KR101428566B1 - Recombinant fluorescent translational reporter vector for detecting promoter strength or expression level of target protein and uses thereof - Google Patents

Abstract

The present invention provides MCS1 (multiple cloning site 1), RBS (ribosomal binding site) gene for promoter insertion in the 5 '- > 3' direction, MCS2 a multi-cloning site 2) and a recombinant fluorescent transcription reporter vector operatively linked with a fluorescent protein coding gene, Escherichia coli transformed with the recombinant fluorescent transcription reporter vector, and the recombinant fluorescent transcription reporter vector, a method for screening a target of a self-regulating protein using a vector into which a candidate candidate gene for a self-regulating protein is inserted into MCS2 of the recombinant fluorescent transcription reporter vector, and a method for screening a target of the self- , The target of RNase III screened by the target promoter, the target promoter containing the recombinant fluorescent transcription reporter vector Fig or kits, and to analyze the expression of the target protein wherein the recombinant translation fluorescent reporter vector; And a kit for screening a target of a self-regulating protein comprising a self-regulating protein.

Description

Recombinant fluorescent translational reporter vector for detecting promoter intensity or expression amount of a target protein and use thereof {Recombinant fluorescent translational reporter vector for detecting promoter strength or expression level of target protein and uses thereof]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a recombinant fluorescent translational reporter vector for detecting a promoter strength or an expression amount of a target protein and a use thereof. More particularly, 1), a ribosomal binding site (RBS) gene, a multi-cloning site 2 (MCS2) containing a translational initiation codon (ATG) for gene insertion encoding a target protein, and a fluorescent protein coding gene are operably linked A method for analyzing the strength of a target promoter or the expression amount of a target protein using the recombinant fluorescent transcription reporter vector, E. coli transformed with the recombinant fluorescent transcription reporter vector, the recombinant fluorescent transcription reporter vector, Using a vector with the target gene of the self-regulating protein inserted into the MCS2 of the vector, Target, the recombinant fluorescent translated reporter vector for the analysis of expression of the strength or the desired protein of the target promoter, comprising a kit and the recombinant fluorescent translated reporter of the RNase III screened by the method and the method for screening a query targeting vector; And a kit for screening a self-regulating protein target comprising a self-regulating protein.

Gene regulation studies have focused on the development of systems for the detection of target genes or operons by analyte proteins. Color reporter genes are widely used for identification of promoters in various organisms and gene expression analysis as a target (Hughes KT, Maloy SR. 2007 Methods Enzymol. 421: 140-158). Two different types of vectors have been described for constructing protein fusions: multicopy plasmid vectors and single-copy bacteriophage vectors. In general, although plasmid vectors are easier to handle than phage vectors, the use of multicopy vectors may require new host systems that will cause problems and reduce the plasmid copy number.

Bacteriophage vectors for single-copy production have been successfully used for chromogenic reporter assays (Sim et al. 2010 Mol. Microbiol. 75: 413-425). The vectors are easy to quantify, are not sensitive to specific environmental factors, have low background reactivity and increased sensitivity. In addition, numerous highly sensitive detection systems and antibodies are available for his analysis. However, there are some limitations to such chromogenic translation fusion: 1) a limited number of multiple cloning sites in the vector, 2) the need for lac - test strains for analysis, and 3) gene expression by a time- The need for chemical reactions for detection and color detection. Thus, there is a need for an alternative system to be developed for a color reporter system.

Several decades ago, several fluorescent protein genes became available as reporters for bacteria and eukaryotic systems, including the original green fluorescent protein (GFP) obtained from Aqua victoria and some derivatives thereof, (Cormack et al. 1996 Gene 173: 33-38) with both improved GFP (EGFP) or GFP uv . Further, colored derivatives of GFP such as EBFP (blue fluorescence protein), ECFP (cyan fluorescence protein) and YFP (yellow fluorescence protein) have been developed and widely used (Tsien RY.1998 Annu. Rev. Biochem. 67: 509-544 ). Unlike beta -galactosidase or a similar enzyme-based reporter system, a fluorescent protein is detected due to its presence, but not by its activity. Thus, signal generation is independent of a number of considerations that affect enzyme performance, and once formed, the protein is very stable, even if the cell loses its viability.

RNase activity is highly sensitive to environmental responsiveness and self-regulation, but accurate real-time monitoring or cleavage activity analysis is a challenging task. Furthermore, the proven RNase mutations available for activity assays are mostly in the lac + background, requiring multiple genetic manipulations to block direct analysis using available sources. Thus, the fluorescence system will be useful for easy monitoring of RNase activity and for identifying novel regulators in the absence of any chemical reaction when cleavable sequences of RNA are incorporated into the vector.

In the present invention, a strategy has been developed for both the target screening of proteins in vivo and the monitoring of the activity of a promoter or protein in a bacteriophage translation fusion vector expressing a fluorescent protein as a reporter. In addition, we found a heterologous bacillus promoter (BP) that stably expresses in E. coli cells and expresses the reporter gene more efficiently than the T7 promoter without an inducer. An RNase III activity monitoring system was set up by combining the BP and RNase III target signals in the system. The system was verified by monitoring the absence or down regulation of RNase III activity in vivo and revealing novel targets of RNase III from E. coli libraries. In addition, the present invention demonstrated that the RNase III target signal identified from screening is regulated after transcription by RNase III. It is anticipated that the systems and strategies presented herein will be used extensively in the study of both promoters and proteins of interest that remain sensitive to environmental stress and are challenging tasks due to active or quantitative self-regulation.

Korean Patent Laid-Open Publication No. 2006-0036561 discloses 'a promoter gene sequence of taurine transporter and its cloning', and Japanese Patent Laid-Open No. 2001-204474 discloses a 'cloning vector using a green fluorescent protein' The recombinant fluorescent translational reporter vector capable of detecting the intensity of the promoter or the expression amount of the target protein as in the present invention and its use have not been disclosed.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a method for detecting the activity of a protein utilizing a chemical compound capable of analyzing a simultaneous expression pattern of a promoter and a protein, An unnecessary efficient bacterial fluorescent transcription reporter system was developed, and the intensity of the bacillus promoter (BP) was analyzed using the vector. A bacillus promoter (BP) and a candidate gene for RNase III were inserted into the vector and the transformed Escherichia coli And confirming the target of screened RNase III by a method of quantifying the amount of fluorescent protein.

In order to solve the above-described problems, the present invention provides a method for introducing a promoter in a 5 'to 3' direction, comprising: a cloning site 1 (MCS1) for insertion of a promoter; a ribosomal binding site (RBS) ATG), and a recombinant fluorescent translational reporter vector in which a fluorescent protein coding gene is operably linked.

The present invention also provides Escherichia coli transformed with the above recombinant fluorescent transcription reporter vector.

Also, the present invention provides a method for producing a recombinant vector, comprising the steps of: inserting a desired promoter into MCS1 of the recombinant fluorescent transcription reporter vector; or inserting a target protein coding gene into MCS2; And quantifying the amount of the fluorescent protein of the transformant. The present invention also provides a method for analyzing the strength of a target promoter or the expression amount of a target protein.

In addition, the present invention provides a method for producing a recombinant vector, comprising: inserting a target candidate gene of a self-regulating protein into MCS2 of the recombinant fluorescent transcription reporter vector; And a step of screening a target of the self-regulating protein, comprising the step of adding a self-regulating protein to said transformant and then quantifying the amount of fluorescent protein, and a target of RNase III screened by said method.

In addition, the present invention provides a kit for analyzing the strength of a target promoter containing the recombinant fluorescent transcription reporter vector or the expression amount of a target protein.

Further, the present invention relates to the above recombinant fluorescent transcription reporter vector; And a kit for screening a target of a self-regulating protein comprising a self-regulating protein.

The present invention relates to a first translation reporter system in which a fluorescent protein is introduced into a bacterial system, and since it is a single-copy bacteriophage phage vector having a small background signal and sufficient expression of EGFP as a fluorescent protein at about 10 copies, And it can be used as a chromosome fluorescence expression reporter. Furthermore, even small changes can be detected and it can be used as a variety of environmental change reporter system such as self-regulation or activity detection for micro-regulated protein and target detection.

In addition, the Bacillus promoter BP and the ribosome binding site (RBS) can be inserted into the vector, so that efficient expression without restriction on the host Escherichia coli species can be observed. Unlike the T7 promoter required for the conventional IPTG inducer, the BP promoter It is possible to produce a large amount of protein at the same time while reducing the consumption of reagents in various production processes by exhibiting an expression intensity of 6 times or more without any inducer, which is a very useful invention in terms of economy.

Figure 1 shows a schematic representation of the plasmid sequences prepared in the present invention. (A) pRS1553, (B) pRSK1, (C) pRSK2, (D) pRSK3 and (E) pRSK3-BP. The specific restriction enzyme site was designated as the enzyme name. RBS represents a ribosome binding site for translation in (C). MCS1 and MCS2 denote the group of multiple cloning sites 1 and 2, respectively. MCS1 can be used for promoter analysis, while MCS2 can be used mainly for protein function analysis.
Figure 2 shows screening of bacillus promoters expressing strong fluorescence. Screening of bacillus promoters in the pBGRl library by (A) GFP as a reporter or (B) DsRed. (C) DNA sequence analysis results of a strong-intensity clone sequenced by KSKRI21 primer. Each of the regions or the direction of the bold type Bacillus amyl Lowry query Pacific Enschede (Bacillus amyloliquefaciens ) indicates the direction of the promoter region in the corresponding region or the proximity gene in the NCBI database of FZB42. (D) DNA sequence of bacillus promoter (BP). The underlined sequence indicates the corresponding sequence in (C).
Figure 3 shows the characterization of the promoter BP in E. coli cells. (A) the sequence of the T7 promoter. The correct insert Eco RI / Nco I for T7 was indicated. (B) Promoter activity. -IPTG and + IPTG indicate the absence or addition of 1 mM IPTG on LB plates. A single colony derived from a transformant of Escherichia coli BL21 (DE3) strain was redrawn on the plate. (C) Quantitative analysis of (B). The relative intensity of EGFP represents the median value of EGFP expression in colonies measured by Multi Gauge (Fujifilm), and the fluorescence / OD ₆₀₀ is presented. All quantification was performed in triplicate (n = 3) and the values were averaged.
Figure 4 is a graph showing the effect of RNase III on bdm Indicating the regulation of the gene. (A) Effect due to the presence of RNase III on bdm -EGFP expression. Transformants of pRSK3-BP- bdm in BW25113 ( rnc + ) or BW25113 ( rnc14 ) were redrawn on LB / Amp plates. (B) Effect of down-regulation of RNase III activity by YmdB on bdm -EGFP expression. From BW25113 (rnc +) pCA24N (- gfp versions) or ASKA- ymdB (14) were plated with the material on LB / Amp / Cm to the transfection 1 mM IPTG for transformants in the presence or absence of pRSK3-BP- bdm. (C) Quantitative analysis of (B). Image analysis was performed for (A) and (B) by LAS-3000 image analyzer. Relative intensities were defined as the mean EGFP level from 5 independent colonies in three independent experiments and presented representative data.
5 shows a hemL RNase III- dependent expression in vivo. (A) rnc + or rnc14 Expression of pRSK3-BP- hemL- derived EGFP in the background. By colony quantification using the image analyzer LAS-3000, rnc14 than rnc + Relative expression in the background was 2.2-fold higher. (B) qRT-PCR analysis of hemL . qRT-PCR was performed using the homologous gene strains of (A) and the indicated set of primers (+20- + 171, + 536- + 610, and + 1101- + 1281) as indicated by the arrow. The multiples of hemL -change represent the mean exon (n = 3) of qRT-PCR of hemL at rnc14 + to rnc + for each primer set normalized to 16S RNA. (C) pRSK3-BP- hemL hemL The RNA structure predicted by RNADraw in the region (+ 314- +431) was presented.

In order to accomplish the object of the present invention, the present invention provides a method for inserting a gene encoding MCS1 (multi cloning site 1), RBS (ribosomal binding site) gene for promoter insertion in the 5 '→ 3' A recombinant fluorescent translational reporter vector operably linked to MCS2 (multi cloning site 2) and a fluorescent protein coding gene containing codon (ATG).

In order to see the promoter activity in the vector system using the bacteriophage, most of the transcription reporter system was utilized. However, since the present invention utilizes the translation reporter system, the background signal is few and the small change can be detected. Do. Up to now, as a translational reporter system, there has been known a vector having a large size of 10 kb or more, which is limited to 2-3 DNA restriction sites capable of cloning a gene. However, as in the present invention, No vector had a small size of 6kb with 9 enzyme sites. In addition, the promoter sequence of the Bacillus strain detected in the present invention and the artificial ribosome binding site capable of enhancing the expression were inserted into the vector, and the expression was efficiently observed in Escherichia coli, which is not Bacillus species. Because it is not an element in E. coli, it is a system that is less sensitive to environmental changes in E. coli.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell.

The promoter used in the vector of the present invention can be used without limitation as long as it is a promoter capable of inducing expression in host cells containing bacteria.

In the present invention, "MCS (multiple cloning site) " refers to a DNA fragment having various restriction enzyme sites, which is recognized as a specific restriction enzyme and thus cleaved, thereby allowing the insertion of the desired gene into the cleaved MCS region. The MCS that can be used in the vector of the present invention can be used without limitation as long as it is an MCS known in the art and can be obtained from various vectors known in the art having MCS (for example, pUC18, pUC19, etc.).

The term "operably linked" in the present invention means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. That is, the gene coding for the target protein can be linked so that its expression can be regulated by a promoter in the vector. Examples of the gene coding for the target protein used in the present invention include those in which an experimenter wishes to clone in host cells containing bacteria, to express them in host cells containing bacteria, or to screen in host cells containing bacteria You can use all the genes.

In a vector according to an embodiment of the present invention, the MCS1 may include EcoR I, Sma I and Sac I restriction sites in the 5 'to 3' direction, but is not limited thereto.

In a vector according to an embodiment of the present invention, the MCS2 may include Nco I, Kpn I, Xba I, Xho I, Hind III and Bam HI restriction sites in the 5 '→ 3' direction, Do not.

In a vector according to an embodiment of the present invention, the MCS1, RBS, and MCS2 may consist of the nucleotide sequence of SEQ ID NO: 1, but are not limited thereto.

In a vector according to an embodiment of the present invention, the vector may detect the strength of the promoter or the expression amount of the target protein, but is not limited thereto.

In a vector according to an embodiment of the present invention, the fluorescent protein coding gene can include a termination codon at the 3'-end, but is not limited thereto.

In a vector according to an embodiment of the present invention, the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), cycle 3 variant of GFP, enhanced blue fluorescent protein (EBFP) cyan fluorescent protein) or YFP (yellow fluorescent protein), preferably EGFP.

In a vector according to an embodiment of the present invention, the vector may further include, but is not limited to, the Rop gene consisting of the nucleotide sequence of SEQ ID NO: 2.

The Rop (repressor of primer) protein expressed from the rop gene of the present invention is a protein expressed in E. coli having 63 amino acid sequences. It is a protein that regulates the amount of plasmid by ColE1 and pHMB1 replication origin found in the high-copy number plasmid It is known as protein. The mechanism is that ColE1 and pHMB1 encode two complementary RNAs, and RNAI and RNAI, the primer RNA precursors, bind to each other to lower transcription initiation and control intracellular copy number.

In the vector according to one embodiment of the present invention, the vector may be the vector described in E of Fig. 1, but is not limited thereto.

In the vector according to an embodiment of the present invention, the bacillus promoter (BP) comprising the nucleotide sequence of SEQ ID NO: 3 may be inserted into the MCS1, but the present invention is not limited thereto. The bacillus promoter (BP) shows stronger activity than the T7 promoter of bacteriophage, which has been widely used for protein overproduction. The disadvantage of the T7 system is that IPTG must be added for overexpression, but the BP promoter has increased its activity by a factor of 6 or more without the addition of IPTG. In the case of the existing T7 promoter, only E. coli having the DE3 gene can be used as a host cell. However, the bacillus promoter BP isolated from the present invention has an advantage that all E. coli can be used as a host cell regardless of the DE3 gene. Therefore, it is possible to produce a large amount of protein while simultaneously reducing reagent consumption in various production processes.

Also, variants of the promoter sequence are included within the scope of the present invention. The mutant is a nucleotide sequence having a functional characteristic similar to that of SEQ ID NO: 3, although the nucleotide sequence is changed. Specifically, the promoter comprises a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 3 .

The present invention also provides Escherichia coli transformed with the above recombinant fluorescent transcription reporter vector.

The transformation method may be carried out by methods known in the art, for example, but not limited to, heat shock method, calcium phosphate precipitation method, electroporation method, gene gun, (Wu et al., J. Bio. Chem., 267: 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).

The present invention also provides a method for producing a recombinant vector, comprising the steps of: transforming E. coli after inserting a desired promoter into MCS1 of the recombinant fluorescent transcription reporter vector; And quantifying the amount of fluorescent protein of the transformant. The present invention also provides a method for analyzing the strength of a target promoter. The amount of fluorescent protein to be expressed is determined according to the intensity of the target promoter. The more the intensity of the target promoter is, the more the amount of fluorescent protein to be expressed increases. Therefore, the intensity of the target promoter can be analyzed by quantifying the amount of fluorescent protein to be expressed.

The present invention also provides a method for producing a recombinant vector, comprising the steps of: (a) transforming Escherichia coli after inserting a target protein coding gene into MCS2 of the recombinant fluorescent transcription reporter vector; And quantifying the amount of the fluorescent protein of the transformant. Since the translation initiation codon ATG is contained in the Nco I site of MCS2 of the recombinant fluorescent transcription reporter vector, when the target protein coding gene is inserted into the restriction enzyme site after the Nco I site, expression of the desired protein and the fluorescent protein in a fusion form And quantifying the amount of fluorescent protein can analyze the amount of the expressed target protein.

In addition, the present invention provides a method for producing a recombinant vector, comprising: inserting a target candidate gene of a self-regulating protein into MCS2 of the recombinant fluorescent transcription reporter vector; And a step of adding a self-regulating protein to the transformant and quantifying the amount of the fluorescent protein. The present invention also provides a method for screening a target of a self-regulating protein.

In the method of the present invention, the self-regulating protein may be RNase III, TraM (a protein essential for conjugation of F plasmid), Ribosomal protein, acetyltransferase (acetylation control protein), RNase E, PNPase and other RNases such as RNase P , Preferably RNase III. As an example of RNase III, the target candidate gene for RNase III is inserted into MCS2 of the recombinant fluorescent transcription reporter vector of the present invention, and then E. coli is transformed. When the RNase III enzyme is added to the transformant, MCS2 If the inserted target candidate gene is a target of RNase III, it is cleaved by RNase III, so that the fluorescent protein is not expressed. However, when the target candidate gene inserted into MCS2 is not a target of RNase III, it is not cleaved by RNase III Fluorescent proteins are expressed. Therefore, a candidate gene inserted into MCS2 of a colony that does not express a fluorescent protein is targeted for RNase III. In this way, the target of the self-regulating protein including RNase III can be screened.

In addition, the present invention provides a target of RNase III consisting of the nucleotide sequence of SEQ ID NO: 4 screened by the above method.

The present invention also provides a kit for analyzing the strength of a target promoter comprising the recombinant fluorescent transcription reporter vector.

In addition, the present invention provides a kit for analyzing the expression amount of a target protein comprising the recombinant fluorescent transcription reporter vector.

In the kit, the method for analyzing the intensity of the target promoter or the expression amount of the target protein can be performed by quantifying the amount of the fluorescent protein, and thus the kit may include an apparatus for measuring the amount of the fluorescent protein. In addition, the kit may include an inducer such as IPTG depending on the type of the promoter, and may include E. coli as a host cell.

Further, the present invention relates to the above recombinant fluorescent transcription reporter vector; And a kit for screening a target of a self-regulating protein such as RNase III comprising a self-regulating protein such as RNase III.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Strain, oligo, compound  And enzyme

GT PCR Master Mix (Takara)에 의해 수행했다.The microbial strains used in the present invention are E. coli K12 strains DH5a, BL21 (DE3) and BW25113 (Datsenko KA, Wanner BL. 2000 Natl. Acad. Sci. USA 97: 6640-6645). Gram-negative bacteria Bacillus subtilis ) GB03 was used for the production of the promoter library. The RNase III mutant strain ( rnc14 ) in the background of BW25113 was transformed from the HT115 strain (Simons et al., 1987 Gene 53: 85-96) by the general P1 transduction (Miller J H. 1992 Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press ). The plasmids used in the present invention are listed in Table 1. The oligos used in the present invention, synthesized by Bioneer, Inc., are listed in Table 2. Plasmid DNA isolation (Bioneer, Inc), restriction enzyme digestion and ligation with T4 DNA ligase (Liga Fast ^™ Rapid DNA Ligation System, Promega) were performed as described in the manufacturer's protocol. All compounds including IPTG were purchased from Sigma-Aldrich, Inc. AccuPower Pfu PCR Premix (Bioneer) was used for gene amplification for cloning purposes. Colonic PCR by EmeraldAmp

GT PCR Master Mix (Takara).

- The mutation area is shown in bold.

- The underlined sequence represents the restriction enzyme site.

Building the Library

Bacillus standing was prepared with the chromosomal DNA of the subtilis strain GB03 as ^{G-Spin TM Genomic DNA extraction kit} (Intron). The promoter library of GB03 was prepared by Sau 3AI partial cleavage of chromosomal DNA, eluting about 0.3 kbp product from the agarose gel and digesting the Bam HI-linearized pBGR1 (Han et al. 2008 J. Microbiol. Biotechnol. 18 : 1634-1640). The RNase III target-library was prepared as follows: Complete digestion of genomic DNA from Escherichia coli BW25113 with Nco I and elution of the DNA fragment from 0.1 to 1.0 kb in size from the agarose gel followed by removal of the Nco I- Linearization ligation to pRSK3-BP.

DNA  Sequencing

Sequences of the constructs were confirmed by DNA sequencing (SolGent, Inc.) with oligo KSKRI11 or KSKRI12 for pRS or pRSK3 plasmids, respectively. Other vectors were sequenced using a suitable primer.

Detection of fluorescence

)를 이용해 기록했으며, EnVision Software(ver 1.0; PerkinElmer

)로 추가로 분석했다. 실험은 3 회 실험으로 실시했다.
Detection of fluorescent EGFP from colonies was carried out with Fastgene Blue Light Illuminator (Nippon Genetics Europe GmbH) or Luminescent Analyzer LAS-3000 (Fujifilm) with an exposure time of 0.25 seconds. Images were further quantified by Multi Gauge (ver 2.0) (Fujifilm). Expression of EGFP by bacterial growth and temperature stress was monitored at emission wavelengths from 485 to 535 nm and bacterial growth was monitored at 600 nm on 2102 EnVision ^TM Multilabel plate Reader (PerkinElmer

) And recorded using EnVision Software (ver 1.0; PerkinElmer

). The experiment was carried out in three experiments.

RNA  Separation and qPCR  analysis

Total RNA from BW25113 or BW25113 ( rnc14 ) at OD ₆₀₀ = 0.8 was extracted by RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Genomic DNA was removed by two sequential DNase treatments (cleavage on the column using RQ1 RNase-free DNase (Promega) and RNase-Free DNase set (Qiagen)) using the provided kit. Quantification of the extracted RNA was evaluated with a NanoDrop 1000 Spectrophotometer (Thermo Scientific). The integrity of the samples was determined by agarose gel (1.2%) electrophoresis and UV visualization. cDNA synthesis (20 μl reaction) was performed from an RNA sample (1 μl reaction) using a ReverTraAce qPCR RT Kit (Toyobo) as described in the manufacturer's instructions using random hexamer primers as primer oligomers. Cycle conditions were set as follows: 40 cycles of denaturation at 95 ° C for 3 minutes of initial template denaturation followed by denaturation at 95 ° C for 10 seconds, annealing at 60 ° C for 10 seconds, and extension at 72 ° C for 30 seconds. Following this cycle, a melting curve analysis was performed at a temperature ranging from 65 ° C to 95 ° C every 5 seconds at 0.5 ° C. This was done by analyzing the 16S rRNA and hemL mRNAs designed by PrimerQuest (www.idtdna.com) PCR was performed using 1 μl of the cDNA mixture by the IQ SYBR Green Supermix Kit (Bio-Rad) along with the gene specific primers and the CFX96 real-time PCR detection system (Bio-Rad). Baseline and limit values were automatically determined for all plates using the CFX manager software (ver 2.0, Bio-Rad) and the data obtained were analyzed using the -ΔΔCT calculation method using Microsoft Excel.

Example  1. Construction of translational fusion reporter plasmid

A translational fusion reporter system based on pRS1553 (Pepe et al. 1997 J. Mol. Biol. 270: 14-25) was constructed by the following steps. First, the sequence between the +132 to +137 regions of pRS1553 was mutated to have the Bam HI site. Second, the ribosome binding site and a portion of the protein coding region of LacZ were removed by Bam HI cleavage. Third, the vector was re-ligated to yield pRSK1, wherein the eighth codon of LacZ is the first LacZ coding region. For the production of a universal translation fusion vector for reporter analysis, the multi-cloning site of the promoter (MCS1; Eco RI- Sma I- Sac I), a ribosome binding site (Thomas et al 1982 Gene 19: 211-219 from strain). , and another multiple cloning site (MCS2; Nco I- Kpn I- Xba I- Xho I- Hin dIII- Bam HI) insert the annealing of an oligonucleotide synthesis between the Eco RI and Bam HI sites of pRSK1 containing Lt; RTI ID = 0.0 > pRSK2 < / RTI > as a result. The novel translational fusion plasmid pRSK2 is advantageous compared to the known chromosome translation fusion plasmid pRS414 (Simons et al. 1987 Gene 53: 85-96), which has only three multiple cloning sites for promoter analysis. The details of the vector are shown in Fig.

The vector or chromosome fusion of lacZ is widely used and the system of the present invention can also be used for lacZ expression analysis. However, when introducing a fluorescent reporter gene based on pRSK1 or pRSK2 for analysis of promoter activity, useful. For that purpose, EGFP is known as soluble and expresses fluorescence in E. coli with high quantum yield, so the present inventors used the protein as a target fluorescent protein. PRSK3 was constructed by introducing an EGFP open reading frame region with a stop codon at the end of the C-terminal region into the Bam HI / Hin dIII site of pRSK2 (Fig. 1D).

Example  2. Interspecific  Screening and analysis of bacterial-derived promoters

General environmental stresses such as bacterial growth are a problem in the development of generic modules for reporter screening systems. For example, a sudden increase in the expression of reporter by growth prevents the understanding of subtle differences in the gene of interest. In order to elucidate the promoter sequences of strong promoter activity that are less sensitive to the environment, a general module of a gene expression system using a novel promoter of Bacillus in Escherichia coli was constructed by devising a search for a promoter region from non-host bacteria. A bi-directional promoter-trap system (Han et al. 2008 J. Microbiol. Biotechnol. 18: 1634-1640) was used to screen the Bacillus subtilis promoter to reveal the promoter sequences that meet our requirements . The system was useful for monitoring the DsRed or GFP expression promoter. For this purpose, Bacillus subtilis The Sau 3AI -treated chromosomal DNA library of GB03 (size 300-500 bp) was prepared and incorporated into the Bam HI-linearized pBGR1 vector. Three independent screenings of transformants expressing more GFP or DsRed than the control vector in E. coli (about ¹⁰⁷ clones) were performed. Twelve colonies (BGR series) were selected as positive clones from screening of GFP or DsRed, but only four colonies (BGR1, 2, 4 and 12) were found to be genuine positive after re-transformation of plasmid DNA (Figure 2A) , The clones were further analyzed by DNA sequencing. It was found that three colonies (BGR2, BGR4 and BGR12) strongly emitting fluorescence contain plasmids having the same sequence (Fig. 2B). The sequence is Bacillus amyloluria quupaciens amyloliquefaciens ) A portion of the intergenic region between ywcA and gspA located at 84 bp on the 5 'side of gspA corresponding to FZB42 , and is shown in Figure 2C. We have found that the BLAST search results of these species are due to the lack of a complete Bacillus subtilis GB03 genomic sequence in the database and that the inventors have found that the region from the GB03 sequence partially corresponds to the FZB42 sequence. In addition, due to the strongest fluorescence signal, BGR2 was used as a representative of these, and this promoter was named BP as an acronym for the Bacillus promoter.

We further tested whether pRSK3 is efficient for promoter analysis. For that purpose, two promoters, bacteriophage T7 promoter and BP,EcoRI /NcoI orEcoRI /SheetI Lt; RTI ID = 0.0 > Native or synthetic ribosome binding site (RBS) (Figure 3A). To determine whether EGFP is effectively expressed and how the promoter activity differs between the two promoters, pRSK3, pRSK3-T7 or pRSK3-BP plasmids were transformed into E. coli BL21 (DE3) cells, and the effectiveness of each promoter was tested by the fluorescence intensity. The expression of EGFP from colonies in the presence or absence of IPTG was compared by quantification using an image analyzer and the control vector itself was found to only show about 7% of pRSK3-BP in the absence and presence of IPTG ( 3B). Interestingly, the promoter activity of BP was about 6-fold higher than that of the T7 promoter in the absence of IPTG, while the activity was about 2.0-fold lower than that of the T7 promoter in the presence of IPTG (FIG. 3B). The data of the present invention showed that BP itself is much more potent than the T7 promoter sequence and is useful for constitutive gene expression without any inducible material (3C). These results suggest that promoters from species that are distant from the target of interest are more effective in expressing the gene of interest of the target species for the cell signal. Among other species, there may be other promoters of the same purpose that have not yet been revealed.

Example  3. RNase III  activation Monitoring  Use of vectors for

Although the fluorescent reporter system differentiated the promoter activity (Figure 2), the vector was further used for RNase activity using a sequence with a cleavage site inside pRSK3-BP. At present, numerous RNase-active reporters are self-regulating regions of RNase that are fused to the lacZ gene itself (eg, rne :: lacZ or rnc' - lacZ ) (Jain et al. 2002 Mol. Microbiol. 43: 1053 -1064). Thus, it is difficult to assess the effectiveness of cellular factors on RNase activity due to the self-regulatory effect of the reporter itself and the basic level of lacZ activity. To eliminate the inconvenience of the factors that monitor known reporter assays, the known RNase III cleavage target, the bdm gene of E. coli (the amino acid from the initiation codon to the termination codon without the stop codon) (Sim et al. 2010 Mol. Microbiol. 75 : 413-425) was incorporated into the Xba I / Xho I site of the pRSK3-BP vector. The fluorescence from pRSK3-BP- bdm in the rnc14 mutant strain was found to be about 6 times more potent than that of rnc + (Fig. 4A). Overexpression of the known RNase III modulator, YmdB (Kim et al. 2008 Genes. Dev. 22: 3497-3508), however, resulted in 2.6-fold higher EGFP from pRSK3-BP- bdm than in the absence of YmdB in the presence of IPTG (Fig. 4B). The data of the present invention suggests that the reporter system of the present invention is suitable for monitoring subtle differences in in vivo RNase III activity and can be used to identify RNase III modulators acting at specific cellular stresses.

Example  4. New  Escherichia coli RNase III  Screening of targets

The vector of the present invention could be used for screening novel RNase target RNA. For this purpose, the Nco I cleavage-library of E. coli was constructed by completely cutting the chromosomal DNA with Nco I restriction enzyme. When analyzing the number of NcoI recognition sites on E. coli K12 MG1655 chromosomal DNA, we found that 612 cleavage sites were available. After E. coli chromosomal DNA was completely digested with Nco I, the 0.1-1 kbp band of the library was further eluted, followed by ligation to Nco I-linearized pRSK3-BP and transformation into the rnc + strain. From about 10,000 colonies from the independent screening, non-fluorescent colonies on the LED detector were first screened, followed by retransforming them with the rnc14 mutation, followed by selection of more robust fluorescent colonies than pRSK3 itself (data not shown). Eight colonies were selected from the initial screening. The coding region of hemL of E. coli MG1655 chromosome (8 hits; 174452-174569) was found to be incorporated into the Nco I site of the pRSK3-BP plasmid, respectively. Since re-transformation of each plasmid shows the same result, this is not caused by genetic mutation in the chromosome (data not shown). Interestingly, these regions have not been reported with the RNase III target of E. coli. The hemL gene, which is a gene encoding glutamate-1-semialdehyde aminomutase , Expression of EGFP in the rnc14 background is about 2.0-fold higher (Figure 5A), suggesting that both genes are targets of RNase III. There was additional doubt as to whether EGFP expression actually originated from transcriptional levels. The steady-state levels of hemL were measured by qRT-PCR of three different pairs of primers in the rnc + or rnc14 background (Fig. 5B). HemL levels were doubled with rnc14 samples in all primer sets. The secondary structure of the RNA was predicted by RNADraw ( www.rnadraw.com ) and it was found that the hemL sequence contains multiple branched-ring structures known as sRNase III targets (Fig. 5C). All data of the present invention suggest that hemL is a post-transcriptional target of RNase III and may affect the expression of HemL and its function. Using the system of the present invention, an unknown cell RNase III target can be uncovered in the bi-directional library even in less than 2 days, even by a factor of two.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Recombinant fluorescent translational reporter vector for          detecting promoter strength or expression level of target protein          and uses <130> PN12046 <160> 25 <170> Kopatentin 2.0 <210> 1 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> MCS1 RBS MCS2 <400> 1 gaattccccg gggagctcca ggaagaaatc catggtaccc tctagactcg agaagcttgg 60 atccgtc 67 <210> 2 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> rop <400> 2 gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta 60 acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg 120 cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt 180 gaaaacctct ga 192 <210> 3 <211> 139 <212> DNA <213> Bacillus subtilis <400> 3 ggatcttaca cattttttct gcgggaatgt gctgtttgcg atttgatatc ttttgcattc 60 tctgttaaaa ttcatttcat agctaatgat tctgacaata aaaaggagca tgacgccatt 120 gacgaatcgc aacgatgcc 139 <210> 4 <211> 118 <212> RNA <213> Escherichia coli <400> 4 ccauggauau ggugcgcaug gugaacuccg gcacugaagc gaccaugagc gccauccgcc 60 uggcccgugg uuuuaccggu cgcgacaaaa uuauuaaauu ugaagggugu uaccaugg 118 <210> 5 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaccatgatt acggattcag gatccgtcgt ttacaacgtc g 41 <210> 6 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cgacgttgta aacgacggat cctgaatccg taatcatggt c 41 <210> 7 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aattccccgg ggagctcagg aagaaattcc atggtaccct ctagactcga gaagcttg 58 <210> 8 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gatccaagct tctcgagtct agagggtacc atggaatttc ttcctgagct ccccgggg 58 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccaagcttat ggtgagcaag ggcg 24 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 cgggatcctt acttgtacag ctcgtcc 27 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cggaattcgg atcttacaca ttttttct 28 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gcgtgagctc ggcatcgttg cgattcgt 28 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 cggaattcgg ttttgcgcca ttcga 25 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tacccacgtg atggtggtgg tgatg 25 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ctgcgcaact gttgggaagg gcg 23 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cgtccatgcc gagagtg 17 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ctgaaaatct ttacagcgca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ccgacataat cgatataggc 20 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gtacttataa tgatctggct tct 23 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cgacgataat acaggcaat 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ctatcaggat gtgatggcct 20 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tcacaacttc gcaaacaccc g 21 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 gctacaatgg cgcatacaaa 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ttcatggagt cgagttgcag 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 tgaactcggt gatgacgttc 20

Claims (19)

(MCS1) (multi cloning site 1) for promoter insertion in the 5 'to 3' direction, an RBS (ribosomal binding site) gene and a translation initiation codon (ATG) for insertion of a gene encoding a target protein 2) and a recombinant fluorescent translational reporter vector in which a fluorescent protein coding gene is operably linked. 2. The recombinant fluorescent translational reporter vector according to claim 1, wherein the MCS1 comprises Eco RI, Sma I and Sac I restriction enzyme sites in the 5 '→ 3' direction. The recombinant fluorescent translational reporter vector according to claim 1, wherein the MCS2 comprises Nco I, Kpn I, Xba I, Xho I, Hind III and Bam HI restriction sites in the 5 '→ 3' direction. The recombinant fluorescent translational reporter vector according to claim 1, wherein the MCS1, RBS, and MCS2 comprise the nucleotide sequence of SEQ ID NO: 1. 2. The recombinant fluorescent translational reporter vector according to claim 1, wherein said vector is capable of detecting a promoter strength or an expression amount of a target protein. The recombinant fluorescent transcription reporter vector according to claim 1, wherein the fluorescent protein coding gene comprises a termination codon at the 3'-terminal. The method of claim 1, wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), cycle 3 variant of GFP, enhanced blue fluorescent protein (EBFP), enhanced cyan fluorescent protein Wherein the vector is a YFP (yellow fluorescent protein). 2. The recombinant fluorescent translational reporter vector according to claim 1, wherein the vector further comprises a Rop gene consisting of the nucleotide sequence of SEQ ID NO: 2. 2. The recombinant fluorescent translational reporter vector according to claim 1, wherein said vector is the vector described in E of Fig. 2. The recombinant fluorescent translational reporter vector according to claim 1, wherein a bacillary promoter (BP) comprising the nucleotide sequence of SEQ ID NO: 3 is inserted into the MCS1. 10. An E. coli transformed with the recombinant fluorescent transcription reporter vector of any one of claims 1 to 10. Transforming E. coli after inserting a desired promoter into MCS1 of the recombinant fluorescent transcription reporter vector of any one of claims 1 to 9; And
And quantifying the amount of fluorescent protein of said transformant. Transforming Escherichia coli after inserting a target protein coding gene into MCS2 of the recombinant fluorescent transcription reporter vector of any one of claims 1 to 10; And
And quantifying the amount of fluorescent protein of said transformant. 10. A method for producing a recombinant fluorescent transcription reporter vector, comprising: inserting a target candidate gene of a self-regulating protein into MCS2 of the recombinant fluorescent transcription reporter vector of any one of claims 1 to 10, and then transforming E. coli; And
A method for screening a target of a self-regulating protein, comprising the step of adding a self-regulating protein to said transformant and quantifying the amount of fluorescent protein. 15. The method of claim 14, wherein the self-regulating protein is RNase III. delete A kit for analyzing the strength of a target promoter comprising the recombinant fluorescent transcription reporter vector of any one of claims 1 to 9. A kit for analyzing the expression amount of a target protein comprising the recombinant fluorescent transcription reporter vector of any one of claims 1 to 10. 10. A recombinant fluorescent translational reporter vector according to any one of claims 1 to 10; And a kit for screening a target of a self-regulating protein comprising a self-regulating protein.

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