JP6256911B2 - New counter selection method - Google Patents

Description

  The present invention relates to a method for removing a vector present in an E. coli cell and selecting a cell not retaining the vector. More specifically, a cell that does not hold the vector containing the marker is selected by including a gene that suppresses the expression of a gene essential for the growth of Escherichia coli on the target vector to be dropped, and by conditional expression of the gene. The counter selection method, and a counter selection gene marker effective for the method.

  As a method for introducing a vector into a cell, there are various physical, chemical and biological methods such as a chemical method (competent cell method), an electroporation method (electroporation method), and a junction transfer method. . In general, selection of the introduced cells and those that are not is carried out by including gene markers such as drug resistance genes and auxotrophic genes on a vector, and selecting based on drug resistance, auxotrophy, and the like.

  On the other hand, the following method is conceivable for removing a vector once introduced into a cell and selecting a cell that does not hold the vector. That is, there is a method of selecting a clone that has lost the trait of this selection marker by using the selection marker used for introduction into the cell. However, once introduced, the vector is generally maintained in the cell relatively stably, and even when cells are continuously cultured under non-selective conditions, the frequency of spontaneous dropout is low. For this reason, it is necessary to screen a large number of clones in order to select missing clones using the marker used at the time of introduction as an index, which is very inefficient.

  Contrary to genes that serve as positive selection markers for simply selecting vectors introduced such as drug resistance genes and auxotrophic gene markers, there are also genes that cause cells to die by expression. For example, genes encoding nucleases and proteases, and genes that are converted into toxic substances by converting a certain substrate are known. Such a toxic gene can be cloned by suppressing the expression, and cell death can be induced by releasing the expression suppression. Based on this principle, it becomes possible to efficiently select a clone from which the vector has been removed from the cell. Thus, a method of selecting a clone that does not express, for example, a clone from which such a lethal factor is eliminated, is called counter selection, utilizing the principle that cell death is induced by expression.

  There are various methods for introducing vectors into cells, depending on the combination of various selection markers and replication origins. On the other hand, the technology for dropping vectors is lacking in both methodologies and marker genes. It has been demanded. In particular, there has been a demand for the development of an effective counter selection marker in E. coli frequently used in genetic engineering experiments.

  Until now, a sacB gene derived from Bacillus subtilis has been well known as a lethal gene used for counter selection in Gram-negative bacteria such as Escherichia coli (Non-patent Document 1). In gram-negative bacteria such as Escherichia coli, levanshu clease, which is a sacB gene product, is known to convert sucrose to levan, and levan accumulates in the periplasmic layer, leading to cell death. Since it does not show lethality in the absence of sucrose, a vector containing the sacB gene can be selected depending on the presence or absence of sucrose. This method is widely used mainly for Escherichia coli, but it has also been reported that levan sucrose enzyme itself has nonspecific cytotoxicity, and when cell death is induced by addition of sucrose, 5 There are also concerns about the occurrence of unexpected mutations and phenotypic changes caused by applying sucrose at a very high concentration of about 15% to 15%, which is different from normal growth conditions. In addition, in the counter selection using the sacB gene, it is necessary to select again with a sucrose medium after selecting with a non-selective medium without sucrose. The need for such multiple steps has been a drawback. As a method for selecting cells in which only the target plasmid DNA functions more quickly, it is desirable that the direct dropout conditions can be applied. It is also desired that selection is possible not only in a solid medium but also in a liquid medium, but it is well known that many false positives are detected by selection in a liquid medium using the sacB gene (non-patent literature). 2). In the case of Escherichia coli most frequently used as a host for genetic engineering experiments, several counter selection gene markers have been reported in addition to the sacB gene (Non-patent Document 3). However, conventional markers have various problems, such as being applicable only to host bacteria having a special genotype, and being not suitable for use in selection in liquid media. Could not be used. Therefore, the development of new methods and markers for counter selection is required.

  In the above method, the method of including a gene that induces cell death in the vector is exemplified. However, the gene itself is not toxic, but by expressing it, the cell is killed by suppressing the expression of a growth essential gene. It can also be achieved. For example, the E. coli genome encodes many genes essential for growth. For example, the fabI gene of lipid synthesis system can be mentioned. If the expression of this gene or the like is suppressed, cells that have lost the vector containing the expression-suppressing factor can be selected.

  The E. coli genome and extragenomic DNA also contain many toxic and antitoxic genes (TA family). Examples include ccdAB gene (Non-patent document 4), relBE gene (Non-patent document 5), mqzEF gene (Non-patent document 6), dinJ / yafQ gene (Non-patent document 7), hipBA gene (Non-patent document 8), hicAB Known genes (Non-patent Document 9), pas gene (Non-patent Document 10), yefM-yoeB gene (Non-patent Document 11), mqsRA gene (Non-patent Document 12), rnlAB gene (Non-patent Document 13), etc. Yes. Toxicity genes related to these mask the toxicity by expressing an excessive amount of antitoxin gene relative to the toxin gene under normal growth conditions. Therefore, by suppressing the expression of the antitoxin gene, the toxin gene functions and cell death can be achieved.

Gay, P. et al., J. Bacteriol. 164: 918-921. 1985 Hiroshi Mizoguchi et al., Biosci. Biothechnol. Biochem., 71 (12), 2905-2911, 2007 JEAN-MARC REYRAT et al., Infection and Immunity, 66, 9, 4011-4017, 1998 Van Melderen et al., Mol. Microbiol. 11, 1151-1157, 1994 Christensen, S.K. et al., Proc. Natl. Acad. Sci. U.S.A. 98, 14328-14333, 2001 Christensen, S.K. et al., J. Mol. Biol. 332, 809-819. 2003 Prysak, M.H. et al., Mol. Microbiol. 71, 1071-1087. 2009 Hansen, S. et al., PLoS One 7 (6), e39185. Jorgensen, M.G. et al., J. Bacteriol. 191, 1191-1199. 2009 Smith, A.S.G. et al., J. Bacteriol. 180, 5458-5462. 1998 Christensen, S.K. et al., Mol. Microbiol. 51, 1705-1717. 2004 Wang, X. et al., Nat. Chem. Biol. 7, 359-366. 2011 Koga, M. et al., Genetics 187, 123-130. 2011.

  While a technique for introducing a vector into a cell has been established, progress in technology for dropping and removing a vector by selecting a cell from which the vector has been dropped is poor. In the conventional vector selection method based on counter selection, there is generally no experimental system performed by liquid culture. That is, the selection method by plate culture has been the mainstream, but this method has a problem that it takes time to select and is inconvenient for processing a large-scale sample.

  The present invention aims to provide a counter selection method using an antisense method, and to provide an effective counter selection marker used in the method, and by providing them, the operability can be improved as compared with the conventional method. .

  In order to solve the above-mentioned problems, the inventors focused on a technique for suppressing the expression of essential genes. In particular, we focused on antisense RNA. Antisense RNA hybridizes to the mRNA of the target gene, and inhibits the expression of the target gene by inhibiting translation and promoting degradation of the mRNA. In general, it is said that the expression can be most efficiently suppressed by setting the target codon around the start codon from the ribosome binding site (RBS) of the target gene mRNA. In the present invention, E. coli was used as the host. Plasmid DNA was used as the vector. Antisense RNA target genes are those that inhibit the growth of Escherichia coli by suppressing expression, in particular, essential genes and anti-toxic genes of the toxic gene / anti-toxic gene system (TA system). In the present invention, the antisense DNA for the target gene is previously encoded in the plasmid DNA. And the expression level of a target essential gene is reduced by expressing antisense RNA. Since cells with a reduced expression level of essential genes cannot grow, the only cells that can grow under the circumstances are those from which plasmid DNA has been removed. There are many TA systems in E. coli that can be used for such a plasmid DNA selection method as shown above, and as many as 302 other genes essential for growth are known (http: //www.shigen nig.ac.jp/ecoli/pec/) (Kato J. et al., Mol. Syst. Biol. 2007; 3: 132). In the present invention, among the essential genes and TA systems encoded by Escherichia coli MG1655 strain (genomic sequence: NC_000913.2), the target can perform effective counter-selection targeting the essential genes fabI gene and various TA systems. Developed the corresponding genetic marker. As a result, particularly good results were obtained when the mqsA gene of the toxic gene mqsR gene and the rnlB gene of the toxic gene rnlA gene were selected as target genes.

  That is, the present invention includes the following plasmid DNA selection method and antisense RNA used in the method, plasmid DNA encoded by the RNA, and E. coli strains containing the plasmid DNA.

Accordingly, the present invention is as follows.
[1] An expression vector expressing a DNA encoding an antisense RNA that targets a gene that is lethal by suppressing expression in E. coli is introduced into E. coli, and the antisense RNA is expressed to hold the vector. A counter selection method comprising killing E. coli and selecting E. coli that does not retain the vector.
[2] The counter selection method according to [1], wherein an expression-inducible promoter is linked to DNA encoding the antisense RNA, and the antisense RNA is expressed by expression induction.
[3] The counter selection method according to [1] or [2], wherein a gene that is lethal by suppressing expression is a gene essential for the growth of E. coli.
[4] The counter selection method according to [3], wherein the gene essential for the growth of E. coli is the fabI gene.
[5] The counter selection method according to [1] or [2], wherein the gene that is lethal by suppressing expression is an anti-toxic gene of Escherichia coli.
[6] The counter selection method according to [5], wherein the anti-toxic gene of E. coli is the mqsA gene or the rnlB gene.
[7] A vector that contains the DNA encoding an antisense RNA and is capable of expressing the antisense RNA, which is used for the counter selection of [1].
[8] The vector according to [7], wherein an expression-inducible promoter is linked to DNA encoding the antisense RNA, and the antisense RNA is expressed upon expression induction.
[9] The vector according to [7] or [8], wherein the antisense RNA targets an essential gene for Escherichia coli growth or an anti-toxic gene of Escherichia coli to suppress expression.
[10] The vector according to [9], wherein the gene essential for the growth of E. coli is the fabI gene, and the anti-toxic gene of E. coli is the mqsA gene or the rnlB gene.

  The establishment of a transformation method for E. coli and the presence of a wide variety of antibiotics and their resistance genes enabled various vectors to be introduced into cells easily and with high throughput, and recombinant DNA experiments were greatly developed. On the other hand, a technique for removing a once introduced vector from a cell is immature. In the era of synthetic biology, the genome has been directly modified. In order to characterize the characteristics of mutant strains bred by methods such as genome modification, it is necessary to introduce a vector into a host bacterium and to allow it to fall off freely. For example, when selecting a mutant strain in which the expression of a foreign gene encoded in the vector is improved by genome modification, the vector introduced from the host strain is dropped, the vector is introduced again, and the characteristics of the mutant strain are determined. It will be determined. The technology that selects only those that do not have the vector, that is, the counter selection technology is an indispensable technology for synthetic biology including genomic engineering, but the counter selection markers that can be used are limited. Is.

  In Escherichia coli most frequently used in recombination experiments, the sacB gene derived from Bacillus subtilis that is toxic in the presence of sucrose has been used as a counter selection marker. However, counter selection using the sacB gene is not suitable for liquid culture, and it has been possible to drop colonies once grown on a medium without sucrose on an agar medium containing sucrose. Thus, even the most frequently used sacB gene has disadvantages in terms of labor and efficiency.

It is a figure which shows the lethal effect of host Escherichia coli by a triclosan (0.2 micromol) and antisense RNA. It is a figure which shows the counter selection effect (pHSG299) by antisense RNA with respect to fabI gene. 2A shows the ratio of each drug-resistant cell present in the culture medium 5 hours after the start of counter selection, and FIG. 2B shows the ratio of each drug-resistant cell present in the culture medium 10 hours after the start of counter selection. Indicates the ratio of each drug-resistant cell present in the culture solution 15 hours after the start of counter selection. It is a figure which shows the counter selection effect (pHSG396) by antisense RNA with respect to fabI gene. FIG. 3A shows the ratio of each drug-resistant cell present in the culture medium 5 hours after the start of counter selection, and FIG. 3B shows the ratio of each drug-resistant cell present in the culture medium 10 hours after the start of counter selection. Indicates the ratio of each drug-resistant cell present in the culture solution 15 hours after the start of counter selection. It is a figure which shows the lethal effect of host Escherichia coli by the intracellular quantity of the antisense RNA with respect to a mqsA gene. It is a figure which shows the counter selection effect (pHSG299) by antisense RNA with respect to a mqsA gene. FIG. 5A shows the ratio of each drug resistant cell present in the culture medium 5 hours after the start of counter selection, and FIG. 5B shows the ratio of each drug resistant cell present in the culture medium 10 hours after the start of counter selection. Indicates the ratio of each drug-resistant cell present in the culture solution 15 hours after the start of counter selection. It is a figure which shows the counter selection effect (pHSG396) by antisense RNA with respect to a mqsA gene. 6A shows the ratio of each drug-resistant cell present in the culture medium 5 hours after the start of counter selection, and FIG. 6B shows the ratio of each drug-resistant cell present in the culture medium 10 hours after the start of counter selection. Indicates the ratio of each drug-resistant cell present in the culture solution 15 hours after the start of counter selection. It is a figure which shows the lethal effect of host Escherichia coli by the intracellular quantity of the antisense RNA with respect to rnlB gene. It is a figure which shows the counter selection effect (pHSG299) using antisense RNA with respect to rnlB gene. 8A shows the ratio of each drug-resistant cell present in the culture medium 5 hours after the start of counter selection, and FIG. 8B shows the ratio of each drug-resistant cell present in the culture medium 10 hours after the start of counter selection. Indicates the ratio of each drug-resistant cell present in the culture solution 15 hours after the start of counter selection. It is a figure which shows the counter selection effect (pHSG396) using antisense RNA with respect to rnlB gene. FIG. 9A shows the ratio of each drug resistant cell present in the culture medium 5 hours after the start of counter selection, and FIG. 9B shows the ratio of each drug resistant cell present in the culture medium 10 hours after the start of counter selection. Indicates the ratio of each drug-resistant cell present in the culture solution 15 hours after the start of counter selection. It is a figure which shows the counter selection effect in the cell in which a compatibility plasmid is coexpressed. It is a figure which shows the counter selection effect at the time of the combination of each plasmid DNA. The horizontal axis represents CFU when 50 μL of the bacterial solution after 15 hours of culturing from the start of counter selection is cultured on a plate (logarithmic graph). fabI × pHSG299 shows the result of counter selection in E. coli JM109 containing pHN1009-fabI and pHSG299, fabI × pHSG396 shows the result of counter selection in E. coli JM109 containing pHN1009-fabI and pHSG396, and mqsA × pHSG299 shows the result of pHN1009- The results of counter selection in E. coli JM109 containing mqsA and pHSG299, the results of counter selection in Escherichia coli JM109 containing pHN1009-mqsA and pHSG396, and rnlB × pHSG299 contain pHN1009-rnlB and pHSG299 The result of counter selection with E. coli JM109, rnlB × pHSG299 shows the result of counter selection with E. coli JM109 containing pHN1009-rnlB and pHSG396. FIGS. 12A, 12B, 12C, and 12D, which show the lethal effect of sucrose, show the effects when sucrose contains 0%, 1%, 5%, and 10%, respectively. It is a figure which shows suppression of the cell growth by expression of a sacB gene. FIGS. 13A, B, and C show the results when LB medium containing 0.01 mM, 0.1 mM, and 1.0 mM IPTG was used, respectively. It is a figure which shows 302 genes essential for the growth identified by colon_bacillus | E._coli K-12 MG1655 strain.

Hereinafter, the present invention will be described in detail.
The present invention kills a host cell (E. coli) holding a specific vector by counter selection, and selectively selects only host cells that are not naturally holding the specific vector. It is a way to survive. Counter selection refers to a method of using a nucleic acid that can induce cell death by expression, and selecting a cell that does not express the gene by dropping the nucleic acid. By the method of the present invention, a specific vector can be dropped and removed from the host cell population as a result. In this respect, the present invention can also be said to be a method for dropping a vector in a cell.

  In the counter selection method of the present invention, the vector to be dropped contains a nucleic acid that can suppress a gene that is lethal by suppressing expression in advance. Expression of an anti-toxic gene in a nucleic acid capable of suppressing the expression of a gene essential for the growth of a host cell, or a host cell toxic gene-anti-toxic gene system (TA system) as a lethal gene by suppressing the expression And nucleic acids that can suppress the above.

  A gene required for growth refers to a gene in which E. coli cannot grow without expression of the gene. When the gene is expressed in a conditional manner, such as by induction with a specific compound, the expression of a gene essential for the growth of the host cell is suppressed, and the host cell carrying the vector is killed.

  In addition, a nucleic acid capable of suppressing the expression of an antitoxic gene in the toxic gene-antitoxic gene system (TA system) of the host cell may be used in place of the nucleic acid that suppresses the expression of a gene essential for host cell growth. it can. By suppressing the expression of the antitoxin gene, the toxin gene acts in the host cell and the host cell is killed.

  The host cell that can be killed by the method using the counter selection of the present invention is not limited as long as it is a host cell into which a vector can be introduced in genetic engineering technology, and includes bacteria, fungi, animal cells, etc. Target cells are cells that die of host cells and have known genes essential for growth and antitoxin genes. In this respect, Escherichia coli has been analyzed for genes essential for growth and antitoxin genes, and the counter selection method of the present invention can be suitably applied.

  In the method of the present invention, a gene targeted by antisense RNA is called a counter selection marker.

  As an essential gene for E. coli expression, for example, in E. coli MG1655 strain (genome sequence: NC — 000913.2), it is reported that there are a total of 4,746 genes encoded in the genome. Of these genes, 302 genes are essential for growth. Examples of genes essential for the expression of E. coli MG1655 strain include databases Pec Profiling of E. coli Chromosome (http://www.shigen.nig.ac.jp/ecoli/pec/), Kato J. et al., Mol. Syst. Biol. (2007) 3: 132), Homma et al., Gene (2002) 294: 25-33) and the like can be selected. FIG. 14 shows 302 genes essential for growth identified in E. coli K-12 MG1655 strain. This is a gene identified in Escherichia coli K-12 MG1655 strain, but other strains may be selected from the same gene or a homologous gene. When the counter selection method of the present invention is applied to Escherichia coli, the 302 gene essential for growth or a homologous gene thereof is a gene whose expression should be suppressed. These genes are known to be essential for the growth of Escherichia coli, and it is obvious that Escherichia coli becomes lethal by suppressing the expression of genes essential for the growth. It can be predicted that any of the above 302 genes can be used as “a gene that is lethal by suppressing expression” in the present invention.

The Escherichia coli strain used in the present invention is not limited, but K-12 strain (NBRC 3301) and B strain (NBRC 13168), which are usually used for research and production of useful materials, or sub-strains derived from these strains. Can be suitably used. K-12 strain and B strain and their sub-strains are MG1655, W3110, W2637, W1485, WG1, 58, 679, MB408, AG1, Hfr3000, 5K, H1443, DP50, W945, PA309, 58-161, P678, HB101 (K-12 and B hybrid), XL1-Blue, XLOLR, SURE, YN2980, AB311, Hfr 3000 X74, Cavalli Hfr, W208, AB284, EMG2, Y10, WA704, JC9387, BB4, BL21, BM25.5, BMH71-18mutS, BW313, C-Ia, C600 Ltd., CJ236 strain, DH1 strain, DH5 strain, DH5α strain, DH10B strain, DP50supF stock, ED8654 shares, ED8767 shares, ER1647 strain, ER2508 strain, HB101 strain, HMS174 shares, HST02 shares, HMS174 shares, HST02 shares, HST04 dam ^- / dcm ^- strain, HST08 Premium strain, JM83 strain, JM101 strain, JM105 strain, JM106 strain, JM107 strain, JM108 strain, JM109 strain, JM110 strain, K802 shares, K803 shares, LE392 strain, MC1061 strain, MV1184 shares, MN1193 shares , NovaBlue, RR1, TAP90, TG1, TG2, TH2, χ1776, Y-1088, Y-1099, Y-1090, REL6 06 shares. Of these, MG1655, W3110, W2637, W1485, WG1, 58, 679, MB408, AG1, Hfr3000, 5K, H1443, DP50, W945, PA309, 58 -161, P678, HB101 (K-12 and B hybrid), XL1-Blue, XLOLR, SURE, YN2980, AB311, Hfr 3000 X74, Cavalli Hfr, W208, AB284, EMG2, Y10, WA704, JC9387, etc. are often used as strains derived from K-12, and REL606, BL21, etc. are often used as strains derived from B. Is a stock.
Among the genes essential for the growth of 302 Escherichia coli, for example, the fabI gene, the accA gene, the infC gene and the like can be suitably selected.

  In addition, as antitoxin genes in the toxic gene-antitoxic gene system (TA system), ccdAB gene, relBE gene, mqzEF gene, dinJ / yafQ gene, hipBA gene, hicAB gene, pas gene, yefM-yoeB gene, mqsRA gene, rnlAB A gene or the like can be suitably selected. This is a gene identified in Escherichia coli K-12 MG1655 strain, but other strains may be selected from the same gene or a homologous gene.

  Expression of a gene essential for host cell growth or an anti-toxin gene is suppressed by using an antisense RNA targeting a gene essential for host cell growth or an anti-toxin gene. That is, a nucleic acid that suppresses the expression of a gene essential for the growth of host cells, or a nucleic acid that suppresses the expression of an antitoxin gene, which is included in the vector to be dropped in advance, includes DNA encoding an antisense RNA. Use it. Antisense RNA is RNA consisting of a gene that is essential for the growth of the host cell, or a sequence complementary to a part of the DNA sequence of the antitoxin gene, and is a gene that is essential for the growth of the host cell or mRNA of the antitoxin gene. Binds complementarily, inhibits translation of the gene and suppresses expression. The antisense RNA is 10 to 400 nucleotides in length, preferably 10 to 250 nucleotides in length, more preferably 10 to 150 nucleotides in length, more preferably 15 to 150 nucleotides in length. Nucleotides, more preferably 50 to 150 nucleotides in length. The position where the antisense RNA binds complementarily is not limited, but a region where the silencing effect of gene expression appears strongly is preferable. As such a region, a region including a ribosome binding region (RBS) of the target gene and an initiation codon is preferable. In addition, it is desirable not to form a secondary structure in the antisense RNA molecule, and therefore it is desirable that there are no sequence portions complementary to each other in the antisense. Furthermore, it is desirable that the entire transcript containing the antisense RNA does not form a secondary structure. Various methods are known as methods for target sequences of antisense RNA, and appropriate sequences can be designed based on sequence information of genes whose expression is to be controlled.

  Antisense RNA transcribed from a vector containing DNA that encodes antisense RNA is degraded by RNA nucleases such as RNaseE in the cell, but both ends of transcripts containing antisense RNA form secondary structures. Thus, those having a structure that inhibits degradation from nucleases such as RNaseE are preferred.

  The base sequences of fabI gene, accA gene and infC gene, which are genes essential for the growth of E. coli, are shown in SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, respectively. Further, the gene base sequences of the antitoxin gene relB gene, mazE gene, dinJ gene, hipB gene, yefM gene, mqsA gene and rnlB gene, respectively, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 38.

  As examples of antisense RNA, antisense RNA that can be used for expression control of fabI gene, relB gene, mqzE gene, dinJ gene, hipB gene, yefM gene, mqsA gene, and rnlB gene are shown below. In the following, a DNA sequence (antisense DNA sequence) encoding an antisense RNA is shown. What is necessary is just to insert so that an antisense sequence may be transcribed from the following DNA sequence into the vector to be removed by the counter selection method of the present invention.

Antisense DNA region corresponding to fabI gene
GGTTGATTATAATAACCGTTTATCTGTTCGTACTGTTTACTAAAACGACGAATCGCCTGATTTTCAGGCACAACAAGCATCAACAATAAGGATTAAAGCTATGGGTTTTCTTTCCGGTAAGCGCATTCTGGTAACCGGTGTTGCCAGCAAACTATCCATCGCCTACGGTATCGCTCAGGCGATGCA (SEQ ID NO: 1)

Antisense DNA region corresponding to relB gene
GCGATACTTGTAATGACATTTGTAATTACAAGAGGTGTAAGACATGGGTAGCATTAACCTGCGTATTGACGATGAACTTAAAGCGCGTTCTTACG (SEQ ID NO: 18)


Antisense DNA region corresponding to mqzE gene
ATATACTGTATCTACATATGATAGCGGTTTGAGGAAAGGGTTATGATCCACAGTAGCGTAAAGCGTTGGGGAAATTCACCGGCGGTGCGGATCCCGGCTACGT (SEQ ID NO: 19)

Antisense DNA region corresponding to dinJ gene
CTACAATTCAAGCTGAATAAATATACAGCACAGGAGATACCCCAATGGCTGCTAACGCGTTTGTTCGCGCCCGAATCGATGAAGATCTGAAGAATCAGGCAGCGGACGTACTGG (SEQ ID NO: 20)

Antisense DNA region corresponding to hipB gene
GATAAAACTTATAATATCCCCTTAAGCGGATAAACTTGCTGTGGACGTATGACATGATGAGCTTTCAGAAGATCTATAGCCCAACGCAATTGGCGAATGCAATGA (SEQ ID NO: 21)

Antisense DNA region corresponding to hicB gene
CGCCAATTAAAAAGGTTAATGACATGCGAGAGACAGTCGAAATTATGCGTTATCCCGTCACTCTTACACCCGCGCCGGAAGGCGGTTATATGGTTTCTT (SEQ ID NO: 22)

Antisense DNA region corresponding to yefM gene
TAACGCTCATCATTGTACAATGAACTGTACAAAAGAGGAGATTGACATGCGTACAATTAGCTACAGCGAAGCGCGTCAGAATTTGTCGGCAACAATGATGAAAG (SEQ ID NO: 23)

Antisense DNA region corresponding to mqsA gene
ATTACGGTAATTCATGACGTACTGATCGTCTCGTTTAAGGAGAAGTAATATGAAATGTCCGGTTTGCCACCAGGGAGAAATGGTTTCTGGCATTAAAGATATT (SEQ ID NO: 24)

  In the counter selection of the present invention, the vector to be removed may contain the DNA encoding the antisense RNA. At this time, the expression of the antisense RNA may be conditionally expressed by, for example, induction with a specific compound. As a method for conditional expression of antisense RNA, a promoter induced by a specific substance may be used. IPTG (isopropyl-β-thiogalactopyranoside) -inducible lac promoter, tac promoter, trc promoter, xylose-inducible xylF promoter, xylA promoter, xylB promoter, arabinose-inducible promoter Examples thereof include araBAD promoter and araB promoter. The promoter may be operably linked to the DNA encoding the antisense RNA in the vector to be removed.

  The expression vector to be used includes a promoter, DNA encoding the above-mentioned antisense RNA, a terminator, a cis element such as an enhancer, if necessary, a splice donor site present on the 5 ′ end side of the intron, and a 3 ′ end side of the intron. A splicing signal consisting of an existing splice accepting site, a poly A addition signal, a drug resistance selection marker, a ribosome binding sequence (SD sequence) and the like may be operably linked. Examples of drug resistance selection markers include ampicillin resistance genes and kanamycin resistance genes.

  Further, without using a vector capable of expressing antisense RNA, antisense RNA may be added from outside the cell at the time when a specific vector is desired to be dropped and taken into the host cell. In this case, for example, antisense RNA may be chemically synthesized and added.

  Furthermore, PNA (peptide nucleic acid) or dsRNA (double stranded RNA) that can suppress a lethal gene by suppressing expression may be used instead of antisense RNA. dsRNA cleaves the mRNA of a gene that is lethal by RNA interference (RNAi) and suppresses the expression of the gene. dsRNA molecules include siRNA molecules and shRNA molecules, and can also form miRNA molecules. When dsRNA is used, DNA encoding dsRNA may be included in the vector to be removed.

  In the present invention, for example, a plasmid containing DNA encoding an antisense RNA for a certain gene is indicated as pH1009-fabI when the gene is a fabI gene and the plasmid is pH1009.

  When the host cell contains a vector other than the vector containing the DNA encoding the antisense RNA, the vector containing the DNA encoding the antisense RNA is dropped by the counter selection method of the present invention, The vector can be recovered. In this case, the vector containing the antisense RNA and the other vector are preferably incompatible. Other vectors include, but are not limited to, pHSG299, pHSG396, pUC18, pUC19, pJexpress404 and the like.

  The method for introducing a vector into a host cell is not limited, and for example, a method using calcium ions [Cohen, SNet al .: Proc. Natl. Acad. Sci., USA, 69: 2110 (1972)], electroporation Law. The vector to be used is not limited, and known expression vectors such as plasmid DNA and phage DNA can be used.

  Host cell culture can be performed according to a known method. For example, DMEM, MEM, RPMI1640, IMDM, or the like can be used as a culture solution, and a serum supplement such as fetal calf serum (FCS), other supplement reagents, or the like may be added.

  The culture temperature when performing the counter-selection of the present invention is not limited, but is 30 to 40 ° C, preferably 35 to 40 ° C, more preferably 37 ° C.

  The counter selection method of the present invention can be performed by liquid culture, but can also be performed by solid culture such as plate culture using an agar medium or the like.

  The present invention targets the genes essential for host cell growth and the anti-toxic genes in the host cell toxic gene-anti-toxic gene system (TA system), and encodes the above-mentioned antisense RNA capable of suppressing the expression thereof. Contains DNA and vector for use in counter selection. The vector may contain an expression-inducible promoter operably linked to DNA encoding the antisense RNA in order to induce and express the antisense RNA. Furthermore, the expression vector includes a promoter, DNA encoding the above-mentioned antisense RNA, a terminator, a cis element such as an enhancer, if necessary, a splice donor site present on the 5 ′ end side of the intron, and the 3 ′ end side of the intron. A splicing signal consisting of a splice acceptor site present in, a poly A addition signal, a drug resistance selection marker, a ribosome binding sequence (SD sequence) and the like may be operably linked.

  The present invention includes a counter selection marker comprising a gene for use in counter selection. Examples of the counter selection marker include genes that cause the host cell to become lethal when expressed, and include genes essential for host cell growth and host cell toxic genes-antitoxic genes in the antitoxic gene system (TA system) It is. Specifically, for example, fabI gene, accA gene, infC gene etc. are mentioned as essential genes for the growth of E. coli, and ccdAB gene, relBE gene as antitoxin genes in toxic gene-antitoxic gene system (TA system) , MazEF gene, dinJ / yafQ gene, hipBA gene, hicAB gene, pas gene, yefM-yoeB gene, mqsRA gene, rnlAB gene and the like.

  Examples of the present invention will be described below, but the examples given here are merely specific examples and do not limit the technical scope of the present invention.

Example 1 Counter selection by using antisense RNA with E. coli essential gene and fabI gene as target gene (1) Construction of expression plasmid encoding antisense RNA with fabI gene as target gene Antisense RNA expression plasmid as PHN1009 constructed by Nakajima et al. Was used (Nobutaka Nakashima et al., Nucleic Acids Research, 2006; Nobutaka Nakashima et al., Nucleic Acids Research, 2009). Here, a plasmid DNA encoding an antisense RNA for the fabI gene, fabI-PT7 asRNA plasmid (referred to as pHN1009-fabI in the present application) was used for transformation of JM109 (Nobutaka Nakashima et al., Nucleic Acids Research, 2006; Nobutaka Nakashima et al., Nucleic Acids Research, 2009). Plasmid DNA was purified from the transformant. For PCR amplification of the RNA region for the fabI gene, primers of SEQ ID NO: 39 (sSN90) and SEQ ID NO: 40 (sSN91) were used as oligonucleotide primers.

(2) Silencing effect of antisense RNA against fabI gene
pHN1009-fabI Observe cell death due to transcription induction of antisense RNA of E. coli strain JM109 containing only one kind of plasmid DNA. The intracellular concentration of antisense RNA can be adjusted by the concentration of isopropyl-β-thiogalactopyranoside (IPTG). Therefore, cell proliferation according to the amount of IPTG added was observed according to the following procedure. Triclosan functions as an inhibitor for the synthesis of FabI. From this, it is known that the expression suppression of fabI by antisense RNA is higher when a low concentration of triclosan is added to the medium than when it is not added. Therefore, triclosan was added under each condition.

First, E. coli JM109 was transformed with pHN1009-fabI. Transformant colonies that grew on the agar plate were inoculated into 2 mL LB medium (50 μg / mL ampicillin) and cultured at 37 ° C. for 17 hours. Thereafter, the precultured bacterial solution was mixed at 1: 10000 in LB medium (50 μg / mL ampicillin) of triclosan 0.2 μM and IPTG 0, 0.1, 0.5, 1.0 mM, and these were dispensed 200 μL each on a microplate. Then, the cells were cultured at 37 ° C. for 15 hours, and the growth turbidity (OD ₆₀₀ ) was measured every 15 minutes.

  As a result, a difference of about 8 hours was observed in the time to start proliferation irrespective of the concentration of IPTG used for the induction of transcription between the case where the transcription of antisense RNA was induced and the case where it was not (FIG. 1). ). This means that growth was suppressed by the expression of antisense RNA. However, it was not completely killed.

(3) Counterselection with antisense RNA for fabI gene In this example, the removal efficiency was quantitatively evaluated by selectively removing plasmids containing antisense DNA from Escherichia coli holding plural types of plasmids. The JM109 strain is used as the E. coli host, and pHN1009-fabI is used as the plasmid containing the antisense DNA. In addition, pHSG299 (Takara Bio Inc., kanamycin resistant) or pHSG396 (Takara Bio Inc., chloramphenicol resistant) was allowed to coexist with pHN1009-fabI-carrying bacteria.

First, Escherichia coli JM109 strain was transformed with pHN1009-fabI, and this recombinant JM109 was made into competent cells according to a standard method (Hanahan method). As a result of transformation of this competent cell with pHSG299 or pHSG396, an efficiency of 1 × 10 ⁶ CFU / μL was obtained when pHSG299 was used, and 1 × 10 ⁵ CFU / μL was obtained with pHSG396.

  100 μL of this competent cell and 40 ng of pHSG299 were mixed and allowed to stand on ice for 15 minutes. Thereafter, a heat shock was applied at 42 ° C. for 45 seconds, and the mixture was allowed to stand again on ice for 1 minute. 1 mL of SOC medium was added thereto, and cultured with shaking at 37 ° C. for 45 minutes. Next, 500 μL of 8 media, (1) LB Lennox, (2) LB Lennox, 0.5 mM IPTG, (3) LB Lennox, kanamycin 25 μg / ml, (4) LB Lennox, kanamycin 25 μg / ml, 0.5 mM IPTG, (5) LB Lennox, 0.2 μM triclosan, (6) LB Lennox, 0.2 μM triclosan, 0.5 mM IPTG, (7) LB Lennox, 0.2 μM triclosan, 25 μg / ml kanamycin, (8) LB Lennox, 0.2 μM triclosan 50 μL of the bacterial solution was inoculated in 25 μg / ml kanamycin, 0.5 mM IPTG, and cultured at 37 ° C. with shaking. After 5 hours, 10 hours, and 15 hours, the ratio of the two plasmids, pHN1009-fabI and pHSG299 or pHSG396, in the Escherichia coli cells, and the culture broth were added to the ampicillin plate (LB), a resistance drug for pHN1009-fabI. Lennox, 50μg / ml ampicillin) and the kanamycin plate that is resistant to pHSG299 or the chloramphenicol plate that is resistant to pHSG396, and count the number of colonies that appeared after overnight culture at 37 ° C. did.

  As a result, when neither drug nor IPTG is added (LB), only IPTG is added, only triclosan is added, and when triclosan and IPTG are added, the plasmid in E. coli JM109 is not affected even if cultured for 15 hours. All were found to be pHN1009-fabI (Figure 2, C). In addition, when only kanamycin was added and when triclosan and kanamycin were added, the intracellular plasmid DNA abundance at the time of culturing for 15 hours was equivalent to pHN1009-fabI and pHSG299 (FIG. 2, C). On the other hand, when kanamycin and IPTG were added, and when triclosan, kanamycin, and IPTG were added, the cells were 100% pHSG299 when cultured for 15 hours (Fig. 2, C). It was also found that pHSG299 occupies most of these conditions even when cultured for 5 hours and 10 hours (FIGS. 2, A and B). That is, by applying the above conditions, pHN1009-fabI could be selectively removed.

  When pHN396 is used (antibiotic concentration is 34 μg / mL), it takes a long time for the lethal effect to appear, but the same conditions as for pHN299 (when adding chloramphenicol and IPTG and triclosan) When chloramphenicol and IPTG were added), the presence of only pHN396 and the loss of pHN1009-fabI were confirmed after 15 hours of culture (FIG. 3, C).

  From the above, it was confirmed that selective counter selection can be performed even in a liquid culture system by using an antisense RNA against an essential gene of Escherichia coli and the fabI gene.

Example 2 Selection of Antitoxic Gene Encoded in E. coli Genome Targeting Antisense RNA (1) Design of Antisense RNA Targeting Antitoxic Gene
relB (Van Melderen et al., Mol. Microbiol. 11, 1151-1157, 1994), mazE (Christensen, SK et al., Proc. Natl. Acad. Sci. USA 98, 14328-14333, 2001), dinJ ( Christensen, SK et al., J. Mol. Biol. 332, 809-819. 2003), hipB (Prysak, MH et al., Mol. Microbiol. 71, 1071-1087. 2009), hicB (Hansen, S. et al., PLoS One 7 (6), e39185.), yefM (Smith, ASG et al., J. Bacteriol. 180, 5458-5462. 1998), mqsA (Christensen, SK et al., Mol. Microbiol. 51, 1705-1717. 2004), rnlB (Wang, X. et al., Nat. Chem. Biol. 7, 359-366. 2011) are all encoded in the genome of E. coli strain MG1655 (NC_000913.2). Is an anti-toxic gene. Antisense RNAs that suppress the expression of these genes were designed respectively. The designed antisense RNA contains the RBS (ribosome binding region) and start codon of these genes, and has a total length of about 100 bp.

(2) Preparation of Antisense RNA Targeting Antitoxic Genes Using the genome of Escherichia coli MG1655 strain (NC — 000913.2) as a template, a region that becomes antisense RNA was extracted by PCR amplification. The primer used for the reaction has an XhoI restriction enzyme cleavage site added to the forward primer and an NcoI restriction enzyme cleavage site added to the reverse primer, so these restriction enzyme cleavage sites are added to both ends of the amplified product. ing. For PCR amplification of antisense RNA regions corresponding to relB, mqzE, dinJ, hipB, hicB, yefM, mqsA, and rnlB genes, as oligonucleotide primers, SEQ ID NO: 2 (relB-R-NcoI), 3 (relB-F -XhoI), 4 (mazE-R-NcoI), 5 (mazE-F-XhoI), 6 (dinJ-R-Nco), 7 (dinJ-F-XhoI), 8 (hipB-R-NcoI), 9 (HipB-F-XhoI), 10 (hicB-R-NcoI), 11 (hicB-F-XhoI), 12 (yefM-R-NcoI), 13 (yefM-F-XhoI), 14 (mqsA-R- NcoI), 15 (mqsA-F-XhoI), 16 (rnlB-R-NcoI), and 17 (rnlB-F-XhoI) were used. The reaction solution (total volume 25 μL) was 1x PCR Buffer for KOD Neo (Toyobo), 0.2 mM dATP, dGTP, dCTP, dTTP (above, Toyobo), 25 pmol primer, 0.5 μL genomic DNA (about 10 ng), 0.5 unit KOD Neo (manufactured by Toyobo). This solution was kept at 98 ° C. for 2 minutes, and then a temperature cycle of 98 ° C. for 10 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 10 seconds was repeated 25 times, and finally the solution was kept at 68 ° C. for 2 minutes. The obtained PCR products were separated by agarose gel electrophoresis, purified according to the manual using a DNA purification kit (NucleoSpin Extract II) manufactured by Mach Liner Gel, and eluted with 30 μL of water. As a result, as the antisense DNA, SEQ ID NOs: 18 (antisense DNA region corresponding to the relB gene), 19 (antisense DNA region corresponding to the mqzE gene), 20 (antisense DNA region corresponding to the dinJ gene), 21 (Antisense DNA region corresponding to hipB gene), 22 (Antisense DNA region corresponding to hicB gene), 23 (Antisense DNA region corresponding to yefM gene), 24 (Antisense DNA region corresponding to mqsA gene) 25 (an antisense DNA region corresponding to the rnlB gene).

(3) Restriction enzyme treatment of PCR-amplified fragment of antisense RNA region 3 μL NEB Buffer 4 (New England Biolabs), 5 units NcoI (New England Biolabs), 5 A unit XhoI (manufactured by New England Biolabs) was added and incubated at 37 ° C. for 2 hours. Then, it refine | purified according to the manual using the DNA purification kit (NucleoSpin Extract II) made from Mach liner gel, and eluted with 20 microliters water.

(4) Restriction enzyme treatment of antisense RNA expression plasmid As in Example 1, pHN1009 is used as an antisense RNA expression plasmid (Nobutaka Nakashima et al., Nucleic Acids Research, 2006; Nobutaka Nakashima et al., Nucleic Acids). Research, 2009). Cloning of antisense DNA is performed with restriction enzymes NcoI and XhoI. For the restriction enzyme reaction solution, plasmid DNA, pHN1009 40 ng (5 μL), 2 μL NEB Buffer 4 (New England Biolabs), 5 units NcoI (New England Biolabs), 5 units XhoI (New England Biolabs) In addition, sterilized water was added so that the total volume was 20 μL. The reaction solution was incubated at 37 ° C. for 5 hours, then purified according to the manual using a DNA purification kit (NucleoSpin Extract II) manufactured by Mach Liner Gel, and eluted with 20 μL of water.

(5) Cloning of antisense DNA to pHN1009 Ligation of the DNA fragment obtained in (3) and the linear plasmid DNA obtained in (4) is performed. 2 μL T4 DNA Ligase Buffer (manufactured by New England Biolabs), 5 μL antisense DNA solution, 5 μL pHN1009 solution, 400 units T4 DNA Ligase, and sterilized water were added to the reaction solution to make a total volume of 20 μL. The reaction solution was incubated at room temperature for 1 hour, and a part thereof was used to transform E. coli JM109. Select colonies on an LB plate containing 50 μg / ml ampicillin, liquid culture in LB medium containing 50 μg / ml ampicillin, add glycerose stock (sterilized glycerol to a final concentration of 20%, freeze at −80 ° C.) The plasmid was purified from a portion of the culture and eluted with 20 μL of water. Thus, Escherichia coli JM109 strain expressing an antisense expression plasmid containing an antisense DNA for each anti-toxic gene was obtained.

(6) Verification of silencing effect The silencing effect of E. coli JM109 strain obtained in (5) by inducing antisense RNA expression is observed.
First, 2 mL LB medium (50 μg / mL ampicillin) was inoculated from the glycerose stock prepared in (5) and cultured at 37 ° C. for 17 hours. Thereafter, the precultured bacterial solution was mixed at 1: 10000 in 1000 μl of IPTG 0 mM and 1.0 mM LB medium (50 μg / mL ampicillin) and cultured for 17 hours. As a result, particularly remarkable silencing effect was observed when antisense RNA against mqsA gene was used (Table 1). In addition to mqsA, an antisense RNA corresponding to rnlB also showed a slight growth inhibitory effect (Table 1).

Example 3 Counterselection by using antisense RNA targeting mqsA gene, antitoxic gene encoded in E. coli genome (1) Silencing effect of antisense RNA against mqsA gene First, (5 ) Was inoculated into 2 mL LB medium (50 μg / mL ampicillin) and cultured at 37 ° C. for 17 hours. Thereafter, the precultured bacterial solution was mixed at 1: 10000 with LB medium (50 μg / mL ampicillin) of IPTG 0, 0.1, 0.5, 1.0 mM, and 200 μL of these were dispensed on a microplate. Thereafter, the cells were cultured at 37 ° C. for 22 hours, and the growth turbidity (OD ₆₀₀ ) was measured every 15 minutes. As a result, it was found that the transcription of antisense RNA by addition of IPTG to the culture broth inhibited cell growth and did not proliferate even after 15 hours of culture (FIG. 4).

(2) Counter selection with antisense RNA for mqsA gene E. coli JM109 strain was used as an E. coli host. PHN1009-mqsA is used as the plasmid to be removed. In order to quantitatively evaluate the drop-off efficiency, pHSG299 (Takara Bio, Kanamycin resistance) and pHSG299 (Takara Bio, Chloramphenicol resistance) were retained together with pHN1009-mqsA.

First, E. coli JM109 was transformed with pHN1009-mqsA, and competent cells of this transformant were prepared by the Hanahan method. When transformation efficiency was measured, it was calculated to be 1 × 10 ⁶ CFU / μL using pHSG299 and 1 × 10 ⁵ CFU / μL using pHSG396. 100 μL of competent cells and 40 ng of pHSG299 were mixed and left on ice for 15 minutes. Thereafter, a heat shock was applied at 42 ° C. for 45 seconds, and the mixture was again left on ice for 1 minute. 1 mL of SOC medium was added thereto, followed by shaking culture at 37 ° C. for 45 minutes. Then, 500 μL of 4 types of media, (1) LB Lennox, (2) LB Lennox, 0.5 mM IPTG, (3) LB Lennox, 25 μg / ml kanamycin (for pHSG299) or 34 μg / mL chloramphenicol (pHSG396 (4) LB Lennox, 25 μg / ml kanamycin (for pHSG299) or 34 μg / mL chloramphenicol (for pHSG396), 50 μL each in 0.5 mM IPTG, and cultured at 37 ° C. with shaking. After 5 hours, 10 hours, and 15 hours, the ratio of two plasmids, pHN1009-mqsA and pHSG299 or pHSG396, in the Escherichia coli cells, and the culture broth were mixed with ampicillin plates (LB Lennox, LB Lennox, 50μg / ml ampicillin) and the same amount on the kanamycin plate that is resistant to pHSG299 or the chloramphenicol plate that is resistant to pHSG396, and estimated by the number of colonies that emerged overnight at 37 ° C. .

  When pHSG299 was used, neither the selection with antibiotics nor the induction of antisense RNA expression with IPTG revealed that the plasmids in E. coli JM109 after 15 hours of culture were all pHN1009 (FIG. 5, C). . Moreover, the same result was obtained even in a medium containing only IPTG (FIG. 5, C). On the other hand, in the state selected with antibiotics, pHSG299 was 50% after 10 hours of culture (Fig. 5, B), but pHN1009-mqsA could be completely removed from the cells even if the culture time was extended. (FIG. 5, C). When IPTG was added after selection by the drug, the intracellular abundance of pHSG299 was about 90% at the time of 10-hour culture (Fig. 5, B), and when further cultivation was continued for 5 hours. pHN1009 was completely removed from the cells (FIG. 5, C).

  In addition, when pHSG396 is used, the concentration effect appears a little late, but under the same conditions as in pHSG299 (when antibiotics and IPTG are added), counter-selection is effective during 15 hours of culture. Results were obtained (Figure 6, C).

  As described above, it was confirmed that the counter selection can be efficiently performed in the liquid medium by using the antisense RNA (SEQ ID NO: 4) against the mqsA gene which is an anti-toxic gene of Escherichia coli.

Example 4 Counter-selection by using antisense RNA that targets rnlB gene, an antitoxic gene encoded in the E. coli genome (1) Design of antisense RNA for rnlB gene In Example 2, antisense to rnlB gene The expression silencing effect of RNA (SEQ ID NO: 25) was weakly observed. The silencing effect may be due to the unknown complementary mechanism that accompanies a decrease in the expression level of the target gene, but in many cases, the relationship between the transcription amount of the target gene and antisense RNA, and their half-life It is considered that the relationship between the structure and the formation of higher-order structures in the cell greatly influence. Therefore, the design was redesigned so that the antisense RNA does not form a secondary structure in the cell. It is considered that the formation of a higher-order structure of antisense RNA decreases the efficiency of hybridization to the target gene mRNA and ultimately causes a decrease in the silencing effect. The basic antisense RNA region design conditions were the same as in Example 2, and the region was about 100 bp including RBS and the start codon. Secondary structure prediction was performed at CentroidFold (http://www.ncrna.org/centroidfold).

(2) Preparation of antisense RNA for rnlB gene Using the genome of E. coli MG1655 strain (NC_000913.2) as a template, oligonucleotides, rnlB-R-NcoI (SEQ ID NO: 26) and rnlB-F-XhoI (SEQ ID NO: 27) ( The region of the rnlB gene was amplified by the polymerase chain reaction (PCR) method using Sigma-Aldrich (manufactured by Sigma-Aldrich) as a primer. NcoI and XhoI restriction enzyme sites are added to these primers, respectively. The reaction solution (total volume 25 μL) was 1x PCR Buffer for KOD Neo (Toyobo), 0.2 mM dATP, dGTP, dCTP, dTTP (above, Toyobo), 25 pmol primer, 0.5 μL genomic DNA (about 10 ng), 0.5 unit KOD Neo (manufactured by Toyobo). This solution was kept at 98 ° C. for 2 minutes, and then a temperature cycle of 98 ° C. for 10 seconds, 53 ° C. for 30 seconds, and 68 ° C. for 10 seconds was repeated 25 times, and finally the solution was kept at 68 ° C. for 2 minutes. The obtained PCR products were separated by agarose gel electrophoresis, purified according to the manual using a DNA purification kit (NucleoSpin Extract II) manufactured by Mach Liner Gel, and eluted with 30 μL of water. In this way, a fragment containing an antisense DNA serving as an antisense RNA for the rnlB gene was obtained. The antisense DNA sequence is SEQ ID NO: 28 (rnlB-antisense RNA).

(3) Restriction enzyme treatment of rnlB gene translation initiation region fragment In the DNA solution obtained in (2), 3 μL NEB Buffer 4 (New England Biolabs), 5 units NcoI (manufactured by New England Biolabs), 5 units XhoI (New England Bionlabs) was added and incubated at 37 ° C. for 2 hours. Then, it refine | purified according to the manual using the DNA purification kit (NucleoSpin Extract II) made from Mach liner gel, and eluted with 20 microliters water.

(4) Restriction enzyme treatment of antisense RNA expression plasmid As in Example 1, pHN1009 is used as an antisense RNA expression plasmid (Nobutaka Nakashima et al., Nucleic Acids Research, 2006; Nobutaka Nakashima et al., Nucleic Acids). Research, 2009). Cloning of antisense DNA is performed with restriction enzymes NcoI and XhoI. Restriction enzyme reaction solutions were plasmid DNA, pHN1009 400 ng (5 μL), 2 μL NEB Buffer 4 (New England Biolabs), 5 units NcoI (New England Biolabs), 5 units XhoI (New England Bionlabs), Sterile water was added to make a total volume of 20 μL. And it kept at 37 degreeC for 5 hours. Then, it refine | purified according to the manual using the DNA purification kit (NucleoSpin Extract II) made from Mach liner gel, and eluted with 20 microliters water.

(5) Cloning of antisense DNA for rnlB gene into pHN1009 Ligation of the antisense DNA fragment obtained in (3) and the linear plasmid DNA obtained in (4) is performed. 2 μL T4 DNA Ligase Buffer (manufactured by New England Biolabs), 5 μL antisense DNA solution, 5 μL pHN1009 solution, 400 units T4 DNA Ligase, and sterilized water were added to the reaction solution to make a total volume of 20 μL, and kept at room temperature for 1 hour. The reaction product was introduced into Escherichia coli JM109 (TaKaRa), selected on an LB plate containing 50 μg / ml ampicillin, a glycerose stock was prepared from the transformant, and the plasmid was purified therefrom, and 20 μL of water was added. Eluted with. Thus, an antisense expression plasmid, pHN1009-rnlB, encoding an antisense DNA against rnlB was obtained.

(6) Silencing effect of antisense RNA against rnlB gene First, 2 mL of LB medium (50 μg / mL ampicillin) was inoculated from the glycerose stock prepared in (5) and cultured at 37 ° C. for 17 hours. Thereafter, the precultured bacterial solution was mixed at 1: 10000 in an LB medium (50 μg / mL ampicillin) having an IPTG concentration of 0, 0.1, 0.5, 1.0 mM, and 200 μL each was dispensed onto a microplate. Thereafter, the cells were cultured at 37 ° C. for 22 hours, and the growth turbidity (OD ₆₀₀ ) was measured every 15 minutes. As a result, when the expression of antisense RNA was induced by the addition of IPTG, cell growth was inhibited, and a delay of about 10 hours was observed in the growth start time compared to the case where no addition was made (FIG. 7).

(7) Counter selection with antisense RNA for rnlB gene E. coli JM109 strain was used as the E. coli host. PHN1009-rnlB is used as the plasmid to be removed. In order to measure the drop-off efficiency, pHSG299 and pHSG299 were retained together with pHN1009-mqsA.

First, E. coli JM109 strain was transformed with pHN1009-rnlB. Furthermore, competent cells of this transformant were prepared by the Hanahan method. When transformation efficiency was measured, it was calculated to be 1 × 10 ⁶ CFU / μL using pHSG299 and 1 × 10 ⁵ CFU / μL using pHSG396. 100 μL of competent cells and 40 ng of pHSG299 were mixed and left on ice for 15 minutes. Thereafter, a heat shock was applied at 42 ° C. for 45 seconds, and the mixture was again left on ice for 1 minute. 1 mL of SOC medium was added thereto, followed by shaking culture at 37 ° C. for 45 minutes. Then, 500 μL of 4 types of media, (1) LB Lennox, (2) LB Lennox, 0.5 mM IPTG, (3) LB Lennox, 25 μg / mL kanamycin (for pHSG299) or 34 μg / mL chloramphenicol (pHSG396) (4) LB Lennox, 25 μg / mL kanamycin (for pHSG299) or 34 μg / mL chloramphenicol (for pHSG396), 50 μL each in 0.5 mM IPTG, and cultured at 37 ° C. with shaking. The pHN1009-rnlB and pHSG299 or pHSG396 two plasmid ratios in E. coli cells after 5 hours, 10 hours, and 15 hours, and the culture broth were added to the ampicillin plate (LB Lennox, LB Lennox, 50μg / ml ampicillin) and the same amount on a kanamycin plate that is resistant to pHSG299 or chloramphenicol that is resistant to pHSG396, and estimated by the number of colonies that emerged overnight at 37 ° C. .

  In the case of coexisting with pHSG299, when neither antibiotic (kanamycin) nor IPTG was added, or when only IPTG was added, it was found that all the plasmids in E. coli JM109 were pHN1009 even after culturing for 15 hours (FIG. 8, C ). In addition, when selection with kanamycin was performed, pHSG299 was 50% after 15 hours of culture (FIG. 8, C). On the other hand, when IPTG was further added after selection with kanamycin, the intracellular abundance of pHSG299 was already 100% after 10 hours of culture (FIG. 8, B), and for another 5 hours. It was found that pHN1009 was completely removed from the cells when the culture was continued (FIG. 8, C).

  In addition, when pHSG396 is used, the concentration effect appears slightly later, but under the same conditions as in pHSG299 (when drugs and IPTG are added), plasmid DNA selection was successful during 15 hours of culture. (Fig. 9, C).

  As described above, it was confirmed that counter selection can be efficiently performed in a liquid medium by using an antisense RNA (SEQ ID NO: 7) against the rnlB gene, which is an antitoxic gene of Escherichia coli.

Example 5 Counterselection effect with compatible plasmid by this method (1) Counterselection with pKn1009-mqsA and compatible pBAD24 (p15A origin) E. coli strain JM109 was used as an E. coli host. PKn1009-mqsA is used as the plasmid to be removed. This plasmid is obtained by replacing the ampicillin resistance gene of pHN1009-mqsA with a kanamycin resistance gene. In order to measure the shedding efficiency, pBAD24 (manufactured by Takara Bio Inc., ampicillin resistance) was retained together with pKn1009-mqsA.

First, E. coli JM109 was transformed with pKn1009-mqsA, and competent cells of this transformant were prepared by the Hanahan method. When the transformation efficiency was measured, it was calculated to be 1 × 10 ⁶ CFU / μL when using pHSG299. 100 μL of competent cells and 40 ng of pBAD24 were mixed and left on ice for 15 minutes. Thereafter, a heat shock was applied at 42 ° C. for 45 seconds, and the mixture was again left on ice for 1 minute. 1 mL of SOC medium was added thereto, followed by shaking culture at 37 ° C. for 45 minutes. Then, 500 μL of 4 types of media, (1) LB Lennox, (2) LB Lennox, 0.5 mM IPTG, (3) LB Lennox, 100 μg / ml ampicillin, (4) LB Lennox, 100 μg / ml ampicillin, 0.5 mM IPTG 50 μL each was inoculated and cultured at 37 ° C. with shaking. The ratio of pKn1009-mqsA and pBAD24 in E. coli cells after 5 hours, 10 hours, and 15 hours, the culture broth and kanamycin plate (LB Lennox, 25 μg / ml kanamycin), which is a pKn1009-mqsA resistant drug, The same amount was plated on an ampicillin plate (LB Lennox, 100 μg / ml ampicillin), which is a pBAD24 resistant drug, and the number of colonies that appeared after overnight culture at 37 ° C. was estimated.

As a result, in the mixed state of two types of plasmids, when the selection with antibiotics did not induce the expression of antisense RNA with IPTG, and when only IPTG was added and antisense RNA was not selected under the conditions with antibiotics When expressed, all the plasmids in E. coli JM109 after 15 hours of culture were found to be pKn1009 (FIG. 10). On the other hand, in the state selected with antibiotics, the intracellular abundance of the two plasmids was equal after 15 hours of culture (FIG. 10). When IPTG was added after selection by the drug, the intracellular abundance of pKn1009-mqsA was about 10 ⁻⁸ after 15 hours of culture (FIG. 10).

  From the above, it has been clarified that in this method, cells expressing only an arbitrary plasmid can be selected even if cells containing a compatible plasmid are mixed.

Example 6 Comparison of Counterselection Effect of Each Antisense RNA (1) Counterselection E. coli JM109 strain was used as the E. coli host. As plasmids to be removed, pHN1009-fabI, pHN1009-mqsA and pHN1009-rnlB were used, respectively. PHSG299 and pHSG396 were used as plasmids to coexist in E. coli.

100 μL of competent cells of E. coli JM109 strain each containing pHN1009-fabI, pHN1009-mqsA and pHN1009-rnlB were mixed with 40 ng of pHSG299 or pHSG396 and left on ice for 15 minutes. Thereafter, a heat shock was applied at 42 ° C. for 45 seconds, and the mixture was again left on ice for 1 minute. 1 mL of SOC medium was added thereto, followed by shaking culture at 37 ° C. for 45 minutes. Thereafter, 200 μL each of inoculated in 2 mL of medium, LB Lennox, kanamycin 25 μg / mL or chloramphenicol 34 μg / mL, 0.5 mM IPTG in a test tube, and cultured at 37 ° C. with shaking. × 1 bacterial solution after 15 ^{hours, × 10 -1, × 10 -2} , × 10 -3, × 10 -4, × 10 -5, × 10 -6, × 10 -7, × 10 -8, Dilute with LB medium to × 10 ^-9 , × 10 ^-10 , × 10 ^-11 , × 10 ^-12 , × 10 ^-13 , × 10 ^-14 and add 50 μl each to ampicillin plate and kanamycin plate or chloramphenicol plate The seedlings were cultured at 37 ° C. At this time, considering the quantitativeness of the data, the same amount was spread on three plates for each condition. After culturing for 17 hours, a petri dish having a dilution ratio in which colonies were well separated was selected from each condition, and the colonies were counted. Then, the number of colonies in 50 μL of the original bacterial solution was calculated.

  These results are shown in FIG. When pHN1009-fabI and pHSG299 were used, it was found that the pHSG299 contained about 10,000,000 times more than pHN1009-fabI in the bacterial solution 15 hours after the start of the counter selection. Further, in the case of pHSG396, it was found that pHSG396 was contained about 10,000 times more than pHN1009-fabI. When pHN1009-mqsA and pHSG299 were used, it was found that pHSG299 was contained approximately 100,000,000 times more than pHN1009-mqsA. In the case of pHSG396, it was found that pHSG396 was contained about 100,000 times more than pHN1009-mqsA. When pHN1009-rnlB and pHSG299 were used, it was found that pHSG299 was contained about 10,000,000 times more than pHN1009-rnlB. Furthermore, even in the case of pHSG396, pHSG396 was contained about 10,000,000 times more than pHN1009-rnlB.

  From these facts, ampicillin resistant clones derived from pHN1009 were slightly mixed even after counter selection depending on the type of antisense RNA, but the contamination rate was at most about 1 / 10,000 even at the highest. Thus, the present invention showed remarkably high efficiency compared to the conventional method, such as operability and convenience of counter selection by liquid culture, as well as reduction of background and improvement of throughput.

Example 7 Comparison of counter selection efficiency using the method and sacB gene (1) Lethal effect
The sacB gene was cloned into an expression plasmid, pTD-tac plasmid. E. coli strain JM109 was transformed with the pTD-tac plasmid and the plasmid cloned with the sacB gene. Transformants were stored at −80 ° C. as glycerol stocks. Here, the conditions under which cells containing the sacB gene are lethal are verified. The lethal effect of sucrose was observed. A pre-cultured bacterial solution was mixed at 1: 10000 in a medium containing sucrose 0%, 1%, 5%, and 10% in LB medium (ampicillin 50 μg / ml, 2 ml), and 200 μL was dispensed onto a microplate. Then, the cells were cultured at 37 ° C. for 15 hours, and the growth turbidity (OD ₆₀₀ ) was measured every 15 minutes. As a result, when grown in a medium containing sucrose regardless of concentration, cells containing the sacB gene started to grow slightly slower, but completely suppressed cell growth under any conditions I could n’t. In addition, it is apparent that the final turbidity of both types of cells is lower as the sucrose is contained than in the medium not containing sucrose (FIG. 12 (A, B, C, D)).

(2) Cytotoxicity due to expression of sacB gene In (1), the expression is leaked without adding IPTG, which is a transcription inducer, to the expression of sacB gene. Therefore, glucose was added to the medium to observe growth in a state where expression was completely suppressed and in a state where expression was leaking. As in (1), the glycerol stock prepared in (1) was streaked onto an LB medium plate (ampicillin 50 μg / ml) and cultured at 37 ° C. Two well-separated colonies were inoculated into LB medium (ampicillin 50 μg / ml, 2 ml) and cultured at 37 ° C. for 17 hours. Thereafter, the precultured bacterial solution was mixed at 1: 10000 with LB medium (ampicillin 50 μg / ml, 2 ml) and 0.9% glucose LB medium (ampicillin 50 μg / ml), and these were dispensed into microplates at 200 μL. Then, the cells were cultured at 37 ° C. for 15 hours, and the growth turbidity (OD ₆₀₀ ) was measured every 15 minutes. As a result, there was almost no difference between the two types of cells in the medium containing glucose (FIG. 12 (A)), but in the medium not containing glucose, cells containing the sacB gene proliferated in two stages. (Fig. 13). The growth is once stopped from about 200 minutes after the start of the culture, and the growth is started again from about 500 minutes. This non-proliferative state is thought to be caused by cytotoxicity due to the expression of the sacB gene. Therefore, in order to observe the cytotoxicity due to the difference in the expression level of the sacB gene, streak the glycerol stock prepared in (1) to the LB medium plate (ampicillin 50 μg / ml) and culture at 37 ° C, as in (1). did. Two well-separated colonies were inoculated into LB medium (ampicillin 50 μg / ml, 2 ml) and cultured at 37 ° C. for 17 hours. Then, mix the precultured bacterial solution at 1: 10000 in LB medium (ampicillin 50μg / ml, 2ml) and LB medium (ampicillin 50μg / ml) containing 0.01mM, 0.1mM, 1.0mM IPTG, and add them to the microplate. 200 μL each was dispensed. Then, the cells were cultured at 37 ° C. for 15 hours, and the growth turbidity (OD ₆₀₀ ) was measured every 15 minutes. As a result, when cultured in a medium containing IPTG, the non-proliferation state as described above was not observed, and it was found that the growth of cells containing the sacB gene was suppressed from the beginning. However, as in (1), the growth was not completely stopped. From this, it was found that the expression of the sacB gene is cytotoxic even when the amount is small.

SEQ ID NO: 2-17, 26, 27 Primer SEQ ID NO: 1, 18-25, 28 Synthesis

Claims (4)

An expression vector that expresses an antisense RNA that targets genes that are essential for the growth of Escherichia coli or an antitoxic gene of Escherichia coli that becomes lethal by suppressing expression is introduced into Escherichia coli. A counter selection method comprising killing Escherichia coli holding the vector by expressing RNA and selecting Escherichia coli not holding the vector.   The counter selection method according to claim 1, wherein an expression-inducible promoter is linked to DNA encoding the antisense RNA, and the antisense RNA is expressed by expression induction. The counter selection method according to claim 1 or 2 , wherein the gene essential for the growth of E. coli is the fabI gene. The counter selection method according to claim 1 or 2, wherein the anti-toxic gene of E. coli is the mqsA gene or the rnlB gene.

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