one introduced into the genome and a common sequence in this order, except that the n and n + 1 sequences are ligated in the same direction as the DNA having the inserted sequence, is prepared, and (2) the vector , A strain in which expression of the marker gene previously introduced into the host genome is lost is induced by homologous recombination between host genomes into which the marker gene has been previously introduced adjacent to one end sequence (n) of the inserted sequence. A method characterized by selecting, (4) transforming the obtained host with a DNA having an insertion sequence, and repeating (1) to (4). 2. The method according to claim 1, wherein the host is a bacterium of the genus Bacillus or an extreme thermophile. 3. The method according to claim 1 or 2, wherein the marker gene has drug resistance activity. 4. A partial sequence of a marker gene previously introduced into a host genome, one terminal sequence (n + 1) of an insertion sequence, and introduced into a host genome for use in the method according to any one of claims 1 to 3. Marker gene different from the one described, in the order of consensus sequence, but the n and n + 1 sequences have insertion sequences
A vector containing DNA ligated in the same direction as DNA.