KR20150142304A - Method for Fine-Tuning Gene Expression Using Synthetic Regulatory Small RNA - Google Patents

Abstract

The present invention relates to a method for fine-tuning gene expression in a prokaryote using a synthetic regulatory small RNA (sRNA), and more specifically, to a synthetic regulatory sRNA which can fine-tune target gene expression by adjusting an expression amount of the sRNA or a binding force between the sRNA and Hfq to fine-tune target gene expression. According to the present invention, the method for fine-tuning gene expression using the synthetic regulatory sRNA adjusts the expression amount of the sRNA or the binding force between the sRNA and Hfq to fine tune a suppression degree of the target gene expression to apply a combination of various target genes to various strains simultaneously, easily, and quickly without a gene deletion process through the synthetic regulatory sRNA which regulates gene expression, which is very suitable for measuring a metabolic capability for each strain and selecting an optimal strain. Also, a target gene for gene expression suppression can be selected easily and quickly, and the selected target gene can be expressed to a desired degree. Therefore, the method can be used in constructing a recombinant strain for efficiently producing various metabolites and establishing an efficient production method.

Description

[0001] The present invention relates to a method for fine-tuning gene expression using synthetic regulatory sRNA,

More particularly, the present invention relates to a method for controlling the expression of a target gene by controlling the amount of sRNA expression or the binding strength of sRNA and Hfq to control the expression of a target gene Lt; RTI ID = 0.0 > sRNA < / RTI >

Concerns about environmental problems and limited resource exhaustion are increasing rapidly in the world in recent years, and there is an increasing interest in building an environmentally friendly and reproducible organism-based production system as an alternative solution. These systems can be produced by modulating the metabolic pathway in an organism and optimizing the metabolic flux to the desired metabolite, which requires a variety of molecular biology techniques. In order to regulate the metabolic pathway, it is necessary to increase the expression of enzymes necessary for the biosynthesis process in order to strengthen the metabolic flow required for the production of metabolites, to prevent the metabolism flow used for cell growth and other metabolites, There is a way to delete the gene. Currently widely used methods of gene deletion are the loss of function by replacing any sequence with homologous sequences with recombinase (Datsenko meat al. , PNAS , 97 (12): 6640-6645, 2000). However, these gene deletion methods have various problems.

Most of all, gene knock-out requires a few weeks - several months, and gene expression can be regulated only on-off. To apply it to several strains, gene deletion must be repeated for each strain This is the most time-consuming process in the study of metabolic engineering which requires many experiments to be confirmed experimentally. A method of mutating the promoter of a gene existing on a chromosome to weaken its activity or substituting it with various promoters capable of regulating gene expression instead of the existing promoter is also used as a method of controlling and suppressing the expression amount of a gene, Since the method is also performed through the same or similar process as the gene deletion, it is difficult to predict the result until the expression amount due to the mutant promoter is directly confirmed, unlike the gene deletion limit, More effort and time may be required.

In order to overcome the limitations of this conventional gene suppression method, a short-length custom synthetic sRNA technique has been developed (Na, D et al ., Nat . Biotechnol ., 31 (2), 170-174, 2013; Yoo, SM et al . Nat . Protoc ., 8 (9), 1694-1707, 2013), it is possible to easily reduce the expression of the target gene using the plasmid without modifying the chromosomal sequence of the target strain and to inhibit the same gene expression simultaneously in various strains simultaneously There is an advantage that it can be easily applied. Moreover, it is quick and easy to make, store and apply them.

The sRNA consists of a base pairing region (BPR) and a Hfq binding sequence for complementary binding to the mRNA of the target gene. In the conventional technique, the binding free energy of the BPR region was calculated to control the expression of the target gene, thereby controlling the base sequence length and the mutation. This is because it is possible to easily design and recombine a synthetic sRNA through a general gene recombination method by designing the BPR part based on the nucleotide sequence information of the target gene. However, in order to finely control the degree of gene expression to a desired level, The BPR part of the sRNA must be redesigned and the sRNA redesigned based on it. Binding free energy is calculated based on the sequence of sRNA and mRNA. In actual binding of sRNA to mRNA, Hfq protein binds to sRNA and mRNA The accuracy of the calculation can be lowered because it helps. In addition, if structural changes occur in the binding sequence due to the secondary structure of sRNA and mRNA, the prediction is missed. In order to overcome these problems, a universal system that can be adjusted to a desired level regardless of the type of target gene must be developed .

Therefore, the inventors of the present invention have tried to construct a universal gene expression regulatory system that meets the above conditions, and as a result, attempted mutation of other factors while fixing BPR, and a method of introducing promoters controlling various gene expression A method of applying a mutation to the Hfq binding site in the sRNA construct was constructed. It was confirmed that the expression of the target gene could be regulated by controlling the expression level of sRNA by introducing various constitutive promoters. When the inducible promoter was introduced, mutation introduction into the Hfq binding site of the sRNA construct resulted in fine regulation of the expression of the target gene And that the present invention has been completed.

It is an object of the present invention to provide a method for fine regulation of gene expression using various customized synthetic sRNAs capable of overcoming the limitations of conventional gene expression suppression methods and simultaneously controlling mRNA expression of a target gene.

(B) a nucleic acid encoding a region that forms a complementary bond with the mRNA of the target gene; (c) an Hfq-binding region represented by SEQ ID NO: 91 derived from the sRNA of Sgrs; A wild-type or mutagenized sRNA construct (scaffold) encoding a Hfq binding site, and (d) a transcription terminator.

The present invention also provides a recombinant microorganism transformed with said vector.

The present invention also provides a method for fine-regulating mRNA expression of a target gene by introducing the vector into prokaryotes or expressing them in prokaryotes.

The present invention also provides a screening method for a target gene for producing a target substance, comprising the following steps.

(a) fine-regulating the expression of any one or more genes present in a target strain to be produced in a target substance biosynthetic pathway by the above method; And

(b) selecting a gene whose expression is regulated as a target gene for production of a target substance when the production yield of the target substance is improved according to the expression microcontrol.

The present invention also provides a method for improving a target substance producing strain comprising the following steps.

(a) fine-regulating the expression of any one or more genes present in a target strain to be produced in a target substance biosynthetic pathway by the above method; And

(b) screening a gene whose expression has been regulated as a target gene for production of a target substance when the yield of the target substance is improved according to the expression micro-regulation; And

(c) introducing the degree of expression of the screened gene to produce a recombinant strain.

The method of controlling the gene expression using the synthetic control sRNA according to the present invention can control the expression level of the target gene by controlling the sRNA expression level or the binding force between sRNA and Hfq, , It is possible to apply various combinations of target genes easily and quickly in various strains without gene deletion process, and thus it is very suitable for the measurement of metabolic ability by strains and the selection of optimal strains. In addition, since it is possible to easily and rapidly select a target gene for inhibiting gene expression and to express the selected gene as much as desired, it can be used for the production of a recombinant strain for efficiently producing various metabolites and for establishing an efficient production method It is very useful.

Figure 1 shows the recombination of sRNA containing the SgrS structure based on the pKK223-3 plasmid commercially available as a plasmid used for the expression of sRNA.
Fig. 2 shows a partial structure of sgrS, which is an sRNA, and shows a potential target to induce mutation. The sequence 'UUUUUUUUU' at the 3 'end of this sequence is also a transcription terminator and Hfq binding site.
Figure 3 is a recombinant expression of the DsRed2 gene based on the pACYC184 plasmid, which is commercially available as a plasmid used for expression of the sRNA target gene.
FIG. 4 shows that the synthetic sRNA containing the SgrS structure effectively suppressed the expression of DsRed2.
Figure 5 shows that the expression-inhibiting activity of synthetic sRNA targeting DsRed2 is due to BPR.
6 is a graph showing that the logarithm of the effect of suppressing the target gene expression and the expression amount of sRNA are linearly proportional.
FIG. 7 shows changes in the sRNA expression-suppressing activity of the stem loop of the Hfq binding site according to the change of the stem sequence.
FIG. 8 shows changes in the sRNA expression-suppressing activity of the Hfq binding site in response to the change in stem length in the stem loop.
FIG. 9 shows changes in the sRNA expression-inhibiting activity according to the loop sequence change in the stem loop of the Hfq binding site.
Fig. 10 shows changes in the sRNA expression-suppressing activity according to the UAUU sequence change in the Hfq binding site.
FIG. 11 shows changes in the sRNA expression-suppressing activity with the change of the length of U tail which is a transcription terminator and Hfq binding site.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The definitions of the main terms used in the description of the present invention and the like are as follows.

As used herein, the term "sRNA (small RNA) " refers to RNA of a short length of 200 nucleotides or less in length, which effectively prevents translation of a specific mRNA through complementary binding.

As used herein, the term "gene " should be considered in its broadest sense and may encompass structural or regulatory proteins. Wherein the regulatory protein comprises a transcription factor, a heat shock protein or a protein involved in DNA / RNA replication, transcription and / or translation. In the present invention, the target gene to be inhibited in expression may exist as an extrachromosomal component.

(B) a nucleic acid encoding a region that forms a complementary bond with a target gene mRNA; (c) an Hfq binding site represented by SEQ ID NO: 91 derived from the sRNA of Sgrs (Hfq a wild-type or mutagenized sRNA construct (scaffold) encoding a nucleic acid binding site, and (d) a transcription terminator.

The promoter of the present invention is to use an inducible promoter that operates only when an inducer is present, a constitutive promoter that always operates, and promoters derived from these promoters (trc promoter or tac promoter) . Regulating the activity of these promoters changes the expression level of sRNA, thereby regulating the expression level of the target gene. For most genes, expression of the target gene can be controlled by controlling the expression level of sRNA by introducing various constitutive promoters. In the case of constitutive promoters, it may be characterized by the use of various promoters provided by the Anderson promoter collection (http://partsregistry.org/Promoters/Catalog/Anderson) produced by Chris Anderson.

The mutant-induced sRNA construct (scaffold) of the present invention regulates the binding of sRNA and Hfq to fine-tune the expression of the target gene. When suppressing the expression of genes that are critical to survival of microorganisms or inhibiting the growth rate, a system in which sRNA is expressed only when necessary under the inducible promoter should be constructed. However, when the inducible promoter is introduced, the expression level can be easily controlled I do not. Therefore, in the present invention, the expression of the target gene may be finely regulated by inducing mutation of the Hfq binding site.

In the present invention, a partial sequence of the sRNA construct of Sgrs (FIG. 2) is represented by SEQ ID NO: 91 and mutagenesis was induced. And the binding between the sRNA and Hfq is regulated by converting the GGUG sequence of SEQ ID NO: 91, which is the first stem region, into GGAC, CCUG and CCUC, or by converting GGUG 4nt into 6nt and 8nt. Also, the 10th to 13th AAAA sequence of SEQ ID NO: 91, which is the first loop site of the sgrs of Sgrs, is converted into CCGG, CCCC, CCUU, GGGG, GGCC, GGUU, UUGG and UUUU, or the sequence of SEQ ID NO: 91 The UAUUs 1 to 4 may be changed to GAUU, UCUU, UACU, UAAU, UAUC, UAUA, GCCC, GCAC, GACC, and GAAC. It is also possible to convert the length of U tail 42 to 49 of SEQ ID NO: 91, which is a transcription terminator of Sgrs and a binding site of Hfq, to 4 to 8.

[SEQ ID NO: 91] 5'-UAUUGGUGUAAAAUCACCCGCCAGCAGAUUAUACCUGCUGGUUUUUUUU-3 '

As used herein, the "nucleic acid" may be RNA, DNA stabilized RNA or stabilized DNA. Herein, the term "encoding" means a nucleic acid sequence complementary to the sRNA which encodes the sRNA.

As used herein, "vector" means a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several to several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector, (C) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA. After ligation, the vector should be transformed into the appropriate host cell. Transformation can be readily accomplished using a calcium chloride method or electroporation (Neumann, et al., EMBO J. , 1: 841, 1982). As the vector used for expression of the sRNA according to the present invention, an expression vector known in the art can be used.

The nucleotide sequence of a nucleic acid is "operably linked" when placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is an element that directly affects the transcription of a sequence and is placed or linked in an appropriate position to be operable in a coding sequence; The ribosome binding site affects the transcription of the sequence and is linked directly to the coding sequence so as to be operable in the coding sequence. The DNA encoding the pre-sequence or secretion leader sequence encodes the entire protein involved in the secretion of the polypeptide and is directly linked to the target gene. Generally, "operably linked" means that the DNA sequences are physically linked, and in the case of a secretory leader sequence, are in contact and present in the reading frame. However, the enhancer need not be in contact. The linkage of these sequences is carried out by ligation (linkage) at convenient restriction sites. If such a site does not exist, a synthetic oligonucleotide adapter or a linker according to a conventional method is used.

In another aspect, the present invention relates to a recombinant microorganism transformed with said vector.

As used herein, the term "transformation" means introducing DNA into a host and allowing the DNA to replicate as an extrachromosomal factor or by chromosomal integration.

Of course, it should be understood that not all vectors function equally well in expressing the DNA sequences of the present invention. Likewise, not all hosts function identically for the same expression system. However, those skilled in the art will be able to make appropriate selections among a variety of vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of the present invention. For example, in selecting a vector, the host should be considered because the vector must be replicated within it. The number of copies of the vector, the ability to control the number of copies, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered.

In another aspect, the present invention relates to a method for fine-regulating mRNA expression of a target gene by introducing the vector into prokaryotes or expressing them in prokaryotes.

In another aspect, the present invention relates to a screening method of a target gene for producing a target substance, comprising the steps of:

(a) fine-regulating expression of any one or more genes among genes participating in a target substance biosynthetic pathway present in a target strain to be produced, according to the method of claim 1; And

(b) selecting a gene whose expression is regulated as a target gene for production of a target substance when the production yield of the target substance is improved according to the expression microcontrol.

In another aspect, the present invention relates to a method for improving a target substance producing strain, comprising the steps of:

(a) fine-regulating expression of any one or more genes among genes participating in a target substance biosynthetic pathway present in a target strain to be produced, according to the method of claim 1; And

(b) screening a gene whose expression has been regulated as a target gene for production of a target substance when the yield of the target substance is improved according to the expression micro-regulation; And

(c) introducing the degree of expression of the screened gene to produce a recombinant strain.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

synthesis sRNA Check the performance of

1-1: Synthesis sRNA  For production and use sRNA  Production of expression plasmids

In order to ensure the convenience of expression of sRNA, a plasmid as shown in Fig. 1 was prepared. In the subsequent experiments, the plasmid of Fig. 1 was modified by site-directed mutagenesis using sRNA or promoter.

The restriction enzymes used in this and the following examples were purchased from New England BioLabs (USA) and Enzynomics (Korea), and PCR polymerase from Solgent (Korea). Primers were purchased from Macrogen (Korea), and other enzymes were purchased from Enzynomics (Korea). Other things are indicated separately.

The multiple cloning site of pBluescript II KS + (Stratagene) was inserted into the HindIII / NaeI site of the pKK223-3 plasmid produced by Pharmacia by performing PCR using the primers of SEQ ID NOS: 1 and 2. The PCR product of the multiple cloning site was digested with PvuII and NaeI, and the pKK223-3 plasmid was digested with HindIII and NaeI, followed by blunt termination using Klenow polymerase to construct two plasmid pKK223-3 KS. A plasmid for sRNA expression was constructed by inserting T1 / TE into the thus constructed plasmid with the sRNA construct, the Pr promoter for its expression, and the transcription terminator. The sequences of the Pr promoter, transcription terminator T1 / TE and sRNA constructs were cloned by PCR using primers of SEQ ID NOS: 3 and 4 in Rlowcat-PrMicC * Ter used in previous studies (Na, D et al , Nat Biotechnol ., 31 (2), 170-174, 2013). PCR products of the Pr promoter-MimC structure-T1 / TE transcription terminator were digested with BamHI and NotI, and pKK223-3 KS was also digested with BamHI / NotI to ligate the two DNA fragments. The sequence of the Pr promoter used is the sequence of BBa_R0051 registered in the Registry of Standard Biological Parts data (http://parts.igem.org/), and the T1 / TE promoter is the sequence of BBa_B0015 in the same database. In the BPR represented by the base pairing region, a 24-mer nucleotide sequence was added from the initiation codon of the DsRed2 fluorescent protein. The plasmid thus constructed was named pKK223-3 KS Pr MDsRed2. BPR was inserted by inverse PCR using the primers of SEQ ID NOS: 5 and 6. The sequence of the SgrS sequence was as shown in Fig. 2 and was also constructed by inverse PCR using the primers of SEQ ID NOS: 7 and 8. The plasmid thus constructed was named pKK223-3 KS Pr SDsRed2. The map of the plasmid having the sequence of SgrS is shown in Fig. 1, and the E. coli strain DH5? Was used for the plasmid production.

≪ Primer Sequence Used >

[SEQ ID NO: 1] 5'-GGCCAATTCAGCTGGTACCGGGCCCCCCCTCG-3 '

[SEQ ID NO: 2] 5'-GGCCAATTGCCGGCGAGCTCCACCGCGGTGG-3 '

[SEQ ID NO: 3] 5'-GGCCTTAAGCGGCCGCTAACACCGTGCGTGTTGAC-3 '

[SEQ ID NO: 4] 5'-CCGGAATTGGATCCTATAAACGCAGAAAGGCCC-3 '

[SEQ ID NO: 5] 5'-CTCGCCATATATTTGTCTTTCTGTTGGGCCATTGCATTGC-3 '

[SEQ ID NO: 6] 5'-CAGTGAGAACGTCATGCAACCATTATCACCGCCAGAGG-3 '

[SEQ ID NO: 7] 5'-GCCAGCAGATTATACCTGCTGGTTTTTTTTCTCGAGCCAGGCATCAAATAAAAC

G-3 '

[SEQ ID NO: 8] 5'-GGGTGATTTTACACCAATAGACAAATATATGGCGAGCAGTGAGAACGTCATGCA

ACCATTATCACCGCCAGAGG-3 '

1-2: Synthesis sRNA  Production of target gene plasmid for performance study

A fluorescent protein DsRed2 was selected as a target gene for easily confirming the performance of sRNA, and a reporter plasmid as shown in Fig. 3 was prepared for the expression of the DsRed2 gene. The multiple cloning site of pBluescript II KS + (Stratagene) was inserted into the XbaI / NaeI site of the plasmid pACYC184 (New England BioLabs, USA) prepared in Pharmacia by PCR using the primers of SEQ ID NOS: 1 and 2. The PCR product of the multiple cloning site was digested with PvuII and NaeI. The plasmid pACYC184 plasmid was digested with XbaI and NaeI, blunt ends were made using Klenow polymerase, and two DNA fragments were ligated to produce plasmid pACYC184 KS. A plasmid carrying a fluorescent protein as a target gene of sRNA was prepared. The lactase promoter, the gene for the fluorescent protein DsRed2, and the T1 / TE sequence as a transcription terminator were inserted into the ApaI / SalI site at the multiple cloning site. The lactose promoter, DsRed2 gene, and T1 / TE transcription terminator used in this study were constructed by PCR using SEQ ID NOS: 9 and 10 in Rlowcat-Plac-DsRed2 (Na, D et al , Nat Biotechnol ., 31 (2), 170-174, 2013). The Lactose promoter was constructed by PCR using pBluescript SK + (Stratagene) as a template, and the DsRed2 gene was constructed by PCR using pDsRed2-N1 (Clontech) as a template. The T1 / TE transcription terminator is the BBa_R0015 sequence registered in the above Registry of Standard Biological Parts database (http://parts.igem.org/). Escherichia coli DH5? Was used for plasmid production.

≪ Primer Sequence Used >

[SEQ ID NO: 9] 5'-GGCCTTAAGGGCCCGTGGATAACCGTATTACCGC-3 '

[SEQ ID NO: 10] 5'-CCGGAATTGTCGACTATAAACGCAGAAAGGCCC-3 '

1-3: Synthesis sRNA Performance survey

Experiments were conducted to investigate the inhibitory effect of sRNA on the expression of a target gene using the plasmid prepared above. The reporter plasmid prepared in Example 1-2 and the SgrS construct prepared in Example 1-1 were simultaneously transformed into DH5α and a plasmid carrying sRNA with DsRed2 as a target protein. Colonies carrying both plasmids were selected using LB plates containing Ampicilin (Ap) and Chrolamphenicol (Cm) antibiotics, and then inoculated into LB medium containing Ap and Cm. Then, the cells were cultured up to stationary phase to measure fluorescence expression. As shown in FIG. 4, the anti-DsRed2 synthetic sRNA having the SgrS structure showed 99% inhibition of expression compared to the control. Both strains have pACYC184 KS Plac DsRed2, and the control and SgrS have pKK223-3 KS and pKK223-3 KS SDsRed2, respectively. As a control group for fluorescence measurement, strains having pACYC184 KS and pKK223-3 KS were used.

1-4: Synthesis sRNA Specificity survey

Experiments were conducted to investigate whether the inhibition of target gene expression by sRNA was specifically effected by BPR. In this experiment, a plasmid (pKK223-3 KS SDsRed2) carrying an sRNA containing a BPR consisting of 24 nucleotides with a SgrS structure was used. The plasmid without BPR was constructed by using the inverse PCR technique using the primers of SEQ ID NOs: 11 and 12 as a template of pKK223-3 KS SDsRed2, which was named pKK223-3 KS SO. This was used for the samples labeled non-targeting sRNA in the following data. The experiment was carried out in the same manner as in Example 1-3. The results are shown in FIG. 5. As shown in FIG. 5, sRNA without BPR showed 2% inhibition of fluorescent protein expression compared to the control, and sRNA with 24 nucleotide BPR showed 98% inhibition of fluorescent protein expression. This shows that inhibition of a specific gene is achieved by BPR rather than the structure of the synthetic sRNA.

≪ Primer Sequence Used >

[SEQ ID NO: 11] 5'-TATTGGTGTAAAATCACCCGCCAGCAG-3 '

[SEQ ID NO: 12] 5'-GCAACCATTATCACCGCCAGAGG-3 '

Through the introduction of various promoters sRNA  Regulation of expression and its effect

An experiment was conducted to control the expression of sRNA by using a promoter showing various gene expression levels. The plasmid and experimental method of Example 1 were used, and the promoters of the Anderson promoter collection were cloned using the primers of SEQ ID NOS: 13 to 46 using the inverse PCR technique. SgrS was used as the structure of the sRNA and the promoters were J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23109, J23111, J23112, J23113, J23114, J23115, J23116, J23117, J23118. The results are shown in FIG. 6, and the intensity of the promoter was proportional to the logarithm of protein inhibition efficiency.

≪ Primer sequence used for promoter cloning >

J23100:

[SEQ ID NO: 13] 5'-GACTGAGCTAGCCGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

[SEQ ID NO: 14] 5'-CTAGGTACAGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

J23101:

[SEQ ID NO: 15] 5'-CTAGGTATTATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 16] 5'-GACTGAGCTAGCTGTAAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23102:

[SEQ ID NO: 17] 5'-CTAGGTACTGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 18] 5'-GACTGAGCTAGCTGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23103:

[SEQ ID NO: 19] 5'-CTAGGGATTATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 20] 5'-GACTGAGCTAGCTATCAGGCGGCCGCCACCGCGGTGGAGC-3 '

J23104:

[SEQ ID NO: 21] 5'-CTAGGTATTGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 22] 5'-GACTGAGCTAGCTGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23105:

[SEQ ID NO: 23] 5'-GACTGAGCTAGCCGTAAAGCGGCCGCCACCGCGGTGGAGC-3 '

[SEQ ID NO: 24] 5'-CTAGGTACTATGCTAGCGATGACGTTCTCACTGCTCGC-3 '

J23106:

[SEQ ID NO: 25] 5'-CTAGGTATAGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 26] 5'-GACTGAGCTAGCCGTAAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23107:

[SEQ ID NO: 27] 5'-CTAGGTATTATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 28] 5'-GGCTGAGCTAGCCGTAAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23109:

[SEQ ID NO: 29] 5'-GACTGAGCTAGCTGTAAAGCGGCCGCCACCGCGGTGGAGC-3 '

[SEQ ID NO: 30] 5'-CTAGGGACTGTGCTAGCGATGACGTTCTCACTGCTCGC-3 '

J23111:

[SEQ ID NO: 31] 5'-CTAGGTATAGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 32] 5'-GACTGAGCTAGCCGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23112:

[SEQ ID NO: 33] 5'-CTAGGGATTATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 34] 5'-GACTGAGCTAGCTATCAGGCGGCCGCCACCGCGGTGGAGC-3 '

J23113:

[SEQ ID NO: 35] 5'-CTAGGGATTATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 36] 5'-GACTGAGCTAGCCATCAGGCGGCCGCCACCGCGGTGGAGC-3 '

J23114:

[SEQ ID NO: 37] 5'-GACTGAGCTAGCCATAAAGCGGCCGCCACCGCGGTGGAGC-3 '

[SEQ ID NO: 38] 5'-CTAGGTACAATGCTAGCGATGACGTTCTCACTGCTCGC-3 '

J23115:

[SEQ ID NO: 39] 5'-CTTGGTACAATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 40] 5'-GGCTGAGCTAGCTATAAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23116:

[SEQ ID NO: 41] 5'-CTAGGGACTATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 42] 5'-GACTGAGCTAGCTGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23117:

[SEQ ID NO: 43] 5'-CTAGGGATTGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 44] 5'-GACTGAGCTAGCTGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

J23118:

[SEQ ID NO: 45] 5'-CTAGGTATTGTGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

[SEQ ID NO: 46] 5'-GACTGAGCTAGCCGTCAAGCGGCCGCCACCGCGGTGGAGC-3 '

sRNA Modulation of target gene inhibition through mutation in the structure of

The sRNA constructs were mutagenized and their activities were changed to control the degree of target gene inhibition. The plasmid and experimental method of Example 1 were used, and as a promoter of sRNA, J23105, which showed 74% inhibition rate in the previous experiment, was used. Experiment was conducted using J23108 only for the experiment of FIG. The plasmid expressing sRNA with the promoter of J23108 was constructed by inverse PCR using SEQ ID NOS: 51 and 52. Mutations were made in the construct through inverse PCR using primers of the sequences listed below. As control, pKK223-3 KS without sRNA construct and pKK223-3 KS SO without BPR were used. (Fig. 11), the sequence of the loop (Fig. 9), the mutation of the UAUU sequence (Fig. 10), the length of the U tail (Fig. 11) Could weaken or enhance sRNA activity. U tail is also a transcription terminator and Hfq binding site. The combination of these mutations may further weaken or enhance the inhibitory activity of sRNA.

≪ Primer sequence used for mutation in Fig. 7 >

Common use primer:

[SEQ ID NO: 47] 5'-AATCACCCGCCAGCAGATTATACCTGCTGG-3 '

GGAC:

[SEQ ID NO: 48] 5'-TTAGTCCAATAATGGCGAGCAGTGAGAAC-3 '

CCUG:

[SEQ ID NO: 49] 5'-TTACAGGAATAATGGCGAGCAGTGAGAAC-3 '

CCUC

[SEQ ID NO: 50] 5'-TTAGTGGAATAATGGCGAGCAGTGAGAAC-3 '

≪ Primer sequence used in the mutation of Fig. 8 >

J23108 Production primer

[SEQ ID NO: 51] 5'-GACTGAGCTAGCTGTCAGGCGGCCGCCACCGCGGTGGAGC-3 '

[SEQ ID NO: 52] 5'-CTAGGTATAATGCTAGCGATGACGTTCTCACTGCTCGCC-3 '

6nt stem:

[SEQ ID NO: 53] 5'-TTACCCACCAATAATGGCGAGCAGTGAGAAC-3 '

[SEQ ID NO: 54] 5'-AATCCCACCCGCCAGCAGATTATACCTGCTGG-3 '

8nt stem:

[SEQ ID NO: 55] 5'-TTACCCCCACCAATAATGGCGAGCAGTGAGAAC-3 '

[SEQ ID NO: 56] 5'-AATCCCCCACCCGCCAGCAGATTATACCTGCTGG-3 '

<Primer sequence used in the mutation of FIG. 9>

CCGG:

[SEQ ID NO: 57] 5'-GGACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 58] 5'-GGTCACCCGCCAGCAGATTATAC-3 '

CCCC:

[SEQ ID NO: 59] 5'-GGACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 60] 5'-CCTCACCCGCCAGCAGATTATAC-3 '

CCUU:

[SEQ ID NO: 61] 5'-GGACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 62] 5'-TTTCACCCGCCAGCAGATTATAC-3 '

GGGG;

[SEQ ID NO: 63] 5'-CCACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 64] 5'-GGTCACCCGCCAGCAGATTATAC-3 '

GGCC:

[SEQ ID NO: 65] 5'-CCACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 66] 5'-CCTCACCCGCCAGCAGATTATAC-3 '

GGUU:

[SEQ ID NO: 67] 5'-CCACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 68] 5'-TTTCACCCGCCAGCAGATTATAC-3 '

UUGG:

[SEQ ID NO: 69] 5'-AAACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 70] 5'-GGTCACCCGCCAGCAGATTATAC-3 '

UUCC:

[SEQ ID NO: 71] 5'-AAACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 72] 5'-CCTCACCCGCCAGCAGATTATAC-3 '

UUUU:

[SEQ ID NO: 73] 5'-AAACACCAATAATGGCGAGC-3 '

[SEQ ID NO: 74] 5'-TTTCACCCGCCAGCAGATTATAC-3 '

<Primer sequence used in the mutation of FIG. 10>

Commonly used primer

[SEQ ID NO: 75] 5'-GACAAATATATGGCGAGCAG-3 '

GAUU:

[SEQ ID NO: 76] 5'-GATTGGTGTAAAATCACCCGCCAGCAG-3 '

UCUU:

[SEQ ID NO: 77] 5'-GTCTTGGTGTAAAATCACCCGCCAGCAG-3 '

UACU:

[SEQ ID NO: 78] 5'-TACTGGTGTAAAATCACCCGCCAGCAG-3 '

UAAU:

[SEQ ID NO: 79] 5'-TAATGGTGTAAAATCACCCGCCAGCAG-3 '

UAUC:

[SEQ ID NO: 80] 5'-TATCGGTGTAAAATCACCCGCCAGCAG-3 '

UAUA:

[SEQ ID NO: 81] 5'-TATAGGTGTAAAATCACCCGCCAGCAG-3 '

GCCC:

[SEQ ID NO: 82] 5'-GCCCGGTGTAAAATCACCCGCCAGCAG-3 '

GCAC:

[SEQ ID NO: 83] 5'-GCACGGTGTAAAATCACCCGCCAGCAG-3 '

GACC:

[SEQ ID NO: 84] 5'-GACCGGTGTAAAATCACCCGCCAGCAG-3 '

GAAC:

[SEQ ID NO: 85] 5'-GACCGGTGTAAAATCACCCGCCAGCAG-3 '

<Primer sequence used in the mutation of FIG. 11>

Common use primer:

[SEQ ID NO: 86] 5'-CTCGAGCCAGGC-3 '

U7:

[SEQ ID NO: 87] 5'-AAAAAAACCAGCAGGTATAATCTGCTGGCG-3 '

U6:

[SEQ ID NO: 88] 5'-AAAAAACCAGCAGGTATAATCTGCTGGCG-3 '

U5:

[SEQ ID NO: 89] 5'-AAAAACCAGCAGGTATAATCTGCTGGCG-3 '

U4:

[SEQ ID NO: 90] 5'-AAAACCAGCAGGTATAATCTGCTGGCG-3 '

<110> Korea Advanced Institute of Science and Technology <120> Method for Fine-Tuning Gene Expression Using Synthetic Regulatory          Small RNA <130> P13-B298 <160> 91 <170> Kopatentin 2.0 <210> 1 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> p1 <400> 1 ggccaattca gctggtaccg ggccccccct cg 32 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> p2 <400> 2 ggccaattgc cggcgagctc caccgcggtg g 31 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> p3 <400> 3 ggccttaagc ggccgctaac accgtgcgtg ttgac 35 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> p4 <400> 4 ccggaattgg atcctataaa cgcagaaagg ccc 33 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> p5 <400> 5 ctcgccatat atttgtcttt ctgttgggcc attgcattgc 40 <210> 6 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> p6 <400> 6 cagtgagaac gtcatgcaac cattatcacc gccagagg 38 <210> 7 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> p7 <400> 7 gccagcagat tatacctgct ggtttttttt ctcgagccag gcatcaaata aaacg 55 <210> 8 <211> 74 <212> DNA <213> Artificial Sequence <220> <223> p8 <400> 8 gggtgatttt acaccaatag acaaatatat ggcgagcagt gagaacgtca tgcaaccatt 60 atcaccgcca gagg 74 <210> 9 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> p9 <400> 9 ggccttaagg gcccgtggat aaccgtatta ccgc 34 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> p10 <400> 10 ccggaattgt cgactataaa cgcagaaagg ccc 33 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p11 <400> 11 tattggtgta aaatcacccg ccagcag 27 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p12 <400> 12 gcaaccatta tcaccgccag agg 23 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23100-f <400> 13 gactgagcta gccgtcaagc ggccgccacc gcggtggagc 40 <210> 14 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23100-r <400> 14 ctaggtacaga tgctagcgat gacgttctca ctgctcgcc 39 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23101-f <400> 15 ctaggtatta tgctagcgat gacgttctca ctgctcgcc 39 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23101-r <400> 16 gactgagcta gctgtaaagc ggccgccacc gcggtggagc 40 <210> 17 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23102-f <400> 17 ctaggtactg tgctagcgat gacgttctca ctgctcgcc 39 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23102-r <400> 18 gactgagcta gctgtcaagc ggccgccacc gcggtggagc 40 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23103-f <400> 19 ctagggatta tgctagcgat gacgttctca ctgctcgcc 39 <210> 20 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23103-r <400> 20 gactgagcta gctatcaggc ggccgccacc gcggtggagc 40 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23104-f <400> 21 ctaggtattg tgctagcgat gacgttctca ctgctcgcc 39 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23104-r <400> 22 gactgagcta gctgtcaagc ggccgccacc gcggtggagc 40 <210> 23 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23105-f <400> 23 gactgagcta gccgtaaagc ggccgccacc gcggtggagc 40 <210> 24 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> J23105-r <400> 24 ctaggtacta tgctagcgat gacgttctca ctgctcgc 38 <210> 25 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23106-f <400> 25 ctaggtatag tgctagcgat gacgttctca ctgctcgcc 39 <210> 26 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23106-r <400> 26 gactgagcta gccgtaaagc ggccgccacc gcggtggagc 40 <210> 27 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23107-f <400> 27 ctaggtatta tgctagcgat gacgttctca ctgctcgcc 39 <210> 28 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23107-r <400> 28 ggctgagcta gccgtaaagc ggccgccacc gcggtggagc 40 <210> 29 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23109-f <400> 29 gactgagcta gctgtaaagc ggccgccacc gcggtggagc 40 <210> 30 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> J23109-r <400> 30 ctagggactg tgctagcgat gacgttctca ctgctcgc 38 <210> 31 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23111-f <400> 31 ctaggtatag tgctagcgat gacgttctca ctgctcgcc 39 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23111-r <400> 32 gactgagcta gccgtcaagc ggccgccacc gcggtggagc 40 <210> 33 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23112-f <400> 33 ctagggatta tgctagcgat gacgttctca ctgctcgcc 39 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23112-r <400> 34 gactgagcta gctatcaggc ggccgccacc gcggtggagc 40 <210> 35 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23113-f <400> 35 ctagggatta tgctagcgat gacgttctca ctgctcgcc 39 <210> 36 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23113-r <400> 36 gactgagcta gccatcaggc ggccgccacc gcggtggagc 40 <210> 37 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23114-f <400> 37 gactgagcta gccataaagc ggccgccacc gcggtggagc 40 <210> 38 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> J23114-r <400> 38 ctaggtacaa tgctagcgat gacgttctca ctgctcgc 38 <210> 39 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23115-f <400> 39 cttggtacaa tgctagcgat gacgttctca ctgctcgcc 39 <210> 40 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23115-r <400> 40 ggctgagcta gctataaagc ggccgccacc gcggtggagc 40 <210> 41 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23116-f <400> 41 ctagggacta tgctagcgat gacgttctca ctgctcgcc 39 <210> 42 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23116-r <400> 42 gactgagcta gctgtcaagc ggccgccacc gcggtggagc 40 <210> 43 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23117-f <400> 43 ctagggattg tgctagcgat gacgttctca ctgctcgcc 39 <210> 44 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23117-r <400> 44 gactgagcta gctgtcaagc ggccgccacc gcggtggagc 40 <210> 45 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23118-f <400> 45 ctaggtattg tgctagcgat gacgttctca ctgctcgcc 39 <210> 46 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23118-r <400> 46 gactgagcta gccgtcaagc ggccgccacc gcggtggagc 40 <210> 47 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> p47 <400> 47 aatcacccgc cagcagatta tacctgctgg 30 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> p48 <400> 48 ttagtccaat aatggcgagc agtgagaac 29 <210> 49 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> p49 <400> 49 ttacaggaat aatggcgagc agtgagaac 29 <210> 50 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> p50 <400> 50 ttagtggaat aatggcgagc agtgagaac 29 <210> 51 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> J23108-f <400> 51 gactgagcta gctgtcaggc ggccgccacc gcggtggagc 40 <210> 52 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> J23108-r <400> 52 ctaggtataa tgctagcgat gacgttctca ctgctcgcc 39 <210> 53 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 6 nt-53 <400> 53 ttacccacca ataatggcga gcagtgagaa c 31 <210> 54 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> 6nt-54 <400> 54 aatcccaccc gccagcagat tatacctgct gg 32 <210> 55 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> 8 nt-55 <400> 55 ttacccccac caataatggc gagcagtgag aac 33 <210> 56 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> 8 nt-56 <400> 56 aatcccccac ccgccagcag attatacctg ctgg 34 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 57 <400> 57 ggacaccaat aatggcgagc 20 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p58 <400> 58 ggtcacccgc cagcagatta tac 23 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p59 <400> 59 ggacaccaat aatggcgagc 20 <210> 60 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p60 <400> 60 cctcacccgc cagcagatta tac 23 <210> 61 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p61 <400> 61 ggacaccaat aatggcgagc 20 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p62 <400> 62 tttcacccgc cagcagatta tac 23 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p63 <400> 63 ccacaccaat aatggcgagc 20 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p64 <400> 64 ggtcacccgc cagcagatta tac 23 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p65 <400> 65 ccacaccaat aatggcgagc 20 <210> 66 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p66 <400> 66 cctcacccgc cagcagatta tac 23 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p67 <400> 67 ccacaccaat aatggcgagc 20 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p68 <400> 68 tttcacccgc cagcagatta tac 23 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p69 <400> 69 aaacaccaat aatggcgagc 20 <210> 70 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p70 <400> 70 ggtcacccgc cagcagatta tac 23 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p71 <400> 71 aaacaccaat aatggcgagc 20 <210> 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p72 <400> 72 cctcacccgc cagcagatta tac 23 <210> 73 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p73 <400> 73 aaacaccaat aatggcgagc 20 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> p74 <400> 74 tttcacccgc cagcagatta tac 23 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> p75 <400> 75 gacaaatata tggcgagcag 20 <210> 76 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p76 <400> 76 gattggtgta aaatcacccg ccagcag 27 <210> 77 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> p77 <400> 77 gtcttggtgt aaaatcaccc gccagcag 28 <210> 78 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p78 <400> 78 tactggtgta aaatcacccg ccagcag 27 <210> 79 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p79 <400> 79 taatggtgta aaatcacccg ccagcag 27 <210> 80 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p80 <400> 80 tatcggtgta aaatcacccg ccagcag 27 <210> 81 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p81 <400> 81 tataggtgta aaatcacccg ccagcag 27 <210> 82 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p82 <400> 82 gcccggtgta aaatcacccg ccagcag 27 <210> 83 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p83 <400> 83 gcacggtgta aaatcacccg ccagcag 27 <210> 84 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p84 <400> 84 gaccggtgta aaatcacccg ccagcag 27 <210> 85 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> p85 <400> 85 gaccggtgta aaatcacccg ccagcag 27 <210> 86 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> p86 <400> 86 ctcgagccag gc 12 <210> 87 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> u7-87 <400> 87 aaaaaaacca gcaggtataa tctgctggcg 30 <210> 88 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> u6-88 <400> 88 aaaaaaccag caggtataat ctgctggcg 29 <210> 89 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> u5-89 <400> 89 aaaaaccagc aggtataatc tgctggcg 28 <210> 90 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> u4-90 <400> 90 aaaaccagca ggtataatct gctggcg 27 <210> 91 <211> 49 <212> RNA <213> Artificial Sequence <220> <223> SgrS-sRNA <400> 91 uauuggugua aaaucacccg ccagcagauu auaccugcug guuuuuuuu 49

Claims (12)

(a) a promoter, (b) a nucleic acid encoding a region that forms a complementary bond with the mRNA of the target gene, (c) a nucleic acid encoding an Hfq binding site represented by SEQ ID NO: 91 derived from sRNA of sgrs Of a wild type or mutated sRNA construct (scaffold) and (d) a vector comprising a transcription terminator.
The mutant-induced sRNA construct according to claim 1, wherein the mutation-induced sRNA construct comprises (i) an sRNA construct in which any one or more bases of (1) to (4) are substituted, (ii) (Iii) an sRNA structure in which two or four bases are inserted in positions 5 to 8, (iv) an sRNA structure in which four bases in positions 10 to 13 are substituted, And (v) an sRNA construct in which one to four bases 42 to 49 have been deleted.
[Claim 2] The method according to claim 1, wherein the promoter is any one selected from the group consisting of a trc promoter, a tac promoter, an Anderson promoter collection as a constitutive promoter, or an arabinose operon promoter as an inducible promoter, or a lactose operon promoter Vector illustration.
2. The vector according to claim 1, wherein the region forming the complementary binding with the target gene mRNA is 10 to 100 bp.
2. The method of claim 1, wherein the mRNA of the target gene is selected from the group consisting of DsRed2, LuxR, AraC, kanamycin resistance gene, tyrR, tyrosine regulator, phosphoenolpyruvate carboxylase, csrA, phosphate isomerase), glt (citrate synthase), accA (acetyl-CoA carboxyltransferase, alpha-subunit), accB (biotinylated biotin-carboxyl carrier protein), accC (acetyl-CoA carboxylase) ), aceE (subunit of E1p component of pyruvate dehydrogenase complex), aceF (pyruvate dehydrogenase), ackA (propionate kinase / acetate kinase activity), adiY (adiY is a positive DNA-binding transcriptional regulator of arginine) decarboxylase argB (argininosuccinate synthase), argH (argininosuccinate lyase), asnC (transcriptional regulator that activates the expression of asnA, a gene involved in the synthesis of asparagine), argB (acetylglutamate kinase) aspA (as partate ammonia-lyase), CRP (CRP transcriptional dual regulator), csiD (predicted protein. CsiD is the product of a gene induced by carbon starvation), csiR (DNA-binding transcriptional repressor), cytR (transcription factor required for transport and utilization of ribonucleosides and deoxyribonucleosides), dcuA (The dcuA transporter is one of three transporters known to be for example, the responsible for the uptake of C4-dicarboxylates such as fumarate under anaerobic conditions, deoB (phosphopentomutase), deoC (deoxyribose-phosphate aldolase), deoR (the transcriptional repressor DeoR, FADR (FadR Fatty Acid Degradation Regulon), is a multifunctional dual regulator that exerts negative control over the fatty (FAD), fadD (fatty acyl-CoA synthetase) acid degradative regulon [Simons80, Simons80a] and acetate metabolism), fbp (fructose-1,6-bisphosphatase), fnr (FNR is the primary transcriptional regulator that me dietary transition from aerobic to anaerobic growth, fruR (FruR is a dual transcriptional regulator that plays a pleiotropic role to modulate the direction of carbon flow through the different metabolic pathways of energy metabolism, independently of the CRP regulator), ftsL cell division protein FtsL), essential cell division protein FtsQ, essential cell division protein FtsW, essential cell division protein FtsZ, Fur-Fe + 2 DNA-binding transcriptional dual regulator, gabD (GABA APC transporter), glcC (GntR family), gadA (glutamate decarboxylase A subunit), gadA (GABA APC transporter) transcriptional regulator, glc operon transcriptional activator), glpK (glycerol kinase), glpR (sn-Glycerol-3-phosphate repressor), glpX (fructose 1,6-bisphosphatase II), gltA (citrate synthase), hfld (IHF, integration host factor, is a global regulatory protein), ilvB (acetohydroxybutanoate synthase / acetolactate synthase), ilvC (acetohydroxy acid isomeroreductase), ilvD (acetolactate synthase II, large subunit, N-ter fragment (pseudogene)), ilvG2 (acetolactate synthase II, large subunit, C-ter fragment (pseudogene)), ilvH (acetolactate synthase / acetohydroxybutanoate synthase) , ilvL operon leader peptide, ilvM (acetohydroxybutanoate synthase / acetolactate synthase), ilvN (acetohydroxybutanoate synthase / acetolactate synthase), ilvX (Predicted small protein), lexA (LexA represses the transcription of several genes involved in the cellular response to DNA damage), lpxC (UDP-3-O-acyl-N-acetylglucosamine deacetylase), marA (MarA participates in controlling several genes involved in resistance to antibiotics, oxidative stress, (L-aspartate oxidase), narL (MetL transcriptional repressor), metE (MetJ transcriptional repressor), modE (ModE is the principal regulator of the transcription of operons involved in the transport of molybdenum and synthesis of molybdoenzymes and molybdate- (nitrate / nitrite response regulator), pck (phosphoenolpyruvate carboxykinase), PdhR (PdhR, "pyruvate dehydrogenase complex regulator," regulates genes involved in the pyruvate dehydrogenase complex), phoP (PhoP-phosphorylated DNA-binding transcriptional dual regulator. (PnuC NMN transporter), ppsA (phosphoenolpyruvate synthetase), pta (Phosphate acetyltransferase), purA (adenylosuccinate synthetase ), purB (adenylosuccinate lyase), purR (PurR-Hypoxanthine DNA-binding transcriptional repressor. PurR dimer controls several genes involved in purine nucleotide biosynthesis and its own synthesis), puuE (4-aminobutyrate aminotransferase), rbsA (ribose ABC transporter) RbsB (ribose ABC transporter), rbsD (ribose pyranase), rbsK (ribokinase), rbsR (the transcription factor RbsR, for "Ribose Repressor," is negatively autoregulated and controls the transcription of the operon involved in ribose catabolism and transport) (RcsB-BglJ DNA-binding transcriptional activator. RcsB protein for "regulator capsule synthesis B," is a response regulator that belongs to the multicomponent RcsF / RcsC / RcsD / RcsA-RcsB phos (RutR regulates genes directly or indirectly involved in the complex pathway of pyrimidine metabolism), serA ((RutR gene) alpha-ketoglutarate reductase / D-3-phosphoglycerate dehydrogenase, serC (phosphohydroxythreonine aminotransferase / 3-phosphoserine aminotransferase), soxS (dual transcriptional activator and participant in the removal of superoxide and nitric oxide), sroD asparagine synthetase A, asparagine synthetase B, carA (carbamoyl phosphate synthetase), carB (carbamoyl phosphate synthetase), ddlB (D-alanine-D-alanine ligase B), glucose 6-phosphate-1-dehydrogenase, asnA , deoA (thymidine phosphorylase / uracil phosphorylase), deoD (purine nucleoside phosphorylase deoD-type), dpiA (dual transcriptional regulator involved in anaerobic citrate catabolism), fis (GadE) controls the transcription of genes involved in glutamate dependent systems, gadW (GadW controls the transcription of genes " n &quot;, is a small DNA-binding and bending protein whose main role appears to be the organization and maintenance of nucleoid structure) the glabridin gene is involved in glutamate dependent system, gadX controls the transcription of genes involved in glutamate dependent system, glpF (GlpF glycerol MIP channel), ilvY (IlvY DNA-binding transcriptional dual regulator), ivbL (the ilvB operon leader peptide ), lhgO (L-2-hydroxyglutarate oxidase), lpd (Lipoamide dehydrogenase), lrp (Lrp is a dual transcriptional regulator for at least 10% of the genes in Escherichia coli. These genes are involved in amino acid biosynthesis and catabolism, nutrient transport, pili synthesis, and other cellular functions, including 1-carbon metabolism, metB (O-succinylhomoserine lyase / O-succinylhomoserine kinase / homoserine dehydrogenase), mraY (phospho-N-acetylmuramoyl-pentapeptide transferase), mraZ (Unknown function), murE (UDP-N-acetylmuramoylalanyl-D-glutamate 2,6-diaminopimelate ligase) -alanine-adding enzyme, murG (N-acetylglucosaminyl transferase), nac (Nacreulates, without a coeffector, nasA, quinolinate synthase, nsrR, (pantothenate synthetase), panD (aspartate 1-decarboxylase), pgl (6-phosphogluconolactonase), pyrB (aspartate carbamoyltransferase, PyrB subunit), pyrC (dihydroorotase) , pyrL (aspartate carbamoyltransfer rase, PyrI subunit), rob (Rob is a transcriptional dual regulator. Its N-terminal domain shares 49% identity with MarA and SoxS. These proteins activate a common set of about 50 target genes, such as rAp (ribulose phosphate 3-epimerase), thalA / transaldolase A), thrA (aspartate kinase / homoserine dehydrogenase), thrB (homoserine kinase), thrC throne synthase, thrL thr operon leader peptide, tktA transketolase I, tktB transketolase II and torR system, OmpR family, torCAD operon response regulator TorR).
The vector according to claim 1, wherein the sRNA construct is capable of binding to Hfq by mutation.
The vector according to claim 1, wherein the transcription terminator has a U tail length of 4 to 8.
A recombinant microorganism transformed with the vector of claim 1.
A method for fine-regulating mRNA expression of a target gene by introducing the vector of claim 1 into a prokaryote or expressing it in prokaryotes.
10. The method of claim 9, wherein the prokaryote is selected from the group consisting of E. coli, Rhizobium, Bifidobacterium, Rhodococcus, Candida, Erwinia, Enterobacter, But are not limited to, Pasteurella, Mannheimia, Actinobacillus, Aggregatibacter, Xanthomonas, Vibrio, Pseudomonas, Azotobacter, Acinetobacter, Ralstonia, Agrobacterium, Rhizobium, Rhodobacter, Zymomonas, Bacillus, Staphylococcus, A bacterial strain such as Staphylococcus, Lactococcus, Streptococcus, Lactobacillus, Clostridium, Corynebacterium, Streptomyces, Bifidobacterium, ) And between &Lt; RTI ID = 0.0 &gt; Cyclobacterium. &Lt; / RTI &gt;
A screening method for a target gene for producing a target substance comprising the steps of:
(a) fine-regulating the expression of any one or more genes among genes participating in a target substance biosynthetic pathway present in a target strain to be produced by the method of claim 9; And
(b) selecting a gene whose expression is regulated as a target gene for production of a target substance when the production yield of the target substance is improved according to the expression microcontrol.
A method for improving a target substance producing strain comprising the steps of:
(a) fine-regulating the expression of any one or more genes among genes participating in a target substance biosynthetic pathway present in a target strain to be produced by the method of claim 9; And
(b) screening a gene whose expression has been regulated as a target gene for production of a target substance when the yield of the target substance is improved according to the expression micro-regulation; And
(c) introducing the degree of expression of the screened gene to produce a recombinant strain.

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