JP5794558B2 - Modified lethal gene and vector containing the gene - Google Patents

Description

  The present invention relates to a vector containing a mutant lethal gene that is optimal as a cloning vector or to construct a gene library, a mutant lethal gene as a constituent material of the vector, and use thereof.

  In the preparation of a transformant into which a foreign gene has been introduced, a technique for linking a foreign gene fragment to a vector by a combination of a restriction enzyme and a ligase is an established technique. However, in such a gene recombination technique, a restriction enzyme is used. In the past, in order to select a recombinant vector containing the target gene fragment, the vector was purified from many transformants, and one of them was It took time and effort to examine the presence or absence of foreign gene insertion for each one. This is a big problem not only when inserting a single gene fragment, but also when creating a library of various gene fragments, etc. There is a method using a marker gene such as β-galactosidase. This method identifies and selects transformants carrying a vector carrying a foreign gene fragment based on the presence or absence of marker gene expression. An indicator for detecting whether or not a marker gene is expressed is used. There is a problem in that it is necessary to add to the medium, and it takes time and effort to select and collect a transformant expressing the marker gene from other transformants on the medium.

  On the other hand, there is a method of using a gene that is lethal to the host as a marker gene in the vector. For example, a foreign gene insertion site is provided in the promoter located at the end of the lethal gene in the vector. There is. In this method, in a transformant that holds a vector in which a foreign gene is correctly inserted at the insertion site, the promoter does not function and a lethal gene is not expressed, so the transformant can grow but is not inserted. Is a simple method in that a lethal gene is expressed and transformants are killed to select transformants (positive selection), and only grown transformants are targeted for selection. It is. However, in the method in which the insertion site is fused to the end of the lethal gene, the toxic gene is expressed, for example, when an insertion sequence having a promoter activity is inserted, even though the insertion has occurred correctly. In some cases, cells may be killed. In particular, there is a problem when a foreign gene to be inserted into a vector is unknown. For example, in the preparation of a metagenomic library, a useful gene may be missed.

  The present applicant has also filed for a technique using a lethal gene as a marker gene (Patent Document 1). In this method, one or a plurality of stop codons are provided upstream of the lethal gene, and the stop is provided depending on the host. By adjusting the number of codons, the toxicity of a lethal gene is adjusted according to the host to provide an optimal marker gene for the host to be used. The problems caused by this have not been studied.

JP 2004-57063 A

  In view of the above-mentioned problems of the prior art, the object of the present invention is to efficiently remove a vector into which a foreign gene produced by self-circulation of a vector or undigested with a restriction enzyme has not been inserted, The transformant carrying the inserted vector is to provide a simple and efficient means that can be selected without exception, or to provide a high-performance gene library using the means. is there.

  Therefore, as a result of intensive studies on a method for efficiently removing a vector that does not contain a foreign gene, the present inventor used a lethal gene that kills the host by its expression. It has been found that the above-mentioned problems can be solved by providing a restriction enzyme cleavage site for inserting a gene and a stop codon to provide a marker for selecting a transformant, and the present invention has been completed.

That is, the present invention is as follows.
(1) A mutant lethal gene, wherein a stop codon and a foreign gene insertion site are provided in the lethal gene.
(2) A recombinant vector, wherein the mutant lethal gene according to (1) is inserted.
(3) A transformant, wherein a host having no stop codon suppression ability is transformed with the vector described in (2) above.
(4) A recombinant vector, wherein a foreign gene fragment is inserted into the foreign gene insertion site of the mutant lethal gene in the vector according to (2) above.
(5) A transformant, wherein a host having no stop codon suppression ability is transformed with the recombinant vector described in (4) above.
(6) A gene library prepared from a sample containing a plurality of gene fragments, wherein each gene fragment in the sample is inserted into the foreign gene insertion site of the mutant lethal gene in the vector of (2) above. The gene library as described above, comprising an assembly of the respective recombinant vectors.
(7) The mutant lethal gene of the above (1) or a lethal gene having a stop codon for producing the gene is introduced into a vector, the host is transformed and selected, and the mutant lethal gene or the gene A method for cloning a lethal gene having a stop codon for producing a host cell, wherein the host is a cell having no stop codon suppression capability.
(8) A method for amplifying a vector in which a foreign gene insertion site and a stop codon are provided in a lethal gene, wherein the transformant according to (3) is cultured.
(9) Ligation is performed by bringing the vector described in (2) above into contact with a sample containing a plurality of gene fragments in the presence of ligase, and the resulting reaction solution is introduced into a host having stop codon suppression capability. A method for producing a gene library, comprising transforming the obtained transformant, culturing the obtained transformant, and collecting a recombinant vector in the grown transformant.

  According to the present invention, an experiment for suppressing the appearance of a self-circularized vector containing no insert, which occurs due to a combination of a restriction enzyme and a ligation found in the conventional method, and selecting a clone containing no insert is performed. Therefore, it is possible to simply select only clones containing the foreign gene fragment with respect to the vector, and thus the gene can be reliably and accurately cloned. In particular, in the present invention, by providing an insertion site for a foreign gene in the body of a lethal gene, an effect superior to that of a cloning vector having a conventional lethal gene as a marker gene is exhibited. That is, a conventional cloning vector having a lethal gene as a marker gene has a cloning site outside the 5 ′ end of the lethal gene, and in this case, even when the gene fragment to be cloned is inserted correctly, for example, When an insertion sequence having promoter activity is inserted, the toxicity of a lethal gene is expressed and the cell may be killed.

  In contrast, when a cloning site is provided in the lethal gene body as in the present invention, the lethal gene can be completely destroyed when the gene fragment is correctly inserted, so that the gene fragment is inserted correctly. The recombinant vector grows perfectly in the host cell and does not cause the above problems. Therefore, the present invention effectively eliminates cloning errors, and this is particularly useful when preparing a gene library or a mutant protein library in which the gene library is translated or expressed, This is extremely effective in forming a large-scale gene library such as a metagenomic library.

Hereinafter, the present invention will be described in more detail.
[Mutant lethal gene]
In the lethal gene sequence, a foreign gene insertion site and a stop codon are provided in the lethal gene sequence as a transformant selection marker. The toxicity of this lethal gene is controlled by the difference in host cells, Only when the gene is correctly inserted, the transformant is selectively grown and selected. The lethal gene to be used is not particularly limited. For example, ccdB, sacB, deoxyribonuclease, ribonuclease, protease, colicin E1, E2, E3, E4, E5, E6, E7, E8, E9, Ia, Ib, D, B, A, M, N, K, cloacin DF13, crevicein A1, A2, A3, pyocin AP41, S1, S2, S3, S4, barnase, pemK, and other lethal genes can be used.
In the lethal gene, stop codons (TAA, TAG, TGA) are arranged in the sequence, but the position needs to be placed at a site where lethality disappears by replacing the original codon with a stop codon. In addition, as the type of stop codon, an appropriate amino acid may be inserted into the stop codon site and a suppressor tRNA for restoring lethality may be used.

  The foreign gene insertion site is prepared by providing a restriction enzyme cleavage site sequence in the lethal gene. In the present invention, the restriction enzyme cleavage site is not particularly limited, and a gene having a blunt end such as SmaI, HincII, AfeI, DraI, EcoRV, FspI, MscI, NruI, PsiI, PvuII is inserted. A sequence or a sequence for right-inserting a gene having a sticky end such as BamHI, EcoRI, HindIII, XhoI, PstI, SphI can be introduced into a lethal gene.

  Such arrangement of the restriction enzyme cleavage site sequence can be performed by changing the base sequence in the lethal gene to the restriction enzyme cleavage site sequence, but in this case, as the base sequence of the lethal gene to be changed, It is preferable to select a sequence that requires minimal changes in the amino acid sequence of the lethal gene because it is less likely to impair the lethality. However, as long as lethality is maintained, the optimum number of bases is not specified, or not only substitution mutations but insertion mutations and deletion mutations may be used. Further, since the purpose is to insert a foreign gene, it is desirable that the restriction site to be placed does not exist at other sites on the vector. For example, when a SmaI recognition site that does not exist in the wild-type lethal gene is newly placed in the lethal gene, the SmaI recognition sequence is 5′-CCCGGG-3 ′. A sequence similar to 5′-CCCGGG-3 ′ is selected from the sequences and changed to 5′-CCCGGG-3 ′.

  Examples of mutations that do not involve amino acid substitution are as follows. That is, it is assumed that a partial sequence encoding proline-glycine exists in the wild-type lethal gene, and its base sequence is 5'-CCGGGG-3 '. When this site is selected and a SmaI restriction enzyme site is newly arranged, it can be converted into a SmaI sequence of 5'-CCCGGG-3 'by only one base substitution. In this case, since the amino acid proline encoded by the base substitution is not changed, it is obvious that the mutant gene after the SmaI site is maintained lethal as in the wild type. On the other hand, when amino acid substitution is introduced by introduction of a restriction enzyme site, self-circulation that fails to insert a foreign gene fragment must be experimentally verified whether the lethality of the generated mutant lethal gene is maintained. It does not lead to removal of vector or undigested vector. In this case, the introduction of the desired restriction enzyme sequence is carried out by site-specific base substitution as in the above case, but it is confirmed whether or not the restriction enzyme sequence has been introduced and the lethality due to the change in the amino acid sequence. You must make sure that it does not disappear.

In the present invention, when preparing a mutant lethal gene, a site-directed mutagenesis method using a primer for introducing a stop codon and a foreign gene insertion site using an existing vector containing the lethal gene as a marker gene as a template Thus, the sequence of the stop codon and the foreign gene insertion site may be provided in the sequence of the lethal gene of these vectors. Examples of the vector used in this case include plasmid vectors pCR Blunt-II TOPO and pCR-Blunt containing a lethal gene ccdB gene for DNA gyrase as a marker gene.
As the site-specific mutagenesis method, for example, a method known per se, such as a quick change method (Stratagene), Transformer Site-Directed Mutagenesis (Clontech), or the like already used as a kit is used. it can.

[Mutant lethal gene insertion recombination vector]
The mutant lethal gene insertion recombinant vector of the present invention is a vector having a mutant lethal gene as a marker, and is particularly useful as a cloning vector or a gene library preparation vector.
As described above, the mutant lethal gene insertion recombinant vector of the present invention can be obtained by incorporating a mutant lethal gene having a stop codon and a foreign gene insertion site into the lethal gene sequence. Plasmids, cosmids, fosmids, phages and the like can be used, and there are no particular restrictions on this point, and a suitable one may be selected as appropriate according to the target host.

[Host cell]
The mutant lethal gene insertion recombinant vector in the present invention contains a stop codon in the lethal gene sequence, and thus the toxicity of the lethal gene is usually lost. However, in prokaryotes and certain eukaryotes, the synthesis of peptide chains may not be stopped by a stop codon because it has an abnormal suppressor tRNA with an anticodon corresponding to the stop codon. Yes, this suppressor tRNA has an amino acid at the 3 ′ end, and the amino acid is incorporated into the site corresponding to the stop codon. Therefore, when a cell having such a suppressor tRNA and having a function of suppressing a stop codon is used as a host and transformation is performed with the mutant lethal gene insertion recombinant vector, the introduced mutant lethal gene is introduced. The stop codon in the lethal gene in the inserted recombinant vector is abolished, the lethal gene toxicity is restored, and the host cell is killed. A cell having a function of suppressing such a stop codon can be used only when the foreign gene is correctly inserted into the foreign gene insertion site of the mutant lethal gene insertion recombinant vector and the function of the lethal gene is lost. Growth is possible.

Examples of cells in the present invention include cells having genotypes such as supE, supF, supB, supC, supG, supL, supM, supN, supO, supD.
On the other hand, when cells having no stop codon suppression ability are used as a host, even if the above mutant lethal gene insertion recombinant vector is introduced, the lethal gene in the vector is a stop codon and its peptide synthesis is interrupted. Therefore, toxicity is not exhibited and the resulting transformant can grow. Examples of cells that do not have the function of suppressing the stop codon include cells lacking genes such as supE, supF, supB, supC, supG, supL, supM, supN, supO, supD. When it is necessary to maintain the mutant lethal gene insertion vector in the cell, a cell that does not have the function of suppressing this stop codon is used.

The host cell to be used is not limited to its taxonomic type as long as it has and does not have the function of suppressing the stop codon, and various cells such as Escherichia coli, Bacillus subtilis, and yeast can be used. .
For example, in the step of preparing a mutant lethal gene insertion recombinant vector of the present invention, a stop codon is introduced into the lethal gene and a foreign gene introduction site is further introduced, or the mutant lethal gene is prepared in the reverse order. Or you may introduce simultaneously. If the foreign gene insertion site already exists in the wild type gene, it may be utilized.

In cloning of lethal genes having stop codons obtained in each of these production steps or mutant lethal genes having stop codons and foreign gene insertion sites, these mutant genes are commonly used such as kanamycin, chloramphenicol, zeocin, ampicillin, etc. It is inserted into a vector having another marker gene such as a drug resistance gene used in the above to obtain a recombinant vector, and the recombinant vector is introduced into a host cell and screened. Cells that do not have a stop codon suppressor that does not invalidate the stop codon in the recombinant Bacterium are used.
That is, in a host cell that does not have a stop codon suppression capability, the stop codon provided in the lethal gene is not invalidated, and the transformant can grow and proliferate. This also makes it possible to amplify a recombinant vector into which a mutant lethal gene or a stop codon inserted lethal gene has been introduced.

  As apparent from the above, the mutant lethal gene insertion recombinant vector of the present invention exhibits toxicity depending on the host to be used. Therefore, a mutant lethal gene insertion recombinant vector is prepared or a transformant into which the vector is introduced In the production step of (1) and the gene cloning step using the produced recombinant vector, a cell having a stop codon suppressor and a cell having no stop codon suppressor are selectively used as host cells to be used.

[Use of mutant lethal gene insertion recombinant vectors]
In using the mutant lethal gene insertion recombinant vector of the present invention, first, a foreign gene insertion site provided in the lethal gene in the recombinant vector is cleaved with a restriction enzyme, and a restriction enzyme recognition sequence corresponding to the foreign gene insertion site. An operation for ligating gene fragments having the two at their ends is performed. The ligation operation may be a conventional means using ligase. The obtained recombinant vector is introduced into a host and transformed. The host used at this time is a cell having a stop codon suppression capability, and as described above, the stop codon in the lethal gene sequence is invalidated in the host, and the foreign gene is correctly inserted into the foreign gene insertion site. Thus, the growth of the lethal gene is possible only when the function of the lethal gene is lost.

On the other hand, when the vector is self-circularized or the foreign gene or the foreign gene insertion site is undigested in the restriction enzyme treatment, the foreign gene is correctly inserted into the foreign gene insertion site in the mutant lethal gene If not inserted, the transformant containing such a vector is killed by the toxicity of the lethal gene, so that only the transformant that grows should be screened.
Such a technique of the present invention is particularly useful as a means for cloning genes, but is particularly useful for preparing gene libraries.

  In order to prepare a gene library using the mutant lethal gene insertion recombinant vector of the present invention, for example, chromosomes or plasmids, cosmids, fosmids, and phages collected from various organisms are used as foreign vectors for the mutant lethal gene insertion recombinant vector. A fragmented sample having a plurality of gene fragments is obtained by cleaving with a restriction enzyme that recognizes the gene insertion site. On the other hand, the mutant lethal gene insertion recombinant vector is cleaved with the same enzyme as the restriction enzyme used for fragmentation at the foreign gene insertion site, and reacted with the fragmented sample in the presence of ligase. After the reaction, the reaction solution is brought into contact with a host cell having a stop codon suppression ability, and a transformant is obtained by introducing a mutant lethal gene insertion recombinant vector to which each gene fragment in the fragmented sample is linked. . Subsequently, the obtained transformant is cultured in a medium. At this time, the mutant lethal gene insertion recombinant vector causes self-circulation, or the gene fragment or the foreign gene insertion site is not digested in the restriction enzyme treatment. In some cases, a transformant having a vector in which a gene fragment is not correctly inserted into the foreign gene insertion site of a mutant lethal gene is killed because the lethal gene exhibits toxicity.

On the other hand, the transformant that grows has a recombinant vector correctly recombined with each fragmented gene, and each gene fragment is obtained by collecting a recombinant vector containing each fragmented gene from the transformant. A gene library that is an assembly of vectors can be constructed.

EXAMPLES The present invention will be specifically described below with reference to examples, but the technical scope of the present invention is not limited to these examples.

Example 1
(1) Modification of lethal gene The ccdB gene (SEQ ID NO: 1) was used as the lethal gene. Amber termination of the 21st amino acid of the ccdB gene (glutamine in the wild type) using the plasmid containing the foreign gene at the insertion site of the foreign gene of the commercially available plasmid vector pCR Blunt-II-TOPO (Invitrogen) containing the gene Replaced with a codon. Site-specific base substitution was performed according to the quick change method. The reaction solution (total volume 20 μl) was 1 × KOD-plus version 2 buffer (Toyobo), 1.5 mM magnesium sulfate, each 0.2 mM deoxyribonucleotide mixture, template plasmid 1 ng, 1 unit KOD-plus version 2 DNA polymerase (Toyobo) ), Upstream (SEQ ID NO: 2) and downstream (SEQ ID NO: 3) primers of 10 pmol each. After the solution was kept at 98 ° C. for 2 minutes, a temperature cycle of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 4 minutes was repeated 18 times.

Sequence number 2: 5'-CGTCTGTTTGTGGATGTATAGAGTGATATTATTGACACG-3 '
Sequence number 3: 5'-CGTGTCAATAATATCACTCTATACATCCACAAACAGACG-3 '
After completion of the reaction, 10 units of restriction enzyme DpnI (NEB) was added to the solution and reacted at 37 ° C. for 2 hours to eliminate the template. After the reaction, 1 μL was collected, added to E. coli competent cell MV1184 manufactured by Takara Bio Inc., and transformed according to the product manual. The cells were spread on an LB agar medium containing 50 μg / ml kanamycin and cultured at 37 ° C. overnight. The grown colonies were picked, the plasmid was purified with a plasmid purification kit manufactured by Qiagen, and the sequence of the mutant ccdB gene contained in the plasmid was confirmed by DNA sequencing.

(2) Next, PCR was performed to amplify a mutant ccdB gene in which the 21st glutamine was replaced with an amber codon.
In the reaction solution (total amount 20 μl), 1 × KOD-plus version 2 buffer (Toyobo Co., Ltd.), 1.5 mM magnesium sulfate, each 0.2 mM deoxyribonucleotide mixture, and the 21st glutamine obtained in (1) above were replaced with amber codons. 1 ng of a template plasmid containing the mutated ccdB gene, 1 unit KOD-plus version 2 DNA polymerase (Toyobo Co., Ltd.), upstream (SEQ ID NO: 4) and downstream (SEQ ID NO: 5) primers of 10 pmol each. After the solution was kept at 98 ° C. for 2 minutes, a temperature cycle of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 30 seconds was repeated 25 times.

Sequence number 4: 5'-TTTTCATATGCAGTTTAAGGTTTACACCTATAAAAG-3 '
Sequence number 5: 5'-TTTTTAAGCTTATATTCCCCAGAACATCAGGTTAA-3 '
Amplified products are separated by agarose gel electrophoresis, a band of about 400 base pairs is cut out, DNA is purified by QIAprep spin column (Qiagen), and then NEB restriction enzymes NdeI (10 units) and HindIII (10 units) are used. Digested at 37 ° C. for 2 hours.

(3) Cloning of mutant ccdB gene The mutant ccdB gene containing the amber codon obtained in this way was transformed into pTD-tacNd (Miyazaki
K. Isocitrate dehydrogenase from Thermus aquaticus YT1: purification of the
The gene was inserted into the NdeI-HindIII site of Appl Environ Microbiol. 1996 Dec; 62 (12): 4627-31.). For this purpose, pTD-tacNd was digested with NdeI and HindIII at 37 ° C. for 2 hours, separated by agarose gel electrophoresis, a band of about 2600 base pairs was excised, and DNA was purified with a QIAprep spin column (Qiagen). The vector and the mutant ccdB gene fragment were mixed so that the molar ratio was approximately equal, and the ligation reaction was performed overnight at room temperature in the presence of T4 DNA ligase (NEB). 1 μL of the reaction solution was collected, added to 100 μL of E. coli competent cell MV1184 manufactured by Takara Bio Inc., and transformed according to the product manual. The cells were spread on an LB agar medium containing 34 μg / ml chloramphenicol and cultured at 37 ° C. overnight. The grown colonies were picked, the plasmid was purified with a Qiagen plasmid purification kit, and the sequence of the mutant ccdB gene containing the amber mutation was confirmed again by DNA sequencing.

(4) Introduction of SmaI restriction enzyme site Using the pTD-tacNd plasmid containing the mutated ccdB gene containing the amber mutation as a template, a cloning site for inserting a foreign gene was provided in the ccdB gene. First, a SmaI restriction site (-CCCGGG-) was introduced by single nucleotide substitution at the 28th to 29th amino acid positions (Pro (CCG) -Gly (GGG)) using the primers of SEQ ID NO: 6 and SEQ ID NO: 7. The original amino acid sequence is proline-glycine, but the encoded amino acid sequence is not changed by the mutation of the base sequence in this experiment. The method was performed by the quick change method as described above. PCR is under the same conditions for both composition and temperature cycle except that the types of template and primer are different. After completion of the reaction, the solution was treated with DpnI at 37 ° C. for 2 hours, 1 μL of the reaction solution was collected, added to 100 μL of E. coli competent cell MV1184 manufactured by Takara Bio, and transformed according to the product manual. The cells were spread on an LB agar medium containing 34 μg / ml chloramphenicol and cultured at 37 ° C. overnight. The grown colonies were picked, the plasmid was purified with a plasmid purification kit manufactured by Qiagen, and the sequence of the mutant ccdB gene containing the amber mutation and the SmaI site was confirmed again by DNA sequencing.

Sequence number 6: 5'-GATATTATTGACACGCCCGGGCGACGGATG-3 '
Sequence number 7: 5'-CATCCGTCGCCCGGGCGTGTCAATAATATC-3 '

(5) Introduction of BamHI restriction enzyme site Using the plasmid synthesized in (4) above as a template, site-specific substitution was performed to further provide a BamHI restriction site (-GGATCC-) in the ccdB gene. At this time, since a restriction enzyme site was introduced and amino acid substitution was involved, a site where a BamHI site could be set with only one amino acid substitution was searched for in the gene. As a result, it was found that a BamHI site can be introduced by allowing substitution of amino acids of Val33, Val46, and Ala69.
That is, the base sequence corresponding to Val-Ile-Pro at amino acid positions 33 to 35 is -G TGATCC CC-, and the codon (GTG) corresponding to VAL33 is the second letter and the third letter is -GG-. A BamHI restriction site can be introduced by substitution with a codon of another amino acid (-N GGATCC CC-; underlined BamHI site). For Val33, there are three types of amino acids to be mutated (Trp (TGG), Arg (CGG, AGG), and Gly (GGG)), so primers (SEQ ID NOs: 8 and 9) covering all the patterns are used. Designed.

Furthermore, since the corresponding base sequence of Val-Ser at amino acid positions 46 and 47 is -GTCTCC-, the codon of Val46 may be replaced with the codon of Gly (GGA), and a primer for that purpose (SEQ ID NO: 10.11) Designed.
On the other hand, although the corresponding nucleotide sequence of Ala-Ser at amino acid position 69 and 70 is -GCCAGT-, since TCC in the cleavage site of BamHI is also a codon of Ser, amino acid substitution is performed by replacing Ala69 (GCC) with Gly ( It is only necessary to substitute GGA). A primer for substituting the corresponding Ala-Ser base sequence (-GCCAGT-) with the sequence of the BamHI cleavage site (-GAATCC-) is shown in SEQ ID NO: 12.13.
Unlike the case of SmaI described above, the introduction of these BamHI restriction sites is a mutation involving amino acid substitution, and therefore it is not obvious whether the lethality of the ccdB gene is retained. Therefore, since it was necessary to verify the presence or absence of lethality for each site, experiments were performed in parallel.

  The combinations of primers used for site-specific substitution were used in the following three ways: SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13. The method was performed by the quick change method as described above. PCR is under the same conditions for both composition and temperature cycle except that the types of template and primer are different. After completion of the reaction, the solution was treated with DpnI at 37 ° C. for 2 hours, 1 μL of the reaction solution was collected, added to 100 μL of E. coli competent cell MV1184 manufactured by Takara Bio, and transformed according to the product manual. The cells were spread on an LB agar medium containing 34 μg / ml chloramphenicol and cultured at 37 ° C. overnight. The grown colonies were picked and the plasmid was purified with a Qiagen plasmid purification kit, and the sequence of the mutant ccdB gene containing the amber mutation, SmaI site and BamHI was confirmed again by DNA sequencing. In particular, for the mutation at amino acid position 33, all of Trp (TGG), Arg (CGG, AGG), and Gly (GGG) are produced in the library by using the primers of SEQ ID NOs: 8 and 9. Colonies were selected, the plasmid was extracted and purified, and the nucleotide sequence was actually determined to determine what nucleotide substitution and amino acid substitution occurred. In this way, all three substitution patterns were obtained.

Sequence number 8: 5'-GGGCGACGGATGNGGATCCCCCTGGCCA-3 '
Sequence number 9: 5'-TGGCCAGGGGGATCCNCATCCGTCGCCC-3 '
Sequence number 10: 5'-CGTCTGCTGTCAGATAAAGGATCCCGTGAACTTTACCC-3 '
Sequence number 11: 5'-GGGTAAAGTTCACGGGATCCTTTATCTGACAGCAGACG-3 '
Sequence number 12: 5'-ATGATGACCACCGATATGGGATCCGTGCCGGTCTCCGT-3 '
SEQ ID NO: 13: 5′-ACGGAGACCGGCACGGATCCCATATCGGTGGTCATCAT-3 ′

Example 2
(1) Preparation of Library The solution containing various plasmids prepared in Example 1 (5) was introduced into Escherichia coli JM109 competent cell (manufactured by Toyobo Co., Ltd.), and the transformant was 34 μg / L chloram. Phenicol was seeded on LB agar plates containing 0.05 mM isopropyl-β-D-thiogalactopyranoside. Among the above mutant ccdB genes, the gene containing Val33Trp is known to be temperature sensitive (Chakshusmathi G, Mondal K, Lakshmi GS, Singh G, Roy A, Ch RB, Madhusudhanan S, Varadarajan R. Design of temperature-sensitive mutants solely from amino acid sequence. Proc Natl Acad Sci US A. 2004 May 25; 101 (21): 7925-30.). That is, it was considered that the colony formation occurred at 37 ° C. without toxicity at high temperatures. However, as a result of experiments, transformants could not be obtained in all cases at 30 ° C. and 37 ° C. This suggested that when no foreign gene was inserted, none of the vectors obtained in (4) could grow and be selected from clones having the inserted fragment.

(2) Library preparation Among the above plasmids, a pTDtacNd vector (hereinafter referred to as pCm-Ccd1R) containing SmaI and BamHI (Val33Arg type) in the ccdB gene is used to insert a foreign gene fragment, Used for library creation.
Using a plasmid vector containing an unknown gene fragment of about 10 kb in length as a substrate, the plasmid was partially digested with AluI or Sau3AI, and a 1 to 3 kb DNA fragment was inserted into the SmaI and BamHI sites of the pCm-Ccd1R vector, respectively. First, 1 μg of a plasmid serving as a substrate was mixed with 1 unit of restriction enzyme AluI (NEB) or Sau3AI (Takara Bio) and reacted at room temperature for 4 minutes. The reaction was terminated by adding ethylenediaminetetraacetic acid having a final concentration of 10 mM, and the reaction solution was kept at 70 ° C. for 10 minutes. Next, the reaction solution was subjected to agarose gel electrophoresis, and a 1 to 3 kb DNA fragment was excised and purified with a QIAquick spin kit (Qiagen). The purified gene fragment was eluted with 30 μL of water. On the other hand, 1 μg of pCm-Ccd1R was semi-coated with 10 units of SmaI or 10 units of BamHI. SmaI was reacted at room temperature for 2 hours, and BamHI was reacted at 37 ° C. for 2 hours. Thereafter, 2 units of alkaline phosphatase (NEB) was added directly to the reaction solution, and the mixture was reacted at room temperature for 1 hour.

(3) 0.2 μg of each insert and vector gene fragment were mixed and ligation reaction was performed at room temperature in the presence of T4 DNA ligase (NEB) in a 10 μL reaction system. After the reaction for 6 hours, 1 μL was collected, and 20 μL of JM109 competent cell (Takara Bio) was added. The transformed cells were seeded on an LB agar medium containing 34 μg / ml chloramphenicol, and statically cultured at 37 ° C. overnight.

  Ten clones grown from each library were randomly selected, planted in an LB liquid medium containing 34 μg / ml chloramphenicol, extracted at 37 ° C. overnight, and the plasmid was extracted and sandwiched between SmaI and BamHI sites. An attempt was made to amplify the insert with an oligonucleotide primer designed as such. The reaction solution (total volume 20 μl) was 1 × KOD-plus version 2 buffer (Toyobo), 1.5 mM magnesium sulfate, each 0.2 mM deoxyribonucleotide mixture, template plasmid 1 ng, 1 unit KOD-plus version 2 DNA polymerase (Toyobo) ), Upstream (SEQ ID NO: 14) and downstream (SEQ ID NO: 15) primers of 10 pmol each. This solution was kept at 98 ° C. for 2 minutes, and then a temperature cycle of 98 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 68 ° C. for 3 minutes was repeated 25 times.

SEQ ID NO: 14: 5′-CTATAAAAGAGAGAGCCGTTATCGTCTG-3 ′
SEQ ID NO: 15: 5′-ATATCGGTGGTCATCATGCGCCA-3 ′

The reaction solution was analyzed by agarose gel electrophoresis, and a 1 to 3 kb fragment was obtained in all clones, confirming that it was correctly cloned into the vector.
In addition, 96 clones were randomly selected from each library and sequence analysis was performed using primers adjacent to the insert region. As a result, the presence of the insert was confirmed for all clones.

Claims (9)

A mutant lethal gene characterized by providing a stop codon and a foreign gene insertion site in the lethal gene,
In the ccdB gene comprising the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, the mutated lethal gene is mutated to the amber stop codon -TAG-, and the 28th to 29th amino acids are changed. The base sequence to be encoded is mutated to the base sequence -CCCGGG- of the SmaI restriction enzyme site or is not mutated and the base sequence of the BamHI restriction enzyme site of the base sequence encoding the 33rd to 35th amino acids A nucleotide sequence containing -TGGATCCCCC-, -CGGATCCCCC-, -AGGATCCCCC- or -GGGATCCCCC-, a mutation of the nucleotide sequence encoding the 46th to 47th amino acids of the BamHI restriction enzyme site into a nucleotide sequence -GGATCC-, and , 69th to 70th net Acids having at least one mutation of mutations into the BamHI restriction enzyme site of the nucleotide sequence of the nucleotide sequence encoding -GGATCC- and characterized in that it is a mutant ccdB gene, mutant lethal gene.   A recombinant vector, wherein the mutant lethal gene according to claim 1 is inserted.   A transformant, wherein a host having no stop codon suppressive ability is transformed with the vector according to claim 2.   A recombinant vector, wherein a foreign gene fragment is inserted into the foreign gene insertion site of the mutant lethal gene in the vector according to claim 2.   A transformant, wherein a host having a stop codon suppressive ability is transformed with the recombinant vector according to claim 4.   A gene library prepared from a sample containing a plurality of gene fragments, wherein each gene fragment in the sample is inserted into a foreign gene insertion site of a mutant lethal gene in the vector according to claim 2. The gene library described above, characterized by comprising an assembly of the following recombinant vectors. Mutations lethal gene of claim 1, and introduced into a vector, the host and screened by transformation, to a method for cloning the mutant lethal gene, host, are no cells stop codon suppression ability A cloning method as described above.   A method for amplifying a vector in which a foreign gene insertion site and a stop codon are provided in a lethal gene, wherein the transformant according to claim 3 is cultured. A ligation reaction is performed by contacting a foreign gene insertion site provided in a lethal gene in the vector according to claim 2 with a restriction enzyme and a sample containing a plurality of gene fragments in the presence of ligase. A gene characterized by introducing the obtained reaction solution into a host having a stop codon suppressive ability, transforming the resultant, culturing the obtained transformant, and collecting a recombinant vector in the grown transformant Library manufacturing method.