KR101222056B1 - Novel Method for Detecting and Quantitating Target Enzyme Activity Using Artificial Genetic Circuitry - Google Patents

Abstract

The present invention relates to a novel search and quantification method of the target enzyme activity using a redesigned gene circuit, and more particularly, to a novel enzyme activity using a redesigned gene circuit GESS (genetic enzyme screening system) for detecting phenolic compounds. A search and quantification method.
By using the enzyme activity detection method according to the present invention, it is possible to detect useful genes in various gene populations, including the environment or metagenomic library, and to quickly and sensitively detect various enzyme activities using sensitivity and expression control for phenol derivatives. As it can be detected and quantified, it can be usefully used for protein engineering technology for enzyme improvement. Especially, it can be applied to molecular evolution technology because it can quantitatively search enzyme activity.

Description

Novel Method for Detecting and Quantitating Target Enzyme Activity Using Artificial Genetic Circuitry

The present invention relates to a novel search and quantification method of the target enzyme activity using a redesigned genetic circuit, and more particularly, to a new search and quantification method of the target enzyme activity using a redesigned genetic circuit for detecting phenolic compounds. .

Biocatalyst is recognized as a key material of "sustainable economic development" such as energy and clean production technology of industrial raw materials, and various efforts are being made to search for enzymes with new chemical reaction, specificity and stability. While there have been known methods for the rapid detection of antibiotic resistance genes, growth essential enzymes, amylases, lipases, proteases and other industrial enzymes in solid media, most of the enzyme functions useful as biocatalysts require time and cost. It depends on the individual activity analysis technology required.

Recently, researches applying directed evolution to secure new gene sources from microbial genomes or metagenomes, or to improve the activity of existing genes and develop them into highly useful biocatalysts have been conducted. It is emerging as an important strategy, and thus requires the emergence of new high-speed technologies for detecting trace amounts of enzyme activity.

To detect new enzyme genes in large gene libraries such as microbial genomes and metagenomes, first, sequence-based screening technology is used to identify DNA sequences, perform PCR reactions, and obtain amplified genes. (Richardson et al., (2002) J. Biol . Chem . 277: 26501-26507; Piel et al., (2002) PNA S 99: 14002-14007). As the genomic information is rapidly increasing, its utility is increasing day by day, and there is an advantage that it can select only the desired gene specifically, but it can be used only when the exact information about the nucleotide sequence of the target gene is known. There is a drawback that the applicable subject is limited to a part of the genetic source.

On the other hand, activity-based screening, which selects genes based on gene function, ie enzyme activity, is also widely used (Courtois).meat get., (2003)Appl . Environ . Microbiol . 69: 49-55; Vogetmeat get., (2003)Appl . Environ. Microbiol . 69: 6235-6242; Gabormeat get.,Environ . Microbiol .(2004) 6: 879-886). The enzymatic activity of this method is mainly used to isolate microorganisms directly from environmental samples such as soil, rivers, plant wastewater, seawater and forests. Only a very small amount, less than%. Recently, DNA was isolated directly from environmental samples without culturing microorganisms (Pettit, Robin K., (2004)Cancer Chemother . Pharmacol . 54: 1-6; Zhoumeat get ., (1996)Appl . Environ . Microbiol .62: 316-322) Strategies for constructing genetic resources in the form of metagenomic libraries have been actively studied. Therefore, there is a great interest in detection techniques for detecting industrially useful genes in the metagenome library (Voget, S.meat get . (2003)Appl . Environ . Microbiol . 69 (10): 6235-6242).

On the other hand, high-speed analysis techniques of enzyme activity include 1) automated multiple analysis using a well plate, 2) observing color development or halo in solid media, and 3) selective separation using nutrient-deficient microorganisms. Is used. These methods have the advantage of being able to select the genes of the desired function clearly because they are based on the specific activity of the enzyme, but there is a restriction in the general purpose of the technology because one detection technology is required to match the individual enzyme activity. . In addition, the effect is further reduced when there is a problem such as low transcription, translation, or expression of foreign genes in the host cell or protein folding, secretion. Indeed, it is difficult to apply the above-mentioned high-speed analysis method because enzymes derived from genetic sources having high genetic diversity such as novel microorganisms and metagenomes and whose genetic characteristics are not known are very low. Accordingly, there has been a continuous demand for the derivation of a new high-speed search principle capable of detecting even very small amounts of enzyme activity with high sensitivity.

In this context, applying the principle of yeast 2-hybrid transcriptional activation, research on redesigned genetic circuitry that detects enzymatic activities such as intracellular proteases by genetic engineering techniques has received attention and published (Baker, K. et al . (2002) PNAS 99 (26): 16537-16542). Pseudomonas A redesign of the putida metabolic pathway has been reported to detect foreign enzyme activity using benzaldehyde produced intracellularly by the action of foreign benzylformate decarboxylase as a nutrient source (Henning, H. et. al . (2006) Appl . Environ . Microbiol . 72 (12): 7510-7517). Recently, new techniques for detecting enzymatic activity of foreign genes in recombinant E. coli by introducing transcriptional regulators derived from other microorganisms into recombinant E. coli have been actively studied. For example, if benzoate is made from benzaldehyde by the enzymatic action of exogenous xylC (benzaldehyde dehydrogenase) using a mutated regulatory protein (nahR) derived from P. putida , lacZα (α-fragment of β-galactosidase) or Genetic circuits have been developed to activate the expression of tetracycline resistance (tet ^r ) genes (Witholt et al., (2006) PNAS 103: 1693-1698). In addition, when dehydrochlorinase acts on γ-hexachlorohaxane (γ-HCH or lindane) to produce trichlorobenzene (TCB) using a mutated transcriptional regulatory protein (xylR) derived from P. putida , it activates the expression of lacZ in E. coli. New screening techniques for enzyme activity have also been reported (Mohn, WW et al . (2006) Environ . Microbiol . 8 (3): 546-555). In addition, efforts are being made to develop protein engineering techniques to control substrate specificity of regulatory proteins. For example, a molecular evolution by constructing a mutant library of regulatory protein HbpR that binds 2-hydroxybiphenyl (2-HBP) and FACS sorting of E. coli that binds 2-chlorobiphenyl (2-CBP) to activate fluorescent protein expression There has also been research to improve substrate specificity (Beggah, S. et. al . (2008) Microb . Biotechnol . 1 (1): 68-78).

As such, researches to discover new enzymes by constructing genetic circuits that detect reaction products based on regulatory proteins have emerged as new innovations in enzyme activity detection (Konarzycka-Bessler, M. and Jaeger, K). .-E. (2006) Trends Biotechnol. 24 (6): 248-250), these techniques are only a search for a small number of specific enzymatic activities with specificity of substrate-products and regulatory proteins. There are limitations that cannot be applied to a universal system. In other words, if a specific enzyme activity is required, there is a restriction that a regulatory protein suitable for the product of the reaction must be secured.

Another novel enzyme detection technique is SIGEX (substrate-induced gene expression screening) reported by Watanabe's research group in 2005 (Watanabe et al. , (2005) Nature Biotech . 23: 88-93). This technique is a principle of searching for promoters whose transcriptional activity is induced by the added substrate, so it is characterized by indirect gene detection technology, rather than direct enzymatic activity search. That is, enzyme functions not related to transcriptional activation are not detected by this method. This technique has the advantage of being able to process large samples in a short time because FACS can be used for analysis, but it is difficult to apply to metagenomic libraries containing large genes of 20 to 30 kb, and GFP activity depends on the gene direction of the library. Reaction may not occur (Ryu & Yun. (2005) Microb. Cell Factories , 4: 8-12).

On the other hand, research on a technique for detecting a phenolic compound is in progress (Korean Patent No. 10-0464068), but there has been no research applied to the search for various enzyme activities using the same.

The present inventors have studied a method for exploring the activity of various different enzymes. As a result, various phenol-releasing compounds that can liberate phenol in many enzymatic reactions are used as substrates. After fabricating the circuit, it was confirmed that the quantitative activity of the reporter, such as expression-induced fluorescence and antibiotic resistance, could be measured, thereby completing the present invention.

One object of the present invention is to (a) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a reporter protein downstream; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a redesigned genetic circuit for detecting a system compound; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting a phenolic compound liberated by an enzymatic reaction to detect activity of an expression-induced reporter protein.

Another object of the present invention is (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a microorganism containing the redesigned genetic circuit for detecting a system compound in chromosomal DNA or cytoplasm; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting a phenolic compound liberated by an enzymatic reaction to detect activity of an expression-induced reporter protein.

Another object of the present invention is (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a redesigned genetic circuit for detecting a system compound; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) provides a method for quantifying the target enzyme activity using a redesigned gene circuit comprising the step of quantifying the activity of the expression-induced reporter protein by detecting a phenolic compound released by the enzymatic reaction.

Another object of the invention is (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a microorganism containing the redesigned genetic circuit for detecting a system compound in chromosomal DNA or cytoplasm; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) provides a method for quantifying the target enzyme activity using a redesigned gene circuit comprising the step of quantifying the activity of the expression-induced reporter protein by detecting a phenolic compound released by the enzymatic reaction.

In order to achieve the above object, the present invention, (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces the expression of a reporter protein downstream; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a redesigned genetic circuit for detecting a system compound; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting a phenolic compound liberated by an enzymatic reaction to detect activity of an expression-induced reporter protein.

The present invention also relates to (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a microorganism containing the redesigned genetic circuit for detecting a system compound in chromosomal DNA or cytoplasm; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting a phenolic compound liberated by an enzymatic reaction to detect activity of an expression-induced reporter protein.

The present invention also relates to (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a redesigned genetic circuit for detecting a system compound; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) provides a method for quantifying the target enzyme activity using the redesigned gene circuit comprising the step of quantifying the activity of the expression-induced reporter protein by detecting a phenolic compound released by the enzymatic reaction.

The present invention also relates to (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a microorganism containing the redesigned genetic circuit for detecting a system compound in chromosomal DNA or cytoplasm; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) provides a method for quantifying the target enzyme activity using the redesigned gene circuit comprising the step of quantifying the activity of the expression-induced reporter protein by detecting a phenolic compound released by the enzymatic reaction.

By using the enzyme activity detection method according to the present invention, it is possible to detect useful genes in various gene populations, including the environment or metagenomic library, and to quickly and sensitively detect various enzyme activities using sensitivity and expression control for phenol derivatives. As it can be detected and quantified, it can be usefully used for protein engineering technology for enzyme improvement, and in particular, it can be applied to molecular evolution technology because it can quantitatively search for enzyme activity.

Figure 1 (a) shows the principle of detecting the enzyme activity (GESS: genetic enzyme screening system) using the redesigned genetic circuit according to the present invention, (b) according to the redesigned genetic circuit according to the present invention It is a schematic representation of the GESS vector, and shows the structure of the gene expression control region enlarged (TRF: transcriptional regulation factor, means a phenolic compound degrading enzyme regulatory protein according to the present invention) one; OpR: the TRF of the phenolic compound-decomposing enzyme regulatory proteins binding to sites leading to a downstream reporter gene expression, Px: a promoter which regulates expression of the phenolic compound-decomposing enzyme regulatory proteins; the P _R is the expression of the reporter Regulating promoter).
Figure 2 shows the construction of the redesigned genetic circuit according to the present invention, showing the construction of pGESS-T1 containing the fluorescent protein GFP as a reporter.
Figure 3 shows the construction of pGESS-T2 with RBSε and transcription terminator attached to the gene circuit in order to increase the expression sensitivity of pGESS-T1 and induce stable expression of regulatory and reporter proteins.
4 shows the results of fluorescence imaging of colonies to determine whether pGESS-T1 and pGESS-T2 are expressed in phenol.
5 is a diagram illustrating the effect of the expression amount of the reporter protein of GESS on the expression of the redesigned gene circuit, showing the construction of pGESS-T1-3 containing RBSε.
Figure 6 (a) is the value of the activity of each clone was measured by the fluorescent plate reader in the liquid medium, (b) is a picture of fluorescence image of the colonies in the solid medium.
Figure 7 is to examine the effect of the detection ability of pGESS to phenol when the expression level of dmpR used as a regulatory protein of pGESS, (A): construction process of pGESS-T3, (B): SDS-PAGE As a result of confirming the increase of the expression level of dmpR in the analysis, (C): It is the result of observing the change in activity.
8 is (a): Construction process of pGESS-T4 using GFP _UV as a reporter, (b): Figure showing difference in fluorescence change upon ultraviolet irradiation in the presence of phenol, and (c): Change in fluorescence sensitivity for each temperature The graph shown.
9 is a diagram showing the construction of pGESS-T5 using (A): an antibiotic resistance gene as a reporter, and (B): activity of pGESS-T5 in the presence of phenol.
10 is (a): construction of the fluorescent protein dual reporter pGESS-T6-RFP, and (b): activity is observed by fluorescence colony imaging of GFP and RFP.
Figure 11 (a): Construction of the fluorescent protein dual reporter pGESS-T6-Tet, (b): The change in the resistance of tetracycline and the change in GFP fluorescence sensitivity by phenol concentration was observed by the growth of colonies and fluorescence imaging.
Figure 12 shows the construction of pGESS-T7 in which the mutant dmpR in which amino acid glutamic acid 135 of wild type dmpR of pGESS-T2 was substituted with lysine was inserted.
13 is a result of verifying the increase in the detectability of the genetic circuit through the optimization of the enzyme activity detection reaction, (A) is a figure confirming the improvement of the detection ability for the phenol of pGESS-T2 and pGESS-T7, (B) In addition to the phenol of pGESS-T7, it was confirmed that o-nitrophenol and p-nitrophenol were improved in detection performance.
Figure 14 (A): E. coli strains containing pGESS to streaked in a solid medium containing the inducer phenol in order to build the optimal host E. coli of GESS to observe the difference in fluorescence sensitivity by colony imaging, (B) : Observed the fluorescence spectrophotometer of each pGESS-containing Escherichia coli strains cultured in phenol-added liquid medium, and (C): Figure showing the time when the highest fluorescence amount was detected by changing the induction time of incubation material by culture time. .
15 is (A): colony imaging in a solid medium to verify the performance and quantitative phenol of pGESS, (B): fluorescence spectrum obtained by measuring a sample incubated in a liquid medium, and ( C) Fluorescence flow cytometry (FACS) shows the performance and phenol dependence of phenol.
Fig. 16 confirms the performance and phenol dependence of pGESS using pGESS-T5, and is an illustration of antibiotic resistance when 0-1 mM of phenol was added (●: antibiotic concentration 0 ㎍ / ml, ○: antibiotic concentration 10 μg / ml, ▲: antibiotic concentration 20 μg / ml, Δ: antibiotic concentration 30 μg / ml).
Figure 17 is a (a): pGESS detectability of a variety of phenols was measured by fluorescence sensitivity, and (b): antibiotic resistance.
FIG. 18 shows the results of quantitative detection of various phenols of pGESS. (A): Quantitative phenols having additional residues at ortho- and meta-positions, (B) Quantitative quantities of phenols of pGESS The test is a measure of antibiotic resistance.
19 is a result of measuring the performance of pGESS quantitative detection ability (provisional (B)) using a phenol wastewater containing a large amount of phenolic compounds (provisional (A)), phenolic control (provisional (B)), (B) Is the result of GC / MASS analysis.
Figure 20 shows the results of measuring the activity of various enzymes using pGESS, (A) β-galactosidase (beta-galactosidase) derived from E. coli, (B) tyrosine-phenol lyase (TPL) derived from C. freundii , (C) metagenome-derived putative phosphatase, (d) Pseudomonas sp. The activity of the derived methyl parathion hydrolase (MPH) was analyzed by the fluorescence of pGESS.
FIG. 21 shows (a): tyrosine-phenol lyase activity was measured by fluorescence image of colonies in phenol-containing solid medium using pGESS. (B): fluorescence flow cytometer (FACS) in phenol-containing liquid medium. Figure analyzed (C): Analysis of tyrosine-phenol lyase (TPL) activity using tetracycline antibiotic resistance using pGESS.
Figure 22 is a result of quantitatively measuring the difference in the activity of enzyme using pGESS-T2, (A) pGESS-T2 enzyme activity of TPL (tyrosine-phenol lyase) derived from Citrobacter freundii and TPL derived from Symbiobacterium toebii It is a graph measured using, (b) is a graph measuring the amount of phenol produced by using the HPLC after the same strain the enzyme reaction.

Unless defined otherwise, 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 and the experimental methods described below are well known and commonly used in the art.

The present invention relates to a technique for easily and easily detecting various enzyme activities using a redesigned genetic circuit (GESS: genetic enzyme screening system).

Phenolic is not only widely included in various hydrocarbon resources such as petroleum, coal and lignin, but also widely included in artificially synthesized compounds. Therefore, the enzymatic reactions containing phenol residues as a reactive substance or a product are also very diverse. For example, phenolic compounds in 40,000 enzyme reactions registered in the Brenda database ( www.brenda-enzymes.info ) which provide a wide range of enzyme-related information. Enzyme species that favored reached 2,000 results (about 5% of the total list). The present invention relates to a technique for performing enzymatic reactions using various compounds bonded with phenol groups as synthetic substrates and for detecting genetically engineered free phenols. Therefore, the present invention is a general-purpose enzyme applicable to a wide variety of enzyme reactions. It is unique in that it is a search technique. In particular, nitrophenol compound (o / p-nitrophenol) is colorless when combined with other organic substances, but nitrophenol released from the substrate is colored yellow, so that various synthetic substrates can be used to quickly measure the activity of various hydrolases. Is developed. For example, about 500 kinds of nitrophenyl compounds are commercially available only in Sigma-Aldrich. However, since the substance has low self-absorbance and easily diffuses through the cell wall, the reactants of various cells are mixed with each other, which is not suitable as a method for detecting activity characteristics by cell or colony.

The phenolic substance is also not easily degraded in E. coli cells, so that even a small amount of the reaction product produced by the enzymatic reaction can be highly sensitive. This property is in line with the fact that IPTG (isopropylthiogalactoside), which is not degraded, is used as an expression inducer of the lactose promoter, and shows a strong induction effect even with a small amount of IPTG. Recently, the results of controlling the sensitivity and quantitative range of the promoter by controlling the intracellular stability of the reporter protein have been reported (Neuenschwander, M., et. al . (2007) Nature Biotech . 25 (10): 1145-1147). Phenol is cytotoxic, depending on the derivative, but in many cases up to about 100 ppm did not significantly affect E. coli growth.

As such, since phenol is not easily decomposed in E. coli cells, even a small amount of the reaction product generated by the enzymatic reaction can be sensed with high sensitivity, and various phenol-releasing compounds that can release phenol in the enzymatic reaction can be used as a substrate. In view of the above, the present inventors have designed a redesigned genetic circuit that detects phenols, and added a phenol group-containing substrate suitable for the purpose to the recombinant microorganism which introduced the same to detect phenols produced according to the function of the enzyme gene, thereby inducing expression. By quantifying the reporter's quantitative activity, such as fluorescence and antibiotic resistance, a method of detecting and quantifying enzyme activity was completed.

The present invention, in one aspect, (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of downstream reporter proteins; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating expression of the regulatory protein, a region for binding the regulatory protein to induce expression of a reporter gene downstream, and a promoter for regulating expression of the reporter gene. Providing a redesigned genetic circuit for detecting phenolic compounds; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting a phenolic compound liberated by an enzymatic reaction to detect activity of an expression-induced reporter protein.

Here, the step of preparing a recombinant microorganism in which the clone or gene library and the redesigned gene circuit for detecting the phenolic compound is introduced, provides the microorganism containing the redesigned gene circuit for the detection of the phenolic compound, the clone Or by introducing a gene library.

Thus, in another aspect, the present invention provides a kit comprising: (a) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating expression of the regulatory protein, a region for binding the regulatory protein to induce expression of a reporter gene downstream, and a promoter for regulating expression of the reporter gene. Providing a microorganism containing a redesigned genetic circuit for detecting a phenolic compound in chromosomal DNA or cytoplasm; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting a phenolic compound liberated by an enzymatic reaction to detect activity of an expression-induced reporter protein.

In the step (a), the redesigned genetic circuit for phenolic compound detection may be contained in the cytoplasm using a plasmid in the microorganism, or inserted into the chromosomal DNA.

The present invention, in another aspect, (a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of downstream reporter proteins; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating expression of the regulatory protein, a region for binding the regulatory protein to induce expression of a reporter gene downstream, and a promoter for regulating expression of the reporter gene. Providing a redesigned genetic circuit for detecting phenolic compounds; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting the phenolic compound released by the enzymatic reaction to quantify the activity of the reporter protein in which the expression is induced.

In still another aspect, the present invention provides a method for producing a protein comprising: (a) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating expression of the regulatory protein, a region for binding the regulatory protein to induce expression of a reporter gene downstream, and a promoter for regulating expression of the reporter gene. Providing a microorganism containing a redesigned genetic circuit for detecting a phenolic compound in chromosomal DNA or cytoplasm; (b) providing a clone or gene library comprising a gene encoding an enzyme to be searched; (c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism; (d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And (e) detecting the phenolic compound released by the enzymatic reaction to quantify the activity of the reporter protein in which the expression is induced.

In the present invention, the reporter gene and the promoter for regulating the expression of the reporter gene may be characterized in that they are operably linked.

In the present invention, the site where the phenolic compound degrading enzyme regulatory protein binds to induce the expression of a downstream reporter gene is linked to the phenolic compound degrading enzyme regulatory protein so that the downstream reporter gene can be expressed. It may be characterized by activating a promoter.

In the present invention, a gene encoding a regulatory protein that recognizes the phenolic compound and induces expression of a downstream reporter protein and a promoter that regulates the expression of the regulatory protein may be operably linked. have.

Figure 1 (a) shows the principle of detecting the enzyme activity using the genetic enzyme screening system (GESS) according to the present invention, (b) GESS vector of the redesigned genetic circuit according to the present invention Figure 1 shows a simplified representation of the structure of the gene expression control region enlarged, wherein, TRF is a transcriptional regulation factor (transcriptional regulation factor), the site encoding the phenolic compound degrading enzyme regulatory protein in the present invention (E.g., dmpR: gene encoding the positive regulator of the phenol catabolic pathway) Gene expression control site can be divided into three parts-P _x : promoter for controlling the expression of the phenolic compound degrading enzyme regulatory protein, OpR (Operator for reporter) by combining the phenolic compound degrading enzyme regulatory protein A site that induces expression of a downstream reporter gene, and P _R : a promoter that regulates expression of the reporter. In addition, additionally, the redesigned genetic circuit according to the present invention includes a ribosome binding site (RBS), a forward transcriptional terminator (t), a reverse transcriptional terminator (t), and the like. can do.

As shown in (a) and (b) of FIG. 1 as an example, "Artificial genetic circuit" in the present invention (i) recognizes a phenolic compound to detect the expression of a downstream reporter protein. Genes encoding inducing regulatory proteins; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating expression of the regulatory protein, a region for binding the regulatory protein to induce expression of a reporter gene downstream, and a promoter for regulating expression of the reporter gene. Means genetic constructs.

In the present invention, the "enzyme to be searched" will be an enzyme that can release a phenolic compound by an enzymatic reaction. In addition, the enzyme is, for example, alpha-glucosidase, beta-glucosidase, cellulase, glycosyl ceramidase, psopatase, phytase, esterase, lipase, uretanase, amidase, peptidase , Proteinases, oxidoreductases, phenol-lyases, dihalogenases, isomerases, monooxygenases or deoxygenases.

In the present invention, the "phenolic compound degrading enzyme regulatory protein" is a protein that regulates the expression of the phenolic compound degrading enzyme, and activates a promoter that regulates the expression of the phenolic compound degrading enzyme by detecting phenol, and is connected with such a promoter. It is a protein that induces the expression of a reporter.

Regarding the "phenolic compound degrading enzyme regulating protein", genes showing degradative activity of aromatic organic compounds such as phenol, xylene, toluene, and benzene are mainly found in Pseudomonas genus and Acinetobacter , and these genes include polygenes . It is composed of multifunctional operon to be expressed and is expressed by σ ⁵⁴ dependent transcriptional regulation. Representative transcriptional activators include XylR, DmpR, MopR, PhhR, PhlR, TbuT, etc. Among these, Pseudomonas XylR involved in toluene and xylene catabolism in putida (Ramos & Marques, (1997 ) Annu Rev Microbiol 51:... 341-372) and is involved in the DmpR phenol catabolism best known (Shingler et al . , (1993) J. Bacteriol. 175: 1596-1604. In particular, the use of XylR or DmpR to detect toluene, xylene or phenol contaminated with the natural environment has been studied in the concept of microbial biosensor (Ramirez et al. al . , (2004) USP Pat. No. (US 6,803,224 B2); Wise et al . , (2004) USP Pat. No. (US 6,773,918 B2)). This NtrC family expression regulator is a combination of a domain that recognizes activators such as phenol and xylene (A domain), a domain that has ATPase activity (C domain), and a domain that binds to DNA (D domain). It is composed. Therefore, in the absence of a phenol molecule, the A domain inhibits transcription. However, when the phenol molecule binds to inhibit the A domain, transcriptional activation functions by the C and D domains appear. Recently, studies on improving specificity through the use of specificity of the A domain for the detection of new substances and genetic engineering methods have been known (Pavel, H. et. al . (1994) J. Bacteriol . 176 (4): 7550-7557).

Therefore, the phenolic compound degrading enzyme regulatory protein preferably used in the present invention may be dmpR, or a variant thereof, which is a regulatory protein of P. putida- derived phenolic operon. dmpR is the σ ⁵⁴ -dependent transcriptional activity regulatory portion of the dmp operon gene which shows the degradation activity of aromatic organic compounds such as phenol, xylene, toluene and benzene. P. putida The derived dmp operon consists of 15 genes, among which dmpKLMNOP codes for the enzymes required for phenol hydroxylation, and dmpQBCDEFGHI codes for the metabolic pathway that degrades catechol intermediates. The expression of dmpKLMNOP operon was determined by dmpR, which is a σ ^{5 4} dependent transcriptional regulator. Activated in conjunction with the operator, dmpR The transcriptional regulator of itself is known to be σ ⁷⁰ dependent (Lee et al . , (1996) J. Biol . Chem . 271 (29): 17281-17286. In addition, the variant of dmpR is, for example, E135K, where the 135th E of the dmpR amino acid sequence is K-mutated, in addition to E172K, D135N, D135N / E172K, F65L, L184I, F42Y, R109C, L113V, D116N, F122L, K6E / F42S, Q10R / K117M, Q10R, D116G / K117R, and D116V variants have high affinity for phenols, so that various variants including single or multiple mutations at these positions or periphery, as well as variants of various dmpRs Will also be included within the scope of this invention.

In the present invention, as shown in (b) of FIG. 1, the "gene expression control site" is a part for regulating the entire redesigned gene circuit, and (i) the phenolic compound degrading enzyme regulatory protein as the transcriptional regulator Promoter for regulating expression, (ii) the site where the phenolic compound degrading enzyme regulatory protein binds to induce the expression of the downstream reporter gene, and (iii) the promoter for regulating the expression of the reporter gene. In the absence of a phenolic molecule, the phenolic compound degrading enzyme regulatory protein inhibits transcription, but when phenol molecules bind to inhibit the A domain, transcriptional activation function by the C and D domains is shown. ) Binds to the OpR (Operator for Reporter) site, whose activity is regulated with σ ⁵⁴ dependency.

In the present invention, "promoter" refers to a promoter that regulates the expression of a phenolicase degrading enzyme regulatory protein or a promoter that controls the expression of a reporter protein, for example, a promoter of dmpR or dmp operon of Pseudomonas or general protein expression. Promoter may be used, and high expression promoters including trc, T7, lac, ara promoters may be used for high expression of foreign proteins, and in particular, P _{hce, which} is a high expression vector that does not need an inducer, may be used.

Specifically, as shown in (b) of FIG. 1, as a promoter for controlling the expression of the reporter protein, σ- ⁵⁴ -dependent promoter (P _ECO ) of E. coli may be used. In addition to this, Pseudomonas depends on the host of pGESS Applicability to putida (P _PPU ), yeast (P _YST ) and the like will be apparent to those skilled in the art.

In the present invention, the redesigned gene circuit may include not only the promoter, but preferably RBS (Ribosome Binding Site) and / or transcription terminator that facilitates expression of the reporter protein. That is, as a site for regulating the expression of the regulatory protein, in addition to the promoter, it may include RBS and / or transcription terminator.

In general, protein expression starts with AUG (methionine) or GUG (valine), which is an initiation codon in mRNA. Is determined by a rich RBS (or Shine-Dalgarno (SD) sequence), which is known to differ in sequence from species to species (Stryer, L., (1995) Biochemistry, (4) th ed .) WH Freeman, Chapter 34, Protein Synthesis). Redesigned gene circuit built in the present invention and were using transcription factors in Pseudomonas as host E. coli of the gene circuit as well as the survival shape very different from the case of σ ^54-dependent gene expression, the σ ⁵⁴ binding site or the σ ⁵⁴ factor Since it is very different from E. coli, it is possible to use E. coli RBS (RBSε) or RBS (RBSx) which can be integrated into all strains to facilitate the expression of reporter protein in Pseudomonas RBS (RBS _PPU ) or host E. coli. T7 RBS may also be used as an embodiment of the present invention.

In the present invention, the transcription terminator may preferably be rrnBT1T2 or tL3, and besides this, any transcription terminator commonly used in the art may be used to constitute the present invention.

In the present invention, the reporter may be one or more selected from fluorescent proteins and antibiotic resistance genes. As the fluorescent protein, preferably, GFP, GFP _UV , or RFP can be used, and as long as the object of the present invention can be achieved, the fluorescent protein that can be used in the present invention is not limited to these examples. The antibiotic resistance gene may be a conventional antibiotic resistance gene such as genes resistant to antibiotics such as kanamycin, chloramphenicol and tetracycline.

In one embodiment of the present invention, the reporter may be a dual reporter consisting of both a fluorescent protein and an antibiotic resistance gene, a multiple reporter consisting of two or more reporters. In accordance with the present invention, any known method can be used to stepwise transform the metagenome library and phenol sensing redesign gene circuit into a suitable microbial host. In order to increase the efficiency of transformation, electroporation may be preferably used.

In the present invention, the gene encoding the enzyme to be searched may be provided in the form of a clone or gene library, in the form of a single gene, genomic library, metagenomic or metagenomic library applicable in the field of molecular biology research. The single gene may be provided in a form contained in a vector or a microorganism.

In the present invention, the redesigned genetic circuit may be provided in the form of a vector or a microorganism. In the present invention, the microorganism is preferably E. coli, yeast, or plant cells, animal cells and the like.

According to the present invention, the microorganism transformed by the gene circuit is treated with one or more substrates selected from phenol-releasing compounds which may liberate phenols by enzymatic reaction. Preferably, the substrate addition timing may be adjusted to separate the activation stage of the cell growth-redesigned gene circuit in order to optimize the enzymatic reaction. For example, the substrate treatment step may comprise recovering healthy microorganisms grown in nutrient media and treating the recovered microorganisms with the substrate in minimal media.

The nutritional medium or minimal medium will be applicable to a variety of media that can be used generally in the art.

Preferably, when the cell growth (OD ₆₀₀ ) of the transformed microorganism is about 1.5 to 4, the substrate may be treated to optimize the enzyme activation reaction, and more preferably, the enzyme reaction may be performed for 14 to 16 hours. The reaction can be optimized.

Phenolic cognitive transcriptional regulators are dependent on σ ^{54 and} work well in environments with limited nutrients (Sze et. al . , (1996) J. Bacteriol . 178: 37273735). In the case of liquid culture, growth was good in LB medium, but the reactivity of gene circuit was low. On the contrary, M9 medium had high reactivity, but it was inadequate for high-speed search due to slow growth rate. In addition, when the substrate is added at the beginning of the culture, the enzymatic reaction proceeds as it grows, and thus there is an error in comparing the enzyme activity due to the growth difference between cells. In an embodiment of the present invention to recover the cells when the cell concentration (OD ₆₀₀ ) is between about 1.5 to 4 after shaking culture at 37 ℃ using a nutrient-rich LB medium to overcome this (first step) : Growth step), the recovered cells are separated by the step of proceeding the enzyme reaction at 30 ℃ 14 ~ 16 hours using a substrate M9 medium (step 2: redesigned gene circuit activation step) to control the substrate addition To optimize the enzyme reaction.

According to the present invention, a "phenol-releasing compound" is a substrate that can be used to detect intracellular enzymatic activity, and is a compound capable of releasing phenol by enzymatic reaction, and phenol, 2-chlorophenol and 2-iodine by enzymatic reaction. Phenol, 2-fluorophenol, o -cresol, 2-ethylphenol, m -cresol, 2-nitrophenol, catechol, 2-methoxyphenol, 2-aminophenol, 2,3-dichlorophenol, 3-chloro And phenolic compounds such as phenol, 2,3-dimethylphenol, 3-nitrophenol, 4-chlorophenol, p -cresol, 2,5-dichlorophenol, 2,5-dimethylphenol and the like. This is also referred to as a "phenol-tag substrate."

Phenol-releasing compounds that can liberate phenols by enzymatic reactions, ie, phenol-tag substrates, include esters with modified phenol hydroxyl groups (hydroxy, -OH), ether (-OOC-), ether (ether, -OC-), Glycoside (-O-Glc), phospho-ester (-O-PO ₃ ), ortho-, meta-, para-position alkyl (-CH ₃ ), hydroxyl (-OH), Carboxyl (-COOH), amino (-NH ₂ ), thiol (-SH), amide (amide, -NH-CO- or -CO-NH-), sulfide (-S-SH), halogen group ( Phenol derivatives or benzene ring compounds in which -Cl, -Br, -F) are introduced;

Specifically, various phenol derivatives covalently linked to the phenol group may be used as the phenol-tag substrate according to the present invention. For example, ester compound (ester, -OOC-), ether compound (ether, -OC-), glycoside compound (glycoside, -O) prepared by modifying hydroxyl group (hydroxy, -OH) of phenol -Glc), phosphate esters (phospho-ester, -O-PO ₃ ) as a substrate, esterase, lipase, glycosidase, phosphatase It can be used for the purpose of detecting phytase activity (Table 1).

In addition, the phenol-tag substrate is a new methyl group (-CH ₃ ), hydroxyl group (-OH), carboxyl (-COOH), amino group (-NH ₂ ), in one position of ortho-, meta-, para-, It may be a material prepared by introducing hydrogen sulfide (-SH). When a substance having an amide bond (amide, -NH-CO- or -CO-NH-) and a sulfide bond (sulfide, -S-SH) at this position is used as a substrate, the phenolic compound linked through the amide group is amidase ( amidase), peptidase (peptidase) activity can be used for the purpose of sensing, and phenolic compounds linked through a sulfide group can be used for the purpose of detecting the intracellular redox level. In addition, when a phenol compound having a new carbon bond, ie, Ph-C- (R), is used as the phenol-tag substrate, the phenol-lyase activity that liberates phenol may be detected by decomposing the carbon bond. . Benzene ring material may be used as the phenol-tag substrate. In this case, oxidase activity such as monooxygenase and dioxygenase, which are involved in the oxidation of aromatic compounds, may be detected. have. Phenolic compounds bound to halogen such as chlorine (Cl), bromine (Br), and fluorine (F) may also be used as substrates. In this case, enzymatic activity of dehalogenation or isomerization that changes the position of halogen by acting on the above halogenated phenolic compound can be detected according to the present invention.

In the above-mentioned various phenolic compounds, transferases for transferring covalent bonds to other organic molecules or ligase activity for generating new covalent bonds can be detected by the present invention.

Therefore, using the various phenol-tag substrates presented in the present invention, specific hydrolase, oxido-reductase, isomerase, lyase, transfer Enzymes can be detected. In the present invention, the substrate used for detecting intracellular enzymatic activity refers to various synthetic substrates including phenol, o / p-nitrophenol, o / p-chlorophenol, etc. (Table 1). Appropriate enzyme action on these phenol-tagged compounds frees the phenolic substance from the original phenol-tagged compound. For example, in phenyl-β-glucoside When β-galactosidase (lacZ) acts, the phenol component is released according to the enzyme activity (Fig. 1). Table 1 gives examples of phenol-tag substrate characteristics and related enzymatic reactions.

[Table 1]

In other words, the introduction of an enzyme gene into a recombinant microorganism containing a redesigned gene circuit for detecting phenol and processing a phenol-tag substrate will change the concentration of the phenolic compound depending on the function and activity of the enzyme within the cell. Therefore, if a quantitative increase in reporter such as fluorescence and antibiotic resistance due to the expression-inducing function of phenolic compounds is explored using various measurement techniques such as fluorescence spectrometer and antibiotic resistance, a new measurement method can be detected with high sensitivity. Will be provided. In addition, the fluorescent protein and antibiotic resistance protein used as a reporter in the present invention can not only apply a highly sensitive measurement method, but also are characterized in that the foreign gene is expressed in the cell because it is limited to a specific cell without passing through the cell membrane. Will be used individually. Therefore, since each single cell plays the role of an independent reactor and analyzer, it is a means of measuring the activity of reporter induced expression by detecting phenols liberated by enzymatic reaction. It can be measured using a fluorescence flow cytometer (FACS), micro colony fluorescence image analysis, fluorescence spectrum analysis, mass search using antibiotic selection medium.

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.

Genetic redesign according to the present invention

(1) Phenolic Sensing Expression Regulator dmpR  Based redesigned genetic circuit

Enzyme activity detection method according to the present invention requires the construction of a redesigned genetic circuit for detecting phenol released from the substrate. P. putida is a regulatory protein for phenolic compound degrading enzymes that recognizes phenolic compounds . DmpR, a regulatory protein of the derived phenol deferred operon (dmp operon), was cloned into plasmid pUC19, and a reporter protein was used as an enhanced green fluorescence protein (GFP). The cloning process is as follows. First, the pMGFP plasmid (Ha et al . (2007) Appl . Environm. Microbiol . 73 (22): 7408-7414) was treated with EcoR I and HindIII restriction enzymes to obtain 720 bp GFP fragments and then introduced into the pUC19 vector to construct pUGFP (FIG. 2).

P. putida Origin dmp The operon consists of 15 genes, of which dmpKLMNOP codes for the enzymes required for phenol hydroxylation, and dmpQBCDEFGHI codes for the metabolic pathway that degrades catechol intermediates. dmpKLMNOP of operon expression σ ⁵⁴ - dependent transcription factors of the dmpR dmpK top of dmp Activated in conjunction with operator , dmpR The transcriptional regulator of itself is known as σ ⁷⁰ -dependent (Lee, CN et. al . (1996) J. Biol . Chem . 271 (29): 17281-17286. In the present invention, using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 P. putida PCR amplification of colonies resulted in a DNA fragment of 2,157 bp consisting of a dmpR gene (1,692 bp) including an EcoRI restriction enzyme linker, a dmp operator-promoter region, and a portion of the dmpK gene (42 bp) and 12 bp restriction enzyme sequence inserted during the cloning process. SEQ ID NO: 32) was prepared. The dmpK gene is located 405 bp away from the dmpR ( 1,693-2,097 ), and the reporter gene is configured to be linked after the N- terminal 42 bp of dmpK .

SEQ ID NO: 5'-CCGGAATTCGAGCTGATCGAAAGTCGG-3 '

SEQ ID NO: 5'-CCGGAATTCCTAGCCTTCGATGCCGAT-3 '

The N-terminal fragment of dmpK was left to stabilize the transcription enhancer function of the dmp operator-promoter region even when fluorescent proteins and various antibiotic resistance genes were used as reporters. This PCR product was cloned into the EcoR I site of pUGFP to construct pGESS-T1 (5,510 bp; Table 2 below details of pGESS clones) (FIG. 2). Since the inserted DNA can be inserted in both directions, the nucleotide sequence of the obtained clone is analyzed, and the dmpR-Px-phenolic compound degrading enzyme-binding site-P _R -GFP site is inserted from the EcoRI site where the PCR product is correctly inserted. The sequence consisting of (SEQ ID NO: 31) was selected.

Description of the various GESS clones prepared and used in this example is shown in Table 2 below.

TABLE 2 Schematic description of various redesigned genetic circuits used in the examples of the present invention

In general, protein expression starts with AUG (methionine) or GUG (valine), which is the initiation codon in mRNA. It is determined by base-rich RBS (or Shine-Dalgarno (SD) sequence), which is known to differ in sequence from species to species (Stryer, L., 1995, Biochemistry, (4). th ed .) WH Freeman, Chapter 34). Redesigned gene circuit built in the present invention and were using transcription factors in Pseudomonas as host E. coli of the gene circuit as well as the survival shape very different from the case of σ ^54-dependent gene expression, the σ ⁵⁴ binding site or the σ ⁵⁴ factor Because it is very different from E. coli (W, MMSM (1998) FEMS Microbiol . Rev. 22: 127-150), pGESS-T1 with RBS of Pseudomonas , and RBSε (T7 RBS) for Escherichia coli to facilitate expression of reporter proteins in host E. coli (Simons, A. et al. (1984)). PNAS 81: 1624-1628) and pGESS-T2 with transcription terminator were constructed together (FIG. 3). 425 bp rrnBT1T2 transcription terminator from the pHCEIIB vector (Bioreaders, Korea) was amplified by PCR reaction with SEQ ID Nos. 3 and 4 primers, and the dmpR and dmp operator-promoter regions from pGESS-T1 and 2,831 bp of GFP reporter protein portion. Amplification of PCR products with primers of SEQ ID NOs: 5 and 6, followed by homologous recombination at the C-terminus of the GFP reporter protein (Lee Seung-gu et al., Homologous recombination method disclosed in Korean Patent Application No. 10-2005-0116672, Stuart Francis Et al, PCT / EP2000 / 06533, 2000). PGESS-T1-1 is composed of 5,901bp is a clone of the transcription terminator inserted into the reporter protein of pGESS-T1. The tL3 transcription terminator, which was digested with pGESS-T1-1 by EcoRI and PCR amplified from pKD46 vector with SEQ ID NOs: 7 and 8 primers, was ligated to the C-terminus of dmpR and introduced into E. coli by electroporation. PGESS-T2 was constructed (FIG. 3).

SEQ ID NO: 5'-ATGGACAAGCTGTACAAGTAAGCTTCTGTTTTGGCGGATGAGAGAAGA-3 '

SEQ ID NO: 5'-AGCGGATAACAATTTCACACAGAAACAGCTATGACCATGATTACGCCAAGAGTTTGTAGAAACGCAAAAAGG-3 '

SEQ ID NO: 5'-TCTCTCATCCGCCAAAACAGGAATTCCTAGCCTTCGATGCCGATTT-3 '

SEQ ID NO: 5'-TCTTCTCTCATCCGCCAAAACAGAAGCTTACTTGTACAGCTTGTCCAT-3 '

SEQ ID NO: 5'-CCCGAATTCTTCTTCGTCTGTTTCTACTG-3 '

SEQ ID NO: 5'-CCCGAATTCAATGGCGATGACGCATCCTCA-3 '

To confirm the phenolic detection of pGESS-T1 containing E. coli and pGESS-T2 containing E. coli, E. coli DH5α single colony was added LB (1% (w / v) tryptone, added 50 μg / ml of ampicillin, 0.5% (w / v) yeast extract, 1% (w / v) sodium chloride) was inoculated into the liquid medium and incubated for 24 hours at 37 ℃ shake culture. The culture solution was diluted 10 ⁶ times, and then 100 μl of the diluted solution was diluted in 1/10 diluted LB solid medium (dLB solid medium; 0.1% (w / v) tryptone, 0.05) with 50 μg / ml of ampicillin and 1 mM phenol. The fluorescence expression was observed by fluorescence microscopy (Nikon, Japan) while spreading in% (w / v) yeast extract, 0.1% (w / v) sodium chloride, 1.5% (w / v) agar) and incubated at 30 ℃. As a control, the same sample was plated and observed in dLB solid medium without phenol.

As a result, as shown in FIG. 4, the GFP fluorescence was not detected in the sample without adding phenol to both pGESS-T1 and pGESS-T2, but the fluorescence was strongly emitted from colonies grown in a medium containing 1 mM phenol. In addition, the two redesigned gene circuits were confirmed to be controlled by phenol, and pGESS-T2 with transcription promoter and transcription terminator was observed to be stronger than that of pGESS-T1. (FIG. 4).

(2) enzyme activity detection system ( GESS Analysis: Regulation of Translational Activity of Reporter Protein RBS Exchange) on the activation of redesigned genetic circuits

PGESS-T1, which contains only part of the genes of RBS and dmp operon of Pseudomonas , and pGESS-T2, which employs E. coli RBSε and transcription terminator, to facilitate expression of reporter protein in host E. coli As shown in FIG. 4, the fluorescence intensity of the colony showed that the fluorescence intensity of pGESS-T2 using E. coli RBSε and transcription terminator was much higher, and pGESS-T1 was also measured by the fluorescent plate reader. It was confirmed that more than 10 times higher.

In order to control the expression level of the reporter protein, a clone of which the RBSε sequence was inserted into the N-terminal sequence of the reporter gene of pGESS-T1 was quantitatively analyzed for the sensitivity difference of the redesigned gene circuit.

Specifically, PCR amplification of the GFP gene with SEQ ID Nos. 9 and 10 primers having a homologous sequence of 14 bp RBSε, followed by Escherichia coli DH5α containing a pKD-Cm vector together with the DNA of pGESS-T1 digested with BsrGI restriction enzyme. It was introduced and cloned by the homologous recombination method (Lee Seung-gu et al., Homologous recombination method disclosed in Korean Patent Application No. 10-2005-0116672, Stuart Francis et al., PCT / EP2000 / 06533, 2000) (Fig. 5). ).

SEQ ID NO: 5'-GCACAGCTGTTGCACTTTGTCCTGCGCAATCCGCCAACCTGGAGAAGGAGATATACATATGGTGAGCAAGGGCGAGGAGC-3 '

SEQ ID NO: 10'-GATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACAGAAGCTTACTTGTACAGCTTGTCC-3 '

In order to measure the activity of the enzyme activity detection system according to the expression level of the reporter protein in the liquid medium, E. coli DH5α single colony containing the individual clones was inoculated in LB liquid medium to which 50 µg / ml of ampicillin was added. Shake culture was performed for 24 hours at. M9 medium (1 L Na ₂ HPO _{4 ·} 7H ₂ O 6.78g, KH ₂ PO ₄ 3g, NaCl 0.5g, NH ₄ Cl 1g, 2mM MgSO ₄ , 0.1mM CaCl ₂ , 0.4% (w / v) Glucose, 50 μg / ml of ampicillin and 1 mM phenol were added to 0.01% (w / v) thiamine), and the preculture was inoculated at 1% (v / v), followed by shaking culture at 30 ° C. for 24 hours. GFP fluorescence values were measured with a fluorescence plate reader (Perkin Elmer, Singapore). As a control, the samples were cultured and used in the same manner in M9 minimal medium without phenol (FIG. 6A).

Subsequently, the effect according to the expression amount of the reporter protein was confirmed in the solid medium.E. Coli DH5α single colony containing each clone was inoculated in LB liquid medium containing 50 µg / ml of ampicillin and incubated for 24 hours at 37 ° C. It was. The preculture was diluted 10 ⁶ times, and then 100 μl of the diluted solution was plated on a 10-fold diluted dLB solid medium containing 50 μg / ml of ampicillin and 1 mM phenol and incubated at 30 ° C. with a fluorescence microscope (Nikon, Japan). Fluorescence images of colonies were observed. As a control, the same sample was plated and observed in dLB solid medium without phenol (FIG. 6 (b)).

In the results of Fig. 6A, when the transcription terminator is attached to only one of the reporter protein or the phenol cognitive regulatory protein (pGESS-T1-1 and pGESS-T1-2, respectively), the activity is similar to that of pGESS-T1. Even when the transcription terminator was attached only on one side, the clone with the RBSε (E. coli RBS) inserted (pGESS-T1-4) had about 5 times the fluorescence sensitivity compared to pGESS-T1, pGESS-T1-1, or pGESST1-2. Anomaly was detected high. Even though the transcription terminator was included, in the case of the clone without the N-terminal sequence of dmpK and only the promoter of Pseudomonas before the reporter gene (pGESS-T2-1), the activity was detected lower than that of pGESS-T1, but pGESS no transcription termination factor in E. coli RBSε -T1 only for even if the insert (pGESS-T1-3) when viewed by a fluorescence sensitivity increased by about 19 times compared to the T1-pGESS, the gene circuits using Escherichia coli as a host nature Pseudomonas It is thought that the promoter site is due to the difference in sequence between the promoter and RBS necessary for protein expression in E. coli, in particular E. coli RBSε containing a large amount of purine residues. In addition, the transcription terminators mainly used in E. coli expression vectors were inserted into both C-terminus of the reporter protein and the phenol cognitive regulatory protein, and RBSε was inserted into the N-terminus of the reporter protein to increase the expression level of the reporter protein. It was confirmed that the fluorescence sensitivity of about 23 times stronger than pGESS-T1. This result was confirmed to show the same result in colony fluorescence imaging observed in the solid medium (Fig. 6 (b)).

(3) enzyme activity detection system ( GESS ) analysis: Transcriptional regulators  Effect of Expression Regulation on Redesigned Genetic Circuits

The effect of the expression factor dmpR on the expression of the genetic circuit was investigated. Shingler et al reported that the expression level of dmpR in Pseudomonas had little effect on the expression activity of the σ ⁵⁴ dependent promoter (Sze, CC et. al . (1996) J. Bacteriol . 178 (13): 3727-3735), In the present invention, the promoter of the transcriptional regulator dmpR in Escherichia coli is a high expression promoter, P _hce. Poo H. et al . (2002) Biotechnol . Lett . 24 (14) 1185-1189) was investigated to increase the effect of dmpR expression on σ ⁵⁴ -dependent promoter activity.

Specifically, amplification of a 329 bp PCR product, including a 222 bp HCE promoter and a homologous sequence, using the SEQ ID NO: 11 and 12 primers from the pHCEIIB vector, the homologous recombination method at the N-terminus of dmpR of pGESS-T1 (Lee Seung-gu et al., PGESS-T3 clone was prepared by the homologous recombination method disclosed in Korean Patent Application No. 10-2005-0116672, Stuart Francis et al., PCT / EP2000 / 06533, 2000) (Fig. 7 (a)) .

SEQ ID NO: 5'-TCAGGTCCTTGAAATCGGAGTGCTGGATTTCAGGCTTGTACTTGATCGGCATATGATATCTCCTTTTTCCAG-3 '

SEQ ID NO: 12'-CTATAGCGGCGACCCTGATTTCCCCATCTAAAAATAAATAGGGGCCTCGCTTACGATCTCTCCTTCACAGATTC-3 '

Subsequently, the recombinant E. coli DH5α single colony containing pGESS-T1 and pGESS-T3, respectively, was inoculated into LB liquid medium to which 50 µg / ml of ampicillin was added, followed by shaking culture at 37 ° C for 24 hours. After inoculating 1% (v / v) of the preculture solution into 10 ml of LB medium containing 50 µg / ml of ampicillin, adding phenol to 0.1 mM as an inducer, and reacting at 30 ° C. for 20 hours. The degree of fluorescence expression was confirmed. In order to measure the amount of fluorescence, the cultured cells were centrifuged and washed once with PBS buffer solution. Suspend the cell precipitate in 0.5 ml of PBS buffer solution and add an equal amount of cell lytic B solution (Sigma, USA), 0.1 mg / ml lysozyme (Sigma, USA), DNase I (Roche, USA) for 1 hour at 37 ° C. The cells were disrupted by reaction. The supernatant obtained by centrifuging the crushed cells at 15,000 rpm for 20 minutes at 4 ℃ was analyzed with a fluorescence photometer (Varian, Australia) to determine whether the emission wavelength of the GFP is detected at 510nm. As a result, in the case of pGESS-T3, the expression level of dmpR was found to be increased than that of pGESS-T1 (FIG. 7 (B)), and the sensitivity and fluorescence expression intensity were confirmed to be about 2 times improvement compared to pGESS-T1. (Fig. 7 (c)). From these results, it is estimated that the difference in reporter activity according to cell culture characteristics and phenol addition timing is also affected by the expression level of intracellular dmpR.

(4) Construction of redesigned genetic circuits containing various reporter proteins

One) GFP _UV  Redesigned Gene Circuit Containing Reporter

If GFP _UV gene is used as a reporter protein instead of the GFP reporter protein of pGESS-T1 constructed in (1), 10 ² to 10 ³ gene libraries can be easily analyzed at a time by using general image analysis equipment. This technology has the advantage of enabling high-speed search without using expensive equipment such as FACS because GFP _UV is activated by 385nm long wavelength UV irradiation to minimize noise in the visible region.

In the present invention, in order to produce a phenolic surveillance gene circuit using the GFP _UV gene as a reporter, 717 bp of GFP _UV gene was generated by PCR reaction with the primers SEQ ID NO: 13 and 14, and the above-mentioned homology was introduced to introduce into the pGESS-T1 vector. Recombinant gene manipulation was performed, and ampicillin was smeared on an LB solid medium to which 50 µg / ml was added, and then cultured overnight at 37 ° C to select a phenol sensor, pGESS-T4 substituted with the GFP _UV gene. Colonies generated in the selection medium were subjected to colony PCR with SEQ ID No. 3 primer and M13R primer (# S1233S, NEB, UK) located between dmpR and dmpK, and DNA sequencing was performed to confirm gene substitution (FIG. 8 ( end)).

SEQ ID NO: 5'-CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTATTTGTAGAGCTCATCCA-3 '

SEQ ID NO: 14'-ACCTGGAGATGGCCGTGACCAATACCCCCACACCGACTTTCGATCAGCTCATGAGTAAAGGAGAAGAACT-3 '

The prepared pGESS-T4 was introduced into recombinant E. coli DH5α, and inoculated into LB liquid medium containing 50 μg / ml of ampicillin, followed by shaking culture at 37 ° C. for 24 hours. The cultured cells were inoculated in LB liquid medium containing 50 μg / ml ampicillin and 1 μM to 1 mM phenol, followed by incubation at 30 ° C. for 20 hours, and the Gel Doc System (Bio-Rad, USA). Fluorescence image analysis was performed. As the excitation light, a 365 nm long wavelength ultraviolet light bulb (# 170-6887, Bio-Rad, USA) was used, and GFP _UV was detected using a fluorescence filter 520DF30 (# 170-8074, Bio-Rad, USA) for GFP. .

As a result, as shown in (b) and (c) of FIG. 8, the fluorescence intensity was clearly different depending on the presence or absence of phenol, and the fluorescence intensity increased as the concentration of phenol increased. On the other hand, in the conditions which exposed the same sample to white light, the difference between the samples according to phenol concentration was not observed. Therefore, the present invention can be used for the purpose of analyzing unknown enzyme activity using a general image analysis facility without using an expensive analysis device such as FACS.

2) Redesigned Genetic Circuit Using Antibiotic Resistant Protein as Reporter

As a reporter protein of the redesigned gene circuit, it is often useful to utilize not only fluorescent proteins but also antibiotic resistance genes such as chloramphenicol, kanamycin, and tetracycline. In other words, if the antibiotic resistance gene is used as a reporter, no image analysis facility is required, and 10 ⁶ to 10 ⁷ or more colonies can be quickly analyzed on one solid medium.

To construct a phenol-sensing gene circuit using an antibiotic resistance gene as a reporter, PCR amplification of chloramphenicol, kanamycin, and tetracycline resistance genes with SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively, was performed at 660 bp and 813 bp, respectively. DNA fragments of 1,191 bp were prepared, and genetic manipulation was performed to replace the antibiotic resistance genes in the GFP reporter region of the pGESS-T1 clone (FIG. 9A).

Detailed steps of genetic manipulation based on homologous recombination are as follows. First, a host cell is prepared for homologous recombination and a linearized gene is introduced (Zhang et al. al ., (2000) Nature Biotech . 18: 1314-1317; Zhang et al . , (1998) Nature Genetics , 20: 123-128; Lee Seung-gu et al., A method disclosed in Korean Patent Application No. 10-2005-0116672). A single colony of Escherichia coli DH5α (DE3) containing a pKD46-Cm vector encoding a red-recombinase was inoculated in 3 ml of LB liquid medium containing 25 µg / ml of chloramphenicol and Shake incubation for 16 hours at ℃. The culture medium was inoculated in 1% (v / v) of 100 ml LB medium containing chloramphenicol and arabinose to 25 μg / ml and 50 mM, respectively, and shaken at 30 ° C. until the OD ₆₀₀ became 0.5 to 0.6. It was. After the incubation, the culture was left for 20 minutes on ice, the culture was placed in a pre-cooled centrifuge vessel and centrifuged at 5,000 xg for 20 minutes at 4 ℃. After centrifugation, the supernatant was completely removed and suspended by adding cold sterile distilled water to the cell precipitate.The cells were centrifuged under the same conditions as above and do not know the cells again, and then suspended by adding cold 10% (v / v) glycerol. Centrifugation at 5,000xg for 20 minutes at 4 ℃. 10% (v / v) glycerol was added to the centrifuged cell precipitate so that the cell concentration (OD ₆₀₀ ) was 100, and then 50 μl aliquots were used as cells for introducing genes.

10 ng of pGESS-T1 digested with BamHI restriction enzyme with a vector and 100 ng of PCR products of chloramphenicol, kanamycin, and tetracycline resistance gene were added to the cells prepared according to the above method, and subjected to an electric shock (18 kV / cm, 25 Hz). Afterwards, 1 ml of SOC medium was added to induce homologous recombination at 25 ° C. for 20 hours. Each pGESS-T5 clone (pGESS-T5-Cm and pGESS-T5-Tc, pGESS-T5-Km), including chloramphenicol, kanamycin and tetracycline resistance genes, was added to LB solid medium containing 50 μg / ml of ampicillin. After plating, colonies by homologous recombination generated by incubating at 37 ° C. overnight were selected. Colonies generated from each selection medium were first identified by colony PCR with the SEQ ID No. 15 primer of the above (4) located between the regulatory proteins dmpR and dmpK and the M13R primer (# S1233S, NEB, USA). DNA sequencing was performed to confirm gene replacement.

SEQ ID NO: 15'-TTCACCTGCGCCAGCT-3 '

SEQ ID NO: 5'-ACCTGGAGATGGCCGTGACCAATACCCCCACACCGACTTTCGATCAGCTCATGGAGAAAAAAATCACTGG-3 '

SEQ ID NO: 17'-CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTACGCCCCGCCCTGCCACT-3 '

SEQ ID NO: 18'-ACCTGGAGATGGCCGTGACCAATACCCCCACACCGACTTTCGATCAGCTCATGAGCCATATTCAACGGGA-3 '

SEQ ID NO: 19'-CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTAGAAAAACTCATCGAGCA-3 '

SEQ ID NO: 20'-ACCTGGAGATGGCCGTGACCAATACCCCCACACCGACTTTCGATCAGCTCATGAAATCTAACAATGCGCT-3 '

SEQ ID NO: 21'-CGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCAGGTCGAGGTGGCCCGGC-3 '

A single colony of E. coli DH5α containing the three kinds of pGESS-T5 prepared was inoculated in an LB liquid medium containing 50 µg / ml of ampicillin and incubated at 37 ° C for 24 hours. The cultured cells were added to 20 µg / ml of 50 µg / ml ampicillin, chloramphenicol, tetracycline, and kanamycin, respectively, and inoculated in LB liquid medium containing 10 µM to 1 mM of phenol, followed by 20 hours at 30 ° C. The growth of the cells was observed by culturing.

As a result, as shown in (b) of FIG. 9, recombinant E. coli was unable to proliferate in the absence of phenol in the presence of each selected antibiotic, but recombinant E. coli proliferated when the concentration of phenol was 10 μM or more. PGESS-T5 was confirmed that the expression is controlled by phenol. Therefore, the present invention can be used for the purpose of rapidly analyzing 10 ⁶ to 10 ⁷ or more colonies with or without cell growth in a general laboratory without using expensive analysis apparatus such as FACS or image analysis apparatus.

3) Construction of redesigned genetic circuit containing double reporter protein

When using fluorescent protein or antibiotic resistance gene as a reporter, it has the advantage that it can easily analyze a large amount of samples at high speed. However, the characteristics of the host of the genetic circuit or autofluorescence of the components in the nutrient medium used in culture Due to the high intensity of the background signal (false-positive) signal generation is inevitable. To overcome this problem, a redesigned genetic circuit containing a double reporter protein was constructed.

(a) Dual Fluorescent Protein  Reporter gene circuit

GFP and GFP _UV fluorescent proteins have excitation light of 485 nm and 385 nm and emission light of 510 nm, respectively. In contrast, the red fluorescence protein (RFP) has excitation light and emission light of 545 nm and 650 nm, respectively, and the fluorescence spectrum of GFP has a large difference in fluorescence spectra.

To obtain RFP fragments, fluorescence intensity is reported to be more than five times higher than wild-type RFP (Jach, G., et. al . (2006) Nature Methods A mutant mRFP (Q66T) clone in which amino acid number 66 (3 (8): 597-600) was substituted with threonine in glutamic acid was used. Using the pE-mRFP (Q66T) plasmid as a template, a 794bp RFP fragment containing the homologous sequence containing RBSε was PCR amplified by primers SEQ ID NOs: 22 and 23 to digest the GFP reporter gene of pGESS-T2 digested with BsrGI restriction enzyme. PGESS-T6-RFP was cloned at the C-terminus by homologous recombination. Confirmation of pGESS-T6-RFP was confirmed by colony PCR using the SEQ ID No. 24 primer, which is the N-terminus of the GFP gene, and the SEQ ID No. 25 primer, which is the C-terminus of the RFP gene (Fig. 10 (a)).

SEQ ID NO: 22'-GTGACCGCCGCCGGGATCACTCTCGGCATGGACAAGCTGTACAAGTAAAAGGAGATATACATATGGCCTCCTCCGAGGACGTC-3 '

SEQ ID NO: 23'-CTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACAGAAGCTTAGGCGCCGGTGGAGTGGC-3 '

SEQ ID NO: 24'-ATGGTGAGCAAGGGCGAG-3 '

SEQ ID NO: 25'-TTAGGCGCCGGTGGAGTGGC-3 '

E. coli DH5α single colony containing pGESS-T6-RFP was inoculated into LB liquid medium to which 50 μg / ml of ampicillin was added, followed by shaking culture at 37 ° C. for 24 hours. Fluorescence microscope (Nikon, Japan) and by the pre-culture was diluted 10 ⁶ times and then a diluent ssikeul 100㎕ dLB plated on solid medium containing the ampicillin and 1mM phenol 50㎍ / ㎖ added to the culture at 30 ℃ the fluorescent image of the colonies Was observed. As a control, the same sample was plated and observed in dLB solid medium without phenol. As a result, as shown in (b) of FIG. 10, GFP and RFP fluorescence were not detected when there was no inducer phenol, but GFP and RFP fluorescence were strongly detected in the presence of phenol.

(b) Dual reporter gene circuit using fluorescent protein and antibiotic resistance gene

In order to solve the miscellaneous signal of fluorescence protein, a dual reporter using antibiotic resistance gene together with fluorescence protein was constructed. The tetracycline resistance gene was amplified by PCR using 1.2 SEQ ID NOs. 26 and 27 primers and a 1.2 kb Tc fragment containing a homologous sequence containing RBSε from pGESS-T5-Tc, followed by digestion of pGESS-T2 with BsrGI restriction enzyme. PGESS-T6-Tet cloning was performed at the C-terminus of the GFP reporter gene by homologous recombination (FIG. 11A). The identification of pGESS-T6-Tet was confirmed by colony PCR using the N-terminal SEQ ID No. 24 primer of the GFP gene and the SEQ ID No. 28 primer consisting of the C-terminal sequence of the tetracycline resistance gene.

SEQ ID NO: 26'-CCGGGATCACTCTCGGCATGGACAAGCTGTACAAGTAAAAGGAGATATACATATGAAATCTAACAATGCGCTCATCGTCA-3 '

SEQ ID NO: 27'-TTCTGATTTAATCTGTATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACAGAAGCTTCAGGTCGAGGTGGCCCGGC-3 '

SEQ ID NO: 28'-TCAGGTCGAGGTGGCCCG-3 '

A single colony of Escherichia coli DH5α containing pGESS-T6-Tet was inoculated into an LB liquid medium containing 50 µg / ml ampicillin and shaken at 37 ° C for 24 hours. After diluting the culture solution by 10 ⁶ times, 100 μl of the diluted solution was plated in LB solid medium to which 50 μg / ml of ampicillin, 10 μg / ml of tetracycline, and phenol were added to 0 to 100 μM and incubated at 30 ° C. Colony formation according to phenol concentration and fluorescence expression were observed by fluorescence microscope (Nikon, Japan). As a result, pGESS-T6-Tet was not able to observe colony proliferation and fluorescence in the absence of phenol as an inducer, but colony proliferation was observed when the concentration of phenol was above 1 μM. It became more active. Fluorescence intensity was also confirmed to be stronger as the concentration of phenol increased (Fig. 11 (b)).

(5) dmpR Mutant  Construction of redesigned genetic circuit to contain

p-nitrophenol is widely used as a substrate for the simple analysis of the activity of various enzymes, such as alpha-glucosidase, beta-glucosidase, lipase, phosphatase, and is also very useful for detecting enzyme activity using the phenol sensor gene circuit. It can be adopted as a phenol-tag substrate. In the present invention, pGESS-T7 containing a mutant dmpR in which glutamic acid (E), which is amino acid 135 of dmpR, is substituted with lysine (K), was prepared to improve the detectability of pGESS to p-nitrophenol. (Figure 12)

The experimental method is as follows. In the preparation method, DNA sequence was changed so that amino acid No. 135 could be replaced with lysine in glutamic acid, and then a total of 40 bp sequences extended by 20 bp to both sides were identified using PCR primers (SEQ ID NO: 29 and SEQ ID NO: 30). Point mutations were induced. Two PCR primers and pGESS-T2 plasmid as template DNA were used to perform an inverse PCR reaction. The reverse PCR reaction was performed using KOD DNA polymerase (Novagen, USA). PCR reaction was inactivated at 94 ° C for 3 minutes, inactivated at 94 ° C for 30 seconds, primer binding at 55 ° C for 30 seconds, and at 60 ° C at 60 ° C. The procedure of the second DNA synthesis reaction was repeated 25 times, and finally, the DNA linkage reaction was further performed at 72 ° C. for 5 minutes to recover the PCR product of 6,171 bp. The prepared primary PCR product was treated with Proteinase K, purified by column (Qiagen, Germany), and then treated with Dpn I (Roche, Germany) restriction enzyme to remove the plasmid used as template DNA. After the restriction enzyme reaction, the sample was electrophoresed on agarogel, and only the target band was cut out and purified on a column (Qiagen, Germany), and then transformed into E. coli DH5α (ElectroMaxTM-DH5α-ETM, Invitrogen, USA) to 6,171 bp. pGESS-T7 was produced.

SEQ ID NO: 29'-GATCGACTCCTTCGAGGTGAAAATCTGCCAGACCGACCTG-3 '

SEQ ID NO: 30'-CAGGTCGGTCTGGCAGATTTTCACCTCGAAGGAGTCGATC-3 '

Colonies containing pGESS-T2 or pGESS-T7 gene circuits were inoculated into LB medium the day before and inoculated with shaking at 37 ° C. overnight, and 1% (v / v) of the culture solution was inoculated again in LB medium at 37 ° C. When shaken and cultured and cell growth (OD600 / ml) was about 2.5, the cultured cells were recovered to proceed with gene cycle activation. Genetic activation was performed by washing the collected cells with M9 medium once and then suspending them in M9 (acetate) medium containing various concentrations of phenol and shaking at 30 ° C. for 16 hours. 50 [mu] g / ml ampicillin was added to each medium as a selection marker. Strain growth was predicted by measuring absorbance (OD ₆₀₀ ) using a UV / VIS spectrometer (Ultrospec 3000, Pharmacia Biotech, Sweden) and fluorescence intensity was used with a fluorescence plate reader (Multi-label reader, PerkinElmer, USA). Experimental results showed that when mutant dmpR was employed, the concentration of phenol could be detected quantitatively from 0.1 to 10 μM. The sensitivity was increased by about 10 times compared to wild type dmpR, and the quantitative range also increased by about 10 times (Fig. 13 (a)). .

Next, the sensitivity measurement of o-nitrophenol and p-nitrophenol was performed. Experimental method was carried out in the same manner as above, and various concentrations of o-nitrophenol and p-nitrophenol were added and reacted with the genetic circuit. Experimental results showed that the sensitivity of phenol was the best, followed by p-nitrophenol and o-nitrophenol, and all three phenols were able to detect quantitatively 0.1 ~ 10 μM phenol (Fig. 13 (b)).

p-nitrophenol is widely used as a substrate for the simple analysis of the activity of various enzymes, such as alpha-glycosidase, beta-glycosidase, lipase, phosphatase, and is also very useful for detecting enzyme activity using the phenol sensor gene circuit. It can be adopted as a phenol-tag substrate. Thus, phenolic sensors that exhibit high reactivity to p-nitrophenols have particular significance in extending the application of the intracellular enzyme activity detection and screening techniques.

(6) Optimal host E. coli construction of redesigned genetic circuit

In order for GESS to operate as intended, the dmpR of Pseudomonas must be heterologous at appropriate levels and smoothly interact with the σ ^54- dependent transcriptional regulator of Escherichia coli (Sze, CC, e t). al . (1996) J. Bacteriol. 178 (13): 3727-3735; Johanssin, LUM, et al . (2008) Mol . Microbiol. 70 (3): 709-723). Therefore, the signal characteristics of the GESS may vary depending on the growth conditions and inducers of the host microorganism. In other words, the amount and activity of dmpR during cell growth is affected by the growth time. Therefore, the present invention was intended to investigate and optimize how it is affected by i) differences in microbial host and composition of growth medium and ii) phenol addition for stable implementation of the phenol sensing gene circuit.

First, in the present example, the sensitivity of the phenol for each E. coli containing the redesigned genetic circuit was investigated to select a strain suitable for the circuit. To this end, experiments were conducted on DH5α, EPI300, JM109 (DE3), BL21, BL21 (DE3) containing pGESS-T1.

The experimental method was first prepared with LB solid medium containing 0.1mM phenol and 100μg / ml ampicillin, and were treated with Escherichia coli DH5a, EPI300, JM109 (DE3), BL21, BL21 (DE3) and pGESS-T1. EPI300 strains containing pGESS-T5-Cm were incubated together at 30 ° C. for 36 hours. Figure 14 (a) is the result of the genetic circuit reaction of each strain in the solid medium.

The same result was then confirmed in the liquid culture. First, a single colony of pGESS-T1 containing recombinant E. coli JM109 (DE3), DH5α, EPI300, BL21, BL21 (DE3) was inoculated in LB liquid medium containing 100 μg / ml of ampicillin and incubated at 37 ° C. for 24 hours. It was. The cultured strain was inoculated with 0.5% (v / v) of 3ml LB liquid medium containing 100µg / ml of ampicillin, followed by shaking culture at 30 ° C. After incubation for 10 hours (about OD ₆₀₀ = 3-4), 0.1mM phenol was added to induce fluorescence for 20 hours at 30 ° C. In order to measure the fluorescence expression by strain, 1.5 ml of the culture cell solution was centrifuged and washed once with PBS buffer solution, 0.5 ml of cell lytic B solution (Sigma, USA), lysozyme (0.1 mg / ml), DNaseI ( Roche, Germany) was added and the cells were disrupted by reaction at 37 ° C for 1 hour. The supernatant obtained by centrifuging the crushed cells was analyzed with a fluorescence photometer to confirm whether the emission wavelength of GFP was detected at 510 nm. As a result, it was confirmed that B21 strains BL21 and BL21 (DE3) were higher in cell reactivity as well as cell growth than other strains (Fig. 14 (b)).

Next, the effects of phenol addition time, that is, expression induction time of GESS gene circuit, were investigated. pGESS-T1 containing recombinant E. coli DH5α single colony was inoculated in LB liquid medium to which 100 μg / ml of ampicillin was added, followed by shaking culture at 37 ° C. for 24 hours. 100 ml of LB liquid medium containing 100 μg / ml ampicillin was inoculated with 0.5% (v / v) of the preculture solution, followed by shaking culture at 30 ° C., and the culture solution was extracted at 5 ml intervals every 3 hours. Part of the extracted culture solution was measured by absorbance at 600nm to confirm the growth of the cells, and the phenol was added to the remaining culture solution to 0.1mM and reacted for 20 hours at 30 ℃ and analyzed the degree of fluorescence expression. In order to measure the amount of fluorescence, the cultured cells were centrifuged and washed once with PBS buffer solution. Suspend the cell precipitate in 0.5 ml of PBS buffer solution and add an equal amount of cell lytic B solution (Sigma, USA), 0.1 mg / ml lysozyme (Sigma, USA), DNase I (Roche, USA) for 1 hour at 37 ° C. The cells were disrupted by reaction. The supernatant obtained by centrifuging the crushed cells at 15,000 rpm for 20 minutes at 4 ℃ was analyzed with a fluorescence photometer (Varian, Australia) to determine whether the emission wavelength of GFP is detected at 510 nm. As a result, as shown in (c) of Figure 14, the difference in the amount of fluorescence can be confirmed that the difference is more than 10 times largely depending on the cell growth stage, especially when phenol is added in the stationary phase (stationary phase) Was the highest expression level in the gene circuit, which is consistent with the expression characteristic of σ ⁵⁴ .

Validation of Redesigned Genetic Circuits and Quantitative Signal Analysis for Phenolic Compounds

(1) Quantitative fluorescence signal analysis of phenol in redesigned genetic circuit

In order to confirm the quantitative detection of phenol in the redesigned genetic circuit constructed in Example 1, recombinant E. coli BL21 single colony containing pGESS-T1 was inoculated into LB liquid medium to which 50 μg / ml of ampicillin was added. Shaking culture for 24 hours at 37 ℃. After diluting the culture solution 10 ⁶ times, 100 μl of the diluted solution was plated in LB solid medium to which 50 μg / ml of ampicillin and 1 μM to 1 mM phenol were added, followed by culturing at 30 ° C., and fluorescence expression with a fluorescence microscope (Nikon, Japan). Observed.

As a result, as shown in (a) of FIG. 15, fluorescence was not detected in the sample without addition of phenol, but fluorescence colonies were observed when fluorescence was added at 1 μM or more. Also increased.

The sensitivity dependence of the redesigned genetic circuits on the concentration of these inducers, phenol, was also verified with a fluorophotometer. To this end, a single colony of recombinant E. coli containing pGESS-T1 was inoculated into LB liquid medium to which 50 µg / ml ampicillin was added, followed by incubation at 37 ° C for 24 hours. The gastric preculture was inoculated with 50% / ml ampicillin and phenol in an LB liquid medium containing 0.0001-1 mM and incubated at 1% (v / v) for 20 hours at 30 ° C. The culture medium was centrifuged at 3 ml and washed once with PBS buffer solution. The cell precipitate was suspended in 0.5 ml of PBS buffer solution, and the same amount of cell lytic B solution (Sigma, USA), 0.1 mg / ml lysozyme (Sigma, USA) and DNase I (Roche, USA) were added. The cells were disrupted by reaction. The supernatant obtained by centrifuging the crushed cells at 15,000 rpm for 20 minutes at 4 ℃ was analyzed by fluorescence photometer (Varian, Australia) to confirm the emission wavelength of GFP at 510 nm.

As a result, as shown in (b) of FIG. 15, a clear fluorescence signal was observed in the presence of 10 μM or more of phenol, and it was confirmed that the fluorescence intensity increased up to 1 mM in proportion to the phenol concentration. This result shows that the phenol detection gene circuit of the present invention is quantitatively activated in the 0.01 ~ 1mM phenol concentration range.

Next, quantitative analysis of phenol in the redesigned genetic circuit was analyzed by FACS. A single colony of recombinant E. coli DH5α containing pGESS-T1 was inoculated into LB liquid medium containing 50 μg / ml of ampicillin and shaken overnight at 37 ° C. Stomach culture was inoculated 1% in LB liquid medium containing 50 ㎍ / ㎖ ampicillin shaking culture for 6 hours at 37 ℃ and dispensed 2ml each in a test tube. Fluorescence was induced by adding 0-5000 μM phenol to each test tube and shaking for 18 hours at 30 ° C. Intracellular fluorescence induced by phenol at each concentration was analyzed by FACS Calibur system (Becton Dickinson, USA). Detectors were set to detect FSC, SSC, and FL1-H (excitation = 488 nm, emission = 530/30 nm) detectors. The data of 10,000 sample cells were analyzed by CellQuest Pro (Becton Dickinson, USA).

As a result, the fluorescence intensity was clearly distinguished according to the presence or absence of phenol as well as the fluorescence microscope and fluorescence spectrophotometer, and it was confirmed that the fluorescence intensity increased as the concentration of phenol increased (FIG. 15 (c)). Such a signal detection using FACS is more meaningful than other analytical methods because it is possible to use molecular evolution research or high speed analysis of metagenome library using the fast analysis and sorting function of FACS. .

(2) Quantitative Antibiotic Resistance Analysis of Phenol in Redesigned Genetic Circuits

Quantitative antibiotic resistance analysis was performed using the pGESS-T5 gene circuit using the antibiotic resistance gene constructed in (4) of Example 1 as a reporter.

Specifically, a single colony of E. coli JM109 (DE3) containing chloramphenicol, tetracycline, and kanamycin resistance genes as a reporter was inoculated into a liquid medium containing 50 µg / ml ampicillin and 24 at 37 ° C. Time shake cultures. After diluting 10 to ⁶ times the preculture, 100 μl of the dilutions were spread on each selection medium and incubated at 37 ° C. for 3 days, and then the number of colonies generated was counted for resistance. Each selective medium was made by adding 50 μg / ml ampicillin and each of the selective antibiotics (chloramphenicol, tetracycline, or kanamycin) to concentrations 0, 10, 20, 30 μg / ml. In addition, 0, 10 μM, 100 μM, 1 mM was added to the transcription activator, ie, phenol.

As a result, pGESS-T5-Cm using the chloramphenicol resistance gene as a reporter was able to distinguish resistance to chloramphenicol according to the phenol switch. Particularly, when the concentration of phenol was 10 μM, resistance was shown up to 20 μg / ml of chloramphenicol, and when the concentration of phenol increased to 100 μM and 1 mM, the resistance to chloramphenicol also increased to 30 μg / ml (Fig. 16). When the tetracycline resistance gene was used as a reporter, similar quantitative results were observed as in the case of chloramphenicol. This result shows that the phenol sensor gene circuit of the present invention can be effectively used for quantitative analysis of enzyme activity.

(3) Signal analysis of various phenols in redesigned genetic circuit

Phenol derivatives other than phenol, i.e., o-nitrophenol, m-nitrophenol, p-nitrophenol, o-chlorophenol, m-chlorophenol, p-chlorophenol, salicylic acid, 2-amino Phenol, 2-methoxyphenol, o-cresol, m-cresol, p-cresol, catechol, lysosinol, 2-fluorophenol, 2-iodinephenol, 2,4-dimethylphenol, 2,5-dimethyl Phenol, 3,4-dimethylphenol, 2,3-dimethylphenol, 3,5-dimethylphenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,3-dichlorophenol, 2,6-dichlorophenol The sensitivity and sensitivity of 3,4-dichlorophenol, 3,5-dichlorophenol, 2,4-dinitrophenol, 3-methylcatechol, 2-ethylphenol, 3-ethylphenol, and benzene were investigated.

A single colony of Escherichia coli DH5α containing pGESS-T1 was inoculated in LB liquid medium containing 50 μg / ml ampicillin and shaken overnight at 37 ° C. The culture solution was inoculated at 50% / ml ampicillin-added LB liquid medium at 1% (v / v) and incubated at 37 ° C. for about 8 hours until OD ₆₀₀ became 3, and then the phenol derivative was added to the culture medium. Each was added to 100μM shaking culture for 18 hours at 30 ℃. 100 μl of each of the cultures was extracted, and fluorescence distribution of individual cells was examined using FACS. Each sample was repeated three times and the average was calculated.

As a result, as shown in Fig. 17A, among the 31 phenol derivatives used in the experiment, o-chlorophenol, o-nitrophenol, 2-aminophenol, 2-methoxyphenol, catechol, o- It reacted strongly to ten substances such as cresol, m-cresol, 2-ethylphenol, 2-fluorophenol, and 2-iodinephenol, and m-chlorophenol, p-chlorophenol, m-nitrophenol, 2,5-dimethyl Phenol, 2,3-dimethylphenol, 2,5-dichlorophenol, 2,3-dichlorophenol, and p-cresol were weakly reactive and did not react with p-nitrophenol. The material was weakly reactive or unreactive.

Chloramphenicol resistance genes were also detected by the type of substrate in the reporter. First, a single colony of Escherichia coli EPI300 containing pGESS-T5-Cm was inoculated into a liquid medium containing 50 µg / ml of ampicillin and shaken overnight at 37 ° C. The culture solution was inoculated at 50% / ml ampicillin-containing LB liquid medium at 1% (v / v), followed by 30 µg / ml chloramphenicol and 100 μM of phenol derivatives, followed by shaking culture at 37 ° C. for 8 hours. , OD ₆₀₀ was measured to detect the phenol derivatives. At this time, the control group was used by adding 100 μM of phenol without adding chloramphenicol.

The phenol derivatives used were also o-chlorophenol, m-chlorophenol, o-nitrophenol, m-nitrophenol, catechol, 2-methoxyphenol, o-cresol, m-cresol, 2-fluorophenol, 2 -Iodine phenol, 2,3 dimethyl phenol and 2-ethyl phenol are reacted strongly as in the case of using fluorescent proteins, but for substances with a substituent in the para position of phenol such as p-cresol, p-chlorophenol, and p-nitrophenol The reaction was very weak or did not react (b in Figure 17).

The expression level of the fluorescent protein, that is, the degree of activation of the phenol sensor gene circuit, is different depending on the type of the side chain of the phenol derivative and the addition position (Table 3). Therefore, it can be seen that the detection technology considering the activity of the enzyme is possible by selecting an appropriate phenol derivative and an addition group.

Table 3 shows the information on the residues of various phenols applicable to the redesigned gene circuit.

(4) Quantitative Signal Analysis of Various Phenols in Redesigned Genetic Circuits

The signals for phenolic compounds in the redesigned genetic circuits were quantitatively analyzed. A single colony of Escherichia coli DH5α containing pGESS-T1 was inoculated in LB liquid medium containing 50 μg / ml ampicillin and shaken overnight at 37 ° C. The culture solution was inoculated at 50% / ml ampicillin-added LB liquid medium at 1% (v / v), and phenol, o-chlorophenol, m-chlorophenol, o-nitrophenol, and catechol as inducers. , 0.1 μM to 1 mM of lysosinol was added thereto, followed by incubation at 30 ° C. for 24 hours. In order to measure the amount of fluorescence, the above cultured cells were centrifuged and washed once with PBS buffer solution. Suspend the cell precipitate in 0.5 ml of PBS buffer solution and add an equal amount of cell lytic B solution (Sigma, USA), 0.1 mg / ml lysozyme (Sigma, USA), DNase I (Roche, USA) for 1 hour at 37 ° C. The cells were disrupted by reaction. The supernatant obtained by centrifuging the crushed cells at 15,000 rpm for 20 minutes at 4 ℃ was analyzed with a fluorescence photometer (Varian, Australia) to determine whether the emission wavelength of the GFP is detected at 510 nm.

As a result, fluorescence was detected at concentrations of 0.0001 mM for o-chlorophenol, and fluorescence was detected at concentrations of 0.001 mM for phenol and m-chlorophenol, catechol, o-nitrophenol, and lysosinol. It was confirmed that the fluorescence is detected from the concentration of 0.01mM, 0.01mM, 0.1mM (Fig. 18 (a)). The results of FIG. 18A, in which the fluorescence sensitivity was confirmed differently according to the type or position of the substituent, show that a variety of phenol substrates can be selectively used to show an appropriate measurement range as necessary.

The quantitative analysis of phenol derivatives in the redesigned genetic circuit was analyzed for antibiotic resistance. To this end, a single colony of Escherichia coli DH5α containing pGESS-T5-Cm was inoculated into an LB liquid medium containing 50 µg / ml ampicillin and incubated at 37 ° C for 24 hours. The cultured cells were added so that ampicillin and chloramphenicol were 50 µg / ml and 20 µg / ml, respectively, and phenol, o-chlorophenol, o-cresol, 2-aminophenol, salicylic acid, o-nitrophenol and 2-meth After inoculating oxyphenol and catechol in an LB liquid medium added with 0.1 μM to 1 mM, the cells were continuously cultured at 37 ° C. for 24 hours to observe the growth of cells. As a result, even when the chloramphenicol resistance gene was used as a reporter, similarly to the GFP reporter, the fluorescence sensitivity was observed differently depending on the type and position of the substituent (Fig. 18 (b)).

(5) redesign of the genetic circuit Phenolic wastewater  Quantitative signal analysis

In order to confirm the quantitative signal analysis of the phenol sensing gene circuit pGESS-T1, an attempt was made to analyze the concentration of phenolic compounds in wastewater containing various phenolic substances (FIG. 19).

The 50㎍ / ㎖ to the M9 liquid medium containing ampicillin in a single inoculation of E. coli DH5α colonies containing pGESS-T1 were incubated for 24 hours at 37 ℃ Next, the coke waste water (Shanghai, China) in the culture medium 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 times diluted to add and reacted for 24 hours at 30 ℃. The cultured cells were centrifuged and washed once with PBS buffer solution, and then suspended in 0.5 ml of PBS buffer solution, and the same amount of cell lytic B solution (Sigma, USA) and 0.1 mg / ml lysozyme (Sigma, USA), DNaseI (Roche, Germany) was added and reacted for 1 hour at 37 ℃ cells were disrupted. The crushed solution was centrifuged at 15,000 rpm for 20 minutes at 4 ° C. and the supernatant was analyzed by fluorescence photometer (VARIAN, Australia) to confirm whether the emission wavelength of GFP was detected at 510 nm ((A) of FIG. 19). ). As a control, phenol was added in a manner similar to 0.0001 mM, 0.001 mM, 0.01 mM, 0.1 mM, and 1 mM instead of coke wastewater ((B) of FIG. 19).

As a result, it was observed that the fluorescence intensity increased proportionally as the concentration of the added wastewater increased, and compared to the case of adding phenol as a control, coke wastewater contained about 10 mM of phenolic compounds. It could be assumed that there is. These results are consistent with the results of the GC / MS analysis of wastewater, and in the GC / MS analysis, wastewater contains phenol, 2-methylphenol, 3-methylphenol, 2-nitrophenol, 3,5-dimethylphenol, 2,4 Dimethylphenol contained 667.4 ppm, 31.9 ppm, 165.8 ppm, 8.7 ppm, 19.9 ppm, and 5.6 ppm, respectively (FIG. 19 (B)). Therefore, it was confirmed that the generation of a quantitative fluorescence signal proportional to the concentration of phenolic substance using the phenol sensing gene circuit of the present invention.

Foreign Genes Using Redesigned Genetic Circuits Intracellular  Enzyme activity detection

(1) Detection of enzyme activity using redesigned genetic circuit

Using the GESS gene circuit according to the present invention, as an example, E. coli-derived beta-galactosidase, C. freundii Derived tyrosine-phenol lyase (TPL), metagenome derived putative phosphatase, Pseudomonas sp. Derived methyl parathion hydrolase (MPH) activity was detected.

By using phenyl-glucose (phenyl-α / β-glycoside) in which glucoside is linked to the hydroxyl group of phenol as a substrate, various alpha / beta-glycosidase enzyme activities can be detected. Single example of E. coli EPI300 (Epicentre, USA) containing pGESS-T1 redesigned gene circuit and pCC1FOS ™ vector containing E. coli lacZ gene (beta-galactosidase activity, GeneBank: J01636.1) Colonies were inoculated in LB liquid medium to which 50 µg / ml ampicillin and 25 µg / ml chloramphenicol were added, followed by shaking culture at 37 ° C for 24 hours. 1% (v / v) of the preculture was inoculated into 10 ml of LB liquid medium of the same composition, and 0.1mM phenyl-β-glycoside, which is a phenol-tag substrate, was added and reacted at 30 ° C. for 20 hours. It was confirmed by a fluorescence photometer. As a result, the fluorescence of GFP was observed in the cells having beta-galactosidase activity, but the fluorescence was not observed in the cells that did not introduce the beta-galactosidase enzyme using the pGESS redesigned gene circuit of the present invention. It was confirmed that the detection of activity is possible (Fig. 20 (a)).

The GESS redesigned genetic circuit of the present invention can be used to detect phenol-lyases in which phenol is liberated in carbon-bonded substrates, namely Ph-C- (R). The purpose of this study was to investigate the activity of TPL, an enzyme that produces phenol, pyruvic acid, and ammonia by decomposing L-tyrosine, an amino acid containing phenol residues.

The TPL gene of C. freundii (GenBank: X66978.1) was cloned into pEC11a (i.e., the substitution of pET11a (+) ori with ACYC ori) to prepare pEC-TPL, which then contained pGESS-T1. E. coli DH5α was introduced. E. coli cells cultured with shaking at 37 ° C for 24 hours after adding 50 µg / ml ampicillin and 25 µg / ml chloramphenicol to an LB liquid medium containing 1 mM IPTG (isopropyl-thio-β-D-galactopyranoside) Was transferred to M9 liquid medium to which 1 mM tyrosine and IPTG were added, followed by continued incubation at 30 ° C. for 20 hours. 3 ml of the culture cell solution was centrifuged and washed once with PBS buffer solution, and then suspended in 0.5 ml of PBS buffer solution. The same amount of cell lytic B solution (Sigma, USA), lysozyme (0.1 mg / ml), and DNase I (Roche) , Germany) was added and the cells were disrupted by reacting at 37 ° C for 1 hour. The supernatant obtained by centrifuging the crushed cells was analyzed with a fluorescence photometer to confirm whether the emission wavelength of GFP was detected at 510 nm. As a result, the fluorescence activity of the cell lysate was greatly increased by the TPL activity, and it was confirmed that the measurement method using the redesigned gene circuit according to the present invention was also useful for the analysis of phenol-lyase (FIG. 20 (b)). .

In addition, it was possible to detect phosphatase derived from microorganisms using various substrates in which phosphoric acid was linked to the hydroxyl group of phenol. Phosphatases are enzymes that show the activity of hydrolyzing phosphates bound to various types of molecules such as nucleic acids, proteins, alkaloids, and the like. Introduced pGESS-T1 in E. coli DH5α introduced metagene genotype putative phosphatase gene (Genebank GQ250428) was pre-cultured in the same manner as in the case of TPL. 1% (v / v) of the above culture was inoculated into 10 ml of LB medium containing 50 µg / ml ampicillin and 25 µg / ml chloramphenicol, and 0.1 mM phenyl phosphate was added as an inducer at 30 ° C. After reacting for 20 hours, the degree of fluorescence expression was confirmed with a fluorophotometer. As a result, GFP fluorescence color intensity was observed higher in the cells having phosphatase activity than in the cells without phosphatase (Fig. 18 (C)).

In addition, it was confirmed that the present GESS technology detects MPH (methyl parathion hydrolase) activity that decomposes organophosphate, which is widely used as a pesticide. MPH gene (GenBank: AY251554.2) was introduced into E. coli by electroporation, pre-cultured for 24 hours in the same manner as in the case of TPL, and then 0.1 mM of methyl parathion, a phenol-tagged compound, which is the substrate of MPH. It was added to the culture medium and examined whether the recombinant gene circuit (E. coli into which pGESS-T7 was introduced) detects p-nitrophenol produced by digesting each substrate. The cultured cells were centrifuged at 2ml (OD600 = 2) and washed once with PBS buffer solution, 0.5ml of cell lytic B solution (Sigma, USA), lysozyme (0.2mg / ml), DNaseI (Roche, Germany) ) Was added and reacted for 1 hour at room temperature to disrupt the cells. The supernatant obtained by centrifuging the crushed cells was analyzed with a fluorescence photometer to confirm whether the emission wavelength of GFP was detected at 510 nm. As a result, fluorescence was not detected in the control group that could not decompose methylparathion, but fluorescence was strongly detected in the presence of MPH (FIG. 20 (d)).

2) Fluorescence for Detecting Enzyme Activity Imaging Flow cell  Analyzer and Antibiotic Resistance Analysis

In the method of measuring enzyme activity using the GESS redesigned gene circuit according to the present invention, fluorescence image observation of colonies was used. A single colony of recombinant Escherichia coli containing the TPL gene and pGESS-T1 was inoculated into an LB liquid medium containing 50 µg / ml of ampicillin and incubated at 37 ° C for 24 hours. After diluting the gastric preculture by 10 ⁶ times, 100 μl was taken and streaked in LB solid medium containing 0.1 mM phenol and 100 μg / ml ampicillin, and cultured at 30 ° C. for 36 hours. Nikon, Japan) and the colony of E. coli DH5α containing only pGESS-T1 but not the TPL gene as a control group was examined by the same method. As a result, no fluorescence was observed in the absence of the TPL gene, but colony imaging of pGESS-T1-containing E. coli was observed in the presence of the TPL gene (FIG. 21A).

Similar results were seen when the enzyme was analyzed by a fluorescence flow cytometer (FACS). We compared the activity of recombinant E. coli containing TPL gene and pGESS-T1, and recombinant E. coli containing pGESS-T1 but no TPL gene as a control. Each colony was inoculated into LB liquid medium to which 50 μg / ml of ampicillin was added and shaken at 37 ° C. for 24 hours. The gastric preculture was inoculated at 1% (v / v) in an LB liquid medium containing 1 mM tyrosine, 10 uM PLP (pyridoxal-5'-phosphate) and 50 µg / ml ampicillin, and cultured at 30 ° C. for 20 hours. . Cultured intracellular fluorescence was analyzed by FACSAria system (Becton Dickinson, USA). Detectors were set to detect FSC, SSC, and FL1-H (excitation = 488 nm, emission = 530/30 nm) detectors, and data from 10,000 sample cells were analyzed by FACSDiva (Becton Dickinson, USA). As a result, it could be observed that the fluorescence distribution pattern in the TPL-containing sample shifted to the right in which the fluorescence intensity was increased rather than the fluorescence distribution pattern in the TPL-free cell sample (FIG. 21B).

The enzyme activity detection was observed as antibiotic resistance using the GESS redesigned genetic circuit according to the present invention. A single colony of recombinant E. coli containing TPL gene and pGESS-T5-Tc was inoculated into LB liquid medium containing 50 µg / ml ampicillin and incubated at 37 ° C for 24 hours. After diluting the gastric preculture by 10 ⁶ times, 100 µl was taken and plated in LB solid medium containing 0.1 mM phenol, 100 µg / ml ampicillin and 20 µg / ml tetracycline, and incubated at 30 ° C. for 36 hours. The growth pattern of colonies was observed, and the same pre-culture solution was observed by plating on solid medium containing 0.1 mM phenol without tetracycline. As a result, when tetracycline is absent, colony grows throughout the medium because there is no selectivity of GESS. However, when tetracycline is present, only colonies where enzymes decompose substrates to produce phenol can survive. It was possible to confirm the selective detection of (Fig. 21 (c)).

3) Quantitative Verification of Redesigned Genetic Circuits (GESS)

In the case of different enzyme activities, it was investigated whether it could be quantitatively searched using GESS. Enzyme The Citrobacter freundii Derived tyrosine-phenol lyase (hereinafter "TPL (C)") and Symbiobacterium toebii Derived tyrosine-phenol lyase (hereinafter, "TPL (S)") was used, and E. coli without these enzymes was used as a control. Significance was determined by comparing enzyme activity measurements using pGESS-T2 and phenol production values analyzed using HPLC.

The experimental method was as follows. After treatment with Cla I and Tth111 I in pSTV28 (Takara, Japan), a vector in which TPL (C) was introduced into the pSHCE vector prepared by inserting the HCE promoter and terminator derived from pHCEIIB (Takara, Japan) and the pSHCE vector Enzyme-activated strains were prepared by transforming E. coli DH5α containing pGESS-T2 with vectors in which TPL (S) was introduced and control pSHCE vectors in which these TPLs were not introduced.

Each colony was inoculated in an LB liquid medium to which 50 μg / ml of ampicillin and 12.5 μg / ml of chloramphenicol was added and shaken overnight at 37 ° C. The culture solution was inoculated 1% (v / v) in M9 liquid medium containing 1 mM tyrosine, 10 μM PLP (pyridoxal-5'-phosphate), 50 μg / ml ampicillin and 12.5 μg / ml chloramphenicol. Shake culture was carried out for 12 hours at ℃. Strain growth was predicted by measuring absorbance (OD ₆₀₀ ) using a UV / VIS spectrometer (Ultrospec 3000, Pharmacia Biotech, Sweden) and fluorescence intensity was used with a fluorescence plate reader (Multi-label reader, PerkinElmer, USA).

Enzyme activity was measured in the same manner as above, and then inoculated with 1% (v / v) of LB liquid medium to which 50 μg / ml of ampicillin and 12.5 μg / ml of chloramphenicol were added and inoculated for 24 hours at 37 ° C. Incubated. Cells in the culture were recovered by centrifugation (5000 rpm, 10 minutes) and washed once with 50 mM Tris-HCl buffer (pH 7.5). Cells were completely disrupted using an ultrasonic grinder (method: 3 sec crushing, 3 sec stop, 3 min, 20% intensity, Vibra cell, Sonics, USA). Enzyme reaction was performed at 37 ° C. for 12 hours by adding 1 mM tyrosine, 100 μM PLP and cell disruption solution (1 mg / ml) in 100 mM potassium phosphate buffer (pH 8.0). The enzyme activity was measured using HPLC (SCL-10A vp, Shimadzu, Japan), the column was C18 reverse column (C / N. 18R03, Chemco Pak, Japan) and the buffer was 50:50 Acetonitrile-Water. .

Figure 22A shows the activity of TPL (C), TPL (S) and pSHCE analyzed by pGESS. It was confirmed that the activity of TPL (C) was about 2.5 times higher than that of TPL (S). Next, after the enzyme reaction, the amount of phenol analyzed using HPLC was compared (FIG. 22 (b)). As with the result of pGESS, it was confirmed that TPL (C) activity was about 2.5 times higher than TPL (S). Two results show that pGESS can be used to quantitatively analyze enzymes with various enzyme activities.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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Claims (21)

A method for screening for target enzyme activity using a redesigned genetic circuit comprising the following steps:
(a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a redesigned genetic circuit for detecting a system compound;
(b) providing a clone or gene library comprising a gene encoding an enzyme to be searched;
(c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively;
(d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And
(e) detecting the activity of the reporter protein expression-induced by sensing the phenolic compound released by the enzymatic reaction.
A method for screening for target enzyme activity using a redesigned genetic circuit comprising the following steps:
(a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a microorganism containing the redesigned genetic circuit for detecting a system compound in chromosomal DNA or cytoplasm;
(b) providing a clone or gene library comprising a gene encoding an enzyme to be searched;
(c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism;
(d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And
(e) detecting the activity of the reporter protein expression-induced by sensing the phenolic compound released by the enzymatic reaction.
Method for quantifying target enzyme activity using a redesigned genetic circuit comprising the following steps:
(a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a phenol comprising a gene expression regulating site consisting of a promoter binding the regulatory protein to induce expression of a downstream reporter gene and a promoter regulating the expression of the reporter gene. Providing a redesigned genetic circuit for detecting a system compound;
(b) providing a clone or gene library comprising a gene encoding the enzyme to be quantified;
(c) preparing a recombinant microorganism by introducing the clone or gene library and the redesigned gene circuit for detecting the phenolic compound into a host microorganism, respectively;
(d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And
(e) detecting the phenolic compound released by the enzymatic reaction to quantify the activity of the reporter protein induced expression.
Method for quantifying target enzyme activity using a redesigned genetic circuit comprising the following steps:
(a) (i) a gene encoding a regulatory protein that recognizes a phenolic compound and induces expression of a downstream reporter protein; (ii) one or more reporter genes selected from the group consisting of genes encoding fluorescent proteins and antibiotic resistance genes; And (iii) a promoter for regulating the expression of the regulatory protein, a region in which the regulatory protein binds to induce the expression of a downstream reporter gene, and a promoter for regulating the expression of the reporter gene. Providing a microorganism contained in the cytoplasm;
(b) providing a clone or gene library comprising a gene encoding the enzyme to be quantified;
(c) introducing the clone or gene library into a microorganism containing the redesigned genetic circuit for detecting the phenolic compound to produce a recombinant microorganism;
(d) treating the recombinant microorganisms with a phenol-releasing compound which may liberate the phenolic compound by enzymatic reaction; And
(e) detecting the phenolic compound released by the enzymatic reaction to quantify the activity of the reporter protein induced expression.
The method of any one of claims 1 to 4, wherein the reporter gene and a promoter regulating expression of the reporter gene are operably linked.
The promoter of the reporter gene according to any one of claims 1 to 4, wherein the regulatory protein binds to induce expression of a downstream reporter gene so that the regulatory protein binds to allow the downstream reporter gene to be expressed. Method for activating.
The gene according to any one of claims 1 to 4, wherein a gene encoding a regulatory protein which recognizes the phenolic compound and induces expression of a downstream reporter protein and a promoter regulating the expression of the regulatory protein are interoperable. Characterized in that the connection.
The method according to any one of claims 1 to 4, wherein the target enzyme is an enzyme capable of releasing a phenolic compound by an enzymatic reaction.
The enzyme according to any one of claims 1 to 4, wherein the enzyme is alpha-glucosidase, beta-glucosidase, cellulase, glycosyl ceramidase, psopatase, phytase, esterase, lipase, Uretanase, amidase, peptidase, proteinase, oxidoreductase, phenol-lyase, dihalogenase, isomerase, monooxyginase and deoxygenase How to.
The phenolic compound released by the enzymatic reaction according to any one of claims 1 to 4 is phenol, o-chlorophenol, m-chlorophenol, p-chlorophenol, o-nitrophenol, m-nitrophenol , p-nitrophenol, salicylic acid, 2-aminophenol, 2-methoxyphenol, catechol, lysosinol, 3-methylcatechol, 2,4-dimethylphenol, 2,5-dimethylphenol, 3,4- Dimethylphenol, 2,3-dimethylphenol, 3,5-dimethylphenol, 2,4-dichlorophenol, 2,5-dichlorophenol, 2,3-dichlorophenol, 2,6-dichlorophenol, 3,4-dichloro Phenol, 3,5-dichlorophenol, 2,4-dinitrophenol, o-cresol, m-cresol, p-cresol, 2-ethylphenol, 3-ethylphenol, 2-fluorophenol, 2-iodinephenol and And a compound selected from the group consisting of benzene.
The compound according to any one of claims 1 to 4, wherein the phenol-releasing compound comprises: an ester in which a phenol hydroxyl group is modified; Glycosides; Phosphate esters; Phenol derivatives in which an alkyl group, carboxyl group, amino group, thiol group, amide group, sulfide group, nitro group or halogen group is introduced at ortho-, meta- or para- position; And a benzene ring compound.
delete The method of claim 1, wherein the fluorescent protein is selected from the group consisting of Green Fluorescent Protein (GFP), UV-excited Green Fluorescent Protein (GFP _UV ), and Red Fluorescent Protein (RFP). How to feature.
The method of any one of claims 1 to 4, wherein the antibiotic resistance gene is selected from the group consisting of kanamycin gene, chloramphenicol gene and tetracycline gene.
The method of any one of claims 1 to 4, wherein the activity of the reporter protein is measured using a method selected from the group consisting of microbial colony image analysis, fluorescence spectrum analysis, fluorescence flow cytometry (FACS), and antibiotic resistance measurement. How to feature.
The method according to any one of claims 1 to 4, wherein the microorganism is selected from the group consisting of E. coli, Pseudomonas, yeast, plant cells and animal cells.
The method of claim 1, wherein the redesigned genetic circuit comprises a gene encoding a ribosome binding site (RBS).
The method according to any one of claims 1 to 4, wherein the reporter gene is a double reporter gene consisting of a gene encoding a fluorescent protein and an antibiotic resistance gene. According to any one of claims 1 to 4, wherein the regulatory protein that recognizes the phenolic compound and induces the expression of the downstream reporter protein is DmpR (transcriptional regulator for dimethylphenol), XylR (transcriptional regulator for xylene), MopR (transcriptional regulator for phenol metabolism), transcriptional regulator for phenol and monomethylated phenols metabolism (PhhR), transcriptional regulator for methylphenol degradation (PhlR), and transcriptional regulator for toluene-benzene utilization (TbuT) .
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