KR102354131B1 - Method for Detecting of Chemical Compounds from Environment Using Artificial Genetic Circuitry and Cell to Cell Communication - Google Patents

Abstract

The present invention relates to a method for detecting harmful environmental substances present in the natural environment, in particular water or soil, using the interaction between microorganisms and microorganisms or microorganisms and plants equipped with a redesigned genetic circuit, and more particularly, harmful environmental substances To a method for detecting harmful environmental substances using a sender organism equipped with a circuit for detecting
According to the present invention, it is possible to continuously monitor in real time the presence or absence of harmful environmental substances in all environments in which sender and receiver organisms can inhabit, while the sender and receiver organisms are growing, as well as to monitor the distribution of sender and receiver organisms. Accordingly, a wide detection range can be secured.

Description

Method for Detecting of Chemical Compounds from Environment Using Artificial Genetic Circuitry and Cell to Cell Communication

The present invention relates to a method for detecting harmful environmental substances present in the natural environment, particularly water or soil, using interactions between organisms, such as microorganisms and microorganisms or microorganisms and plants loaded with redesigned genetic circuits for intercellular interactions. In more detail, it relates to a method of detecting harmful environmental substances using a sender organism equipped with a circuit for detecting harmful environmental substances and a receiver organism that emits fluorescent substances by sensing a specific signal emitted therefrom.

Soil and groundwater pollution is one of the causes that can cause serious environmental problems, and it can cause pollution problems to humans as well as secondary and tertiary. In addition, in a situation where there is always a risk of pollutant leakage due to the aging of industrial facilities, it is required to secure an efficient hazardous substance monitoring technology with high sensitivity.

Although instrumental chemical methods are used to detect the above chemical contaminants, an environmental biosensor using microorganisms is being used as an alternative due to the disadvantages of expensive equipment and complicated treatment. In particular , as it is known that many microorganisms, including Pseudomonas , have enzyme genes that biodegrade and adapt to toxic substances such as recalcitrant aromatic compounds in the environment, they can be easily contaminated by combining these genes with fluorescent proteins such as GFP. A whole-cell biosensor that checks the presence or absence of a substance has been developed. A detection biosensor for detecting 2,4,6-trinitrotoluene (TNT) using E. coli (YK Sharon et al ., Appl Microbiol Biotechnol , 98:885, 2013) and a toluene detection sensor (J. Garmendia et al. , Microbial) biotechnology, 1:236, 2008) and phenolic detection sensors (A. Leedjarv et al. , Chemosphere , 64:1910, 2006, S. Choi, AC S Synthetic Biology , 4:163, 2014) are examples. The present inventor's research team has also reported a redesigned gene circuit using a transcriptional regulatory protein that detects phenolic compounds (Korea Patent No. 10-1222056), and this system is a phenol-sensing sensor and It is a system that responds to a phenol concentration of 1 μM or more as a whole-cell sensor in which a reporter is placed in a single cell.

However, these biological sensors take a specific sample, sufficiently react with the biological sensor, and then go through a series of processes to check the presence or absence of harmful substances through expensive and sophisticated laboratory analysis equipment. There is a disadvantage in that it cannot be monitored in real time in a specific environment and external environment.

Accordingly, the present inventors divided the sensor part that detects a specific substance in the whole cell sensor and the reporter part activated by the sensor and modularized each. In detail, a sender module that detects harmful environmental substances and produces communication substances and a receiver module that is activated by sensing communication substances produced/secreted by the sender module were respectively built. The present invention was completed by confirming that environmental substances could be monitored.

The purpose of the present invention is to separate/modularize the sensor and the reporter, unlike the existing whole-cell biosensor in which a sensor and a reporter for detecting environmental pollutants are implemented in the same cell, so that it can be implemented in different cells and organisms of the same type and heterogeneity. To provide a system that can monitor hazardous environmental substances in real time even in a specific environment and external environment to which hazardous environmental substances are exposed.

In order to achieve the above object, the present invention provides (a) a hazardous environmental substance detection site: (i) a gene encoding a transcriptional regulatory protein that recognizes a hazardous environmental substance and induces the expression of a communication material production protein, and (ii) the transcription promoters regulating the expression of regulatory proteins; and

(b) Communication material production site: (i) a gene for producing a communication material by receiving a signal from the transcription control protein, (ii) a promoter for regulating the expression of a gene for producing the communication material, and (iii) a communication material production gene It provides a sender module having a redesigned gene circuit for detecting harmful environmental substances and producing communication materials including a gene encoding a reporter protein that confirms the expression of.

The present invention also provides (a) a communication material sensing site: (i) a gene encoding a transcriptional regulatory protein that recognizes an intercellular communication material and induces the expression of a lower reporter protein, and (ii) regulates the expression of the transcriptional regulatory protein a communication material sensing region consisting of a promoter; and

(b) Reporter region: (i) a gene encoding a reporter protein by receiving a signal from a communication material, and (ii) a promoter for regulating the expression of the reporter protein. It provides a receiver module having a redesigned gene circuit.

The present invention also provides a recombinant sender organism for detecting harmful environmental substances containing the sender module and a recombinant receiver organism for detecting harmful environmental substances containing the receiver module.

The present invention also comprises the steps of (a) culturing the recombinant sender organism for detecting harmful environmental substances in the presence of harmful environmental substances; And (b) culturing the recombinant organism and the recombinant receiver organism together and confirming the reporter protein expression of the receiver organism to provide a method for detecting harmful environmental substances comprising the step of detecting harmful environmental substances.

According to the present invention, while the sender and receiver organisms are growing in all environments contaminated with harmful environmental substances, it is possible to continuously monitor the harmful environmental substances in real time, as well as to secure a wide range of detection according to the distribution of the sender living organisms. have.

1 is an overall schematic diagram of a system for detecting harmful environmental substances based on intercellular communication. Modules divided into sender and receiver are each introduced into various living things and can be driven.
(a) of FIG. 2 shows the production process and vector map of the sender module for microorganisms, (b) the result of analyzing the cell image using a fluorescence microscope and FACS analysis to verify the operation of the sender module introduced into E. coli is a result When the sender module for microorganisms was introduced into E. coli and treated with 0.1 mM phenol, the RFP intensity was higher than that of the negative control not treated with phenol.
3 (a) shows the production process and vector map of the receiver module for microorganisms, (b) the result of analyzing cell images using a fluorescence microscope and FACS analysis to verify the operation of the receiver module introduced into E. coli It is the result. It detects the communication material AHL produced by the sender module and shows a higher GFP intensity than the negative control that is not treated with AHL.
In FIG. 4, it is confirmed that the system operates normally by introducing a sender module and a receiver module to Pseudomonas and Escherichia coli, respectively, to transmit heterogeneous communication materials.
Figure 5 (a) schematically illustrates the vector map and construction process of the receiver module for plants. (b) The result of verifying the operation of the receiver module for plants in onion cells. In the cells treated with the communication substance AHL (+effector), a slightly higher fluorescence intensity than that of the untreated negative control group (-effector) can be confirmed.
6 is a result of verifying whether the receiver module for plants operates in plants. The plant receiver module was introduced into Agrobacterium tumefaciens , and it was introduced into tobacco leaves ( Nicotiana benthamiana ) by infiltration method, and it was confirmed that the AHL concentration-dependent expression of the fluorescent protein in the tobacco leaves of the receiver module for plants by treating the communication material AHL was confirmed.

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 those well known and commonly used in the art.

In the present invention, unlike the existing whole-cell biosensor in which a sensor and a reporter for detecting environmental pollutants are implemented in the same cell, the sensor and the reporter are separated and modularized into a sender and a receiver, respectively, and each is implemented in a different organism. A system for monitoring pollutants was established by recognizing the communication material between objects generated in the receiver module. Using a whole-cell sensor using a single species of microbial cells and organisms, real-time monitoring in a specific environment exposed to harmful environmental substances and in a wide range of external environments is very limited. And by inducing interaction between receiver organisms expressing phenotypes such as color development, technology was secured to continuously monitor harmful environmental substances while the growth of the receiver organism continues.

Accordingly, the present invention is from one point of view, (a) a hazardous environmental substance detection site: (i) a gene encoding a transcriptional regulatory protein that recognizes harmful environmental substances and induces expression of a communication material production protein, and (ii) the transcriptional regulation promoters regulating the expression of proteins; and

(b) Communication material production site: (i) a gene for producing a communication material by receiving a signal from the transcription control protein, (ii) a promoter for regulating the expression of a gene for producing the communication material, and (iii) a communication material production gene It relates to a sender module having a redesigned gene circuit for detection of harmful environmental substances and a redesigned gene circuit for production of communication materials, which includes a gene encoding a reporter protein that confirms the expression of, and a recombinant sender organism for detecting harmful environmental substances containing the sender module .

In the present invention, the organism may be characterized in that it is selected from the group consisting of bacteria, yeast, mold, plant cells and plants, and animal cells.

In one aspect of the present invention, the sender module is composed of a part for detecting harmful environmental substances and a part for producing a communication material. The detection site for hazardous environmental substances is a transcriptional regulation protein that detects phenols and aromatic compounds, VOCs (volatile organic compounds), BTEX (benzene, toluene, ethylbenzene, xylene), TNT (trimitrotoluene, RDX (rapid detention explosive), etc.) It is composed of a promoter that regulates the expression of a protein.The communication material production site is composed of a gene that produces a communication material and a promoter that regulates the expression of this gene, which is a transcription control protein that detects the harmful environmental material. is activated by

In the present invention, the gene encoding the harmful environmental substance detection transcriptional regulatory protein is a phenol detection dmpR gene derived from Pseudomonas putida , toluene, 2-/3-/4-xylene, benzyl alcohol, benzoate and 3-methylbenzoate to detect xylSR gene, todST gene, which detects toluene, benzene, 2-/3-xylene, phenol and trichlorethylene, Pseudomonas sp. The stySR gene for detecting styrene, toluene and epoxystyrene from P. azelaica , the HbpR gene for detecting 2-hydroxybiphenyl from P. fluorescens , the NahR gene for detecting naphthalene and salicylate from P. fluorescens, tbuT for detecting toluene from R. picketti It may be characterized as such.

The gene for producing the intercellular communication material is Vibrio fischeri- derived LuxI/LuxR gene, Aeromonas hydrophila- derived AhyI/AhyR, Burkholderia cepacia- derived CepI/CepR, Chromabacteirum violaceum- derived Cvil/CviR, and the like.

In the present invention, the communication material is a material used as a medium for mutual signal transmission between a sender organism and a receiver organism, and includes AHL (N-acyl homoserine lactone), N-butanoly-HSL, N-octanoyl-HSL, N-hexanoyl -HSL, etc. may be characterized.

In the present invention, the communication material production site of the sender module may be composed of a single gene for producing communication material, or may be formed by fusion with a communication material production gene and an indicator and reporter protein for communication material production. As an indicator and reporter protein of communication material production, the fluorescent protein is _{selected from the group consisting of GFP, GFP UV} , sfGFP and RFP, or the antibiotic resistance gene is an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene and a tetracycline resistance gene. It may be characterized in that it is selected from the group consisting of.

In another aspect, the present invention provides (a) a communication material sensing site: (i) a gene encoding a transcriptional regulatory protein that recognizes an intercellular communication material and induces the expression of a lower reporter protein, and (ii) the expression of the transcriptional regulatory protein a communication material sensing region consisting of a promoter that controls; and

(b) reporter site: (i) a gene encoding a reporter protein by receiving a signal from a communication material, and (ii) a communication material sensing site including a promoter for regulating the expression of the reporter protein and a redesigned gene circuit for reporter expression It relates to a receiver module provided with and a recombinant receiver organism containing the receiver module.

In the present invention, the organism may be characterized in that it is selected from the group consisting of bacteria, yeast, mold, plant cells and plants, and animal cells.

In another aspect of the present invention, the receiver module is composed of a communication material sensing region and a reporter production region. The communication material sensing part is a part that detects the communication material produced by the sender organism, and should be appropriately selected according to the communication material production gene of the communication material production part of the sender organism. As an example, if LuxI is selected as the communication material production gene of the sender organism, the communication material sensing gene of the receiver organism should be selected as LuxR.

In the present invention, the gene encoding the transcriptional regulatory protein for sensing the communication material is Vibrio fischeri- derived LuxI/LuxR gene, Aeromonas hydrophila- derived AhyI/AhyR, Burkholderia cepacia- derived CepI/CepR, Chromabacteirum violaceum- derived Cvil/CviR, etc. can be done with

In the present invention, a fluorescent protein and an antibiotic resistance gene may be used as the reporter protein, and the fluorescent protein _{may be selected from the group consisting of GFP, GFP UV} , sfGFP and RFP, and the antibiotic resistance gene is ampicillin It may be characterized in that it is selected from the group consisting of a resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene.

In the present invention, the promoter regulating the expression of the reporter protein may be a region regulating the lux box or lux operon (luxCDABE upper sequence region).

As used herein, the term “vector” refers to a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. A vector may be a plasmid, a phage particle, or simply a potential genomic insert. Upon transformation into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Since a plasmid is currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. However, the present invention includes other forms of vectors that have an equivalent function as known or coming to be known in the art. Typical expression vectors for expression in mammalian cell culture are, for example, based on pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392 (Pharmingen).

The expression "expression control sequence" refers to a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism. Such regulatory sequences include promoters for effecting transcription, optional operator sequences for regulating such transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating the termination of transcription and translation. For example, regulatory sequences suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells include promoters, polyadenylation signals and enhancers. The factor most affecting the expression amount of a gene in a plasmid is a promoter. As a promoter for high expression, the SRα promoter, a cytomegalovirus-derived promoter, etc. are preferably used.

To express the DNA sequences of the present invention, any of a wide variety of expression control sequences can be used in the vector. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter region of phage lambda, fd Regulatory regions of coding proteins, promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, promoters of said phosphatases, such as Pho5, promoters of yeast alpha-crossing systems and prokaryotic or eukaryotic cells or viruses thereof Other sequences of constructs and induction known to regulate the expression of genes of T7 RNA polymerase promoter Φ10 is E. It can be usefully used to express the protein NSP in E. coli.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. It can be a gene and regulatory sequence(s) linked in such a way that an appropriate molecule (eg, a transcriptional activation protein) allows gene expression when bound to the regulatory sequence(s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a preprotein that participates in secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence if it affects transcription of the sequence; or the ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequences are in contact and, in the case of a secretory leader, in contact and in reading frame. However, the enhancer does not need to be in contact. Linking of these sequences is accomplished by ligation (ligation) at convenient restriction enzyme sites. If such a site does not exist, a synthetic oligonucleotide adapter or linker according to a conventional method is used.

As used herein, the term "expression vector" generally refers to a fragment of double-stranded DNA as a recombinant carrier into which a heterologous DNA fragment is inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not found naturally in host cells. An expression vector, once in the host cell, can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be produced.

As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host. Preferably, the expression control sequence and the corresponding gene are included in one expression vector including the bacterial selection marker and the replication origin. If the expression host is a eukaryotic cell, the expression vector must further comprise an expression marker useful in the eukaryotic expression host.

A host cell transformed or transfected with the above-described expression vector constitutes another aspect of the present invention. As used herein, the term “transformation” refers to the introduction of DNA into a host such that the DNA becomes replicable either as an extrachromosomal factor or by chromosomal integrity. As used herein, the term “transfection” means that an expression vector is accepted by a host cell, whether or not any coding sequence is actually expressed.

A host cell of the invention may be a prokaryotic or eukaryotic cell. In addition, a host having a high DNA introduction efficiency and a high expression efficiency of the introduced DNA is usually used. Well-known eukaryotic and prokaryotic hosts such as Escherichia coli, Pseudomonas, Bacillus, Streptomyces, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO and mouse cells, COS 1, COS 7, African green monkey cells such as BSC 1, BSC 40 and BMT 10, and tissue cultured human cells are examples of host cells that can be used. When cloning the cDNA encoding the NSP protein of the present invention, it is preferable to use an animal cell as a host. In the case of using COS cells, since SV40 large T antigen is expressed in COS cells, the plasmid having the replication origin of SV40 is present as multiple-copy episomes in cells, and is usually Higher expression can be expected. The introduced DNA sequence may be from the same species as the host cell, may be of a different species than the host cell, or it may be a hybrid DNA sequence comprising any heterologous or homologous DNA.

Of course, it should be understood that not all vectors and expression control sequences function equally in expressing the DNA sequences of the present invention. Likewise, not all hosts function equally against the same expression system. However, those skilled in the art can make an appropriate selection among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The copy number of the vector, its ability to control the copy number and the expression of other proteins encoded by the vector, for example antibiotic markers, must also be considered. In selecting the expression control sequence, various factors must be considered. For example, the relative strength, controllability and compatibility of the sequences with the DNA sequences of the invention, etc., should be taken into account, particularly with regard to possible secondary structures. The single-celled host is capable of transferring the product encoded by the DNA sequence of the invention from the host to the selected vector, toxicity, secretion characteristics, ability to correctly fold the protein, culture and fermentation requirements, and the product encoded by the DNA sequence of the invention. It should be selected in consideration of factors such as ease of purification.

In another aspect, the present invention comprises the steps of (a) culturing the recombinant sender organism for detecting harmful environmental substances in the presence of harmful environmental substances; and (b) culturing the recombinant sender and receiver organisms together, and detecting the harmful environmental substances by confirming the expression of the reporter protein of the receiver organism.

In the present invention, the receiver module for plants is composed of a communication material sensing region and a reporter production region, and broad host and binary vectors are used to be operable in plant cells. The vector for a broad-spectrum host for plants in the present invention basically contains a pVS1 replication origin and a streptomycin resistance gene as a selective antibiotic marker. The communication material sensing region of the plant receiver module was prepared by fusion of the communication material sensing gene and the Kozac sequence and the VP64 sequence at the N-terminal and C-terminal ends of this gene, respectively.

In the present invention, the Kozac sequence of the communication material sensing region of the plant receiver module functions similarly to the RBS (ribosome binding sequence) of prokaryotic cells such as E. coli, and is intended for efficient gene expression in plants or plant cells.

In the present invention, the VP64 sequence of the communication material sensing site of the plant receiver module is composed of a partial fusion of a protein sequence of VP16 derived from Herpes simplex virus 4 repeatedly. When a heterologous gene is expressed in a eukaryotic cell such as a plant, It is for recruiting RNA polymerase.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Preparation of sender module for microorganisms

The sender module for microorganisms uses the dmpR gene derived from P. putida that detects phenolic compounds and the Pu promoter to construct a hazardous environmental substance detection site, and uses the LuxI gene derived from Vibrio fischeri that produces AHL, a communication material, to produce a communication material. was built. LuxI gene derived from Vibrio fischeri is inserted into pGESSv4 (pGESSv4, ACS Synth. Biol ., 3: 163~171, 2014) containing the dmpR gene derived from P. putida for microorganisms that produce AHL in a phenolic compound-dependent manner The center module was built.

Additionally, turboRFP was fused/expressed at the C-terminus of the communication material production gene to confirm that the communication material production site works properly in the microorganism in which the sender module is inserted.

(1) Obtaining the LuxI gene for the communication material production site of the sender module

The LuxI gene (GenBank: Acc.No. CP000021.2) was synthesized in Bioneer (Korea) to secure the gene (pGENB1-LuxI). The luxI gene was obtained from a synthetic gene through a polymerase chain reaction (PCR). The primers used at this time were subjected to PCR using forward (SEQ ID NO: 1) and reverse primers (SEQ ID NO: 2). PCR was performed in consideration of the primer binding temperature and length of each gene, and was repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 1 minute at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of 633 bp, which is the expected size, was generated.

SEQ ID NO: 1,

5'-atccgccaacctggagaaggagatatacatatgataaaaaaatcggactttttgggcatt-3'

SEQ ID NO: 2,

5'-aaatcttctctcatccgccaaaaacagaagcttaatttgatacagcttttctaaactgatc-3'

(2) Obtaining the dmpR gene for the hazardous environmental substance detection site of the sender module

Next, using pGESSv4 (pGESSv4, ACS Synth. Biol ., 3: 163-171, 2014) containing the dmpR gene derived from P. putida that detects phenolic compounds as a template DNA, forward (SEQ ID NO: 3) and reverse primers (SEQ ID NO: 4) was subjected to PCR to secure the vector by PCR. PCR was performed in consideration of the gene primer binding temperature and size, and repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 3 minutes at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of 5,511 bp, which is the expected size, was generated.

SEQ ID NO: 3,

5'-gatcagtttagaaaagctgtatcaaattaagcttctgttttggcggatgaagagaagattt-3'

SEQ ID NO: 4,

5'-aatgcccaaaaagtccgatttttttatcatatgtatatctccttctccaggttggcggat-3'

Gibson Assembly (Master Mix Assembly Master Mix, NEB, USA) reaction of the vector obtained from luxI and pGESSv4 (pGESSv4, ACS Synth. Biol ., 3: 163~171, 2014) obtained by the PCR is provided in the kit. carried out according to The recombinant plasmid was introduced into Escherichia coli DH5α by heat shock, spread on LB solid medium to which 100 μg/ml of ampicillin was added as a selection marker, and cultured at 37° C. for one day.

(3) obtaining turboRFP (732bp) gene

To check whether the communication material production region of the sender module works properly, turboRFP was fused/expressed at the C-terminus of the communication material production gene.

TurboRFP gene pturboRFP-dest1 (Evrogen, Russia) as a template was carried out PCR using the forward (SEQ ID NO: 5) and reverse primers (SEQ ID NO: 6) to secure the insert containing turboRFP. PCR was performed in consideration of the primer binding temperature and length of the genes, and was repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 1 minute at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of the expected size of 792 bp was generated.

SEQ ID NO: 5;

5'-agctgtatcaaattaaaaggagatatacatatgagcgagctgatcaaggagaacatgcac-3'

SEQ ID NO: 6,

5'-aaatcttctctcatccgccaaaacagaagcttagccatggctgattcgagatctgagtcc-3'

Next, using the recombinant plasmid (Example 1 (2)) that detects the phenolic compound and synthesizes N-acyl homoserine lactone (AHL) as a template DNA, forward (SEQ ID NO: 7) and reverse primers (SEQ ID NO: 8) are used Thus, PCR was carried out to obtain a vector. PCR was performed in consideration of the gene primer binding temperature and size, and repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 4 minutes at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of 6,405 bp, which is the expected size, was generated.

SEQ ID NO: 7,

5'-ggactcagatctcgaatcagccatggctaagcttctgttttggcggatgaagagaagattt-3'

SEQ ID NO: 8;

5'-gtgcatgttctccttgatcagctcgctcatatgtatatctccttttaatttgatacagct-3'

Gibson Assembly (Master Mix Assembly Master Mix, NEB, USA) reaction was performed with the turboRFP prepared above and the vector obtained from Example 1 (2) according to the method provided in the kit. The recombinant plasmid was introduced into Escherichia coli DH5α by performing heat shock, spread on LB solid medium supplemented with 100 μg/ml of ampicillin as a selection marker, and cultured at 37° C. for one day (FIG. 2a).

Example 2: Verification of operation of the sender module for microorganisms

In order to verify the operation of the sender module for microorganisms constructed in Example 1, the sender module for microorganisms was introduced into E. coli cells to construct and verify a recombinant sender microorganism.

E. coli DH5α was transformed with the phenol detection sender module plasmid for microorganisms constructed in Example 1, and then cultured/reacted for 17 hours at 30°C with 0.1 mM phenol in LB liquid medium, followed by a fluorescence microscope (Cell Observer system, Carl Zeiss, USA) was used to analyze the fluorescence expression level of RFP using single-cell imaging and FACS (FACSCalibur, BD, USA).

As a result of imaging analysis, RFP fluorescence could not be observed in the control group to which phenol was not added, but cells in which RFP fluorescence was strongly expressed could be observed when phenol was added (FIG. 2c). Similar results were also confirmed in FACS analysis. Compared to the control group without addition of phenol, when the sender module was activated by adding phenol, it was observed that the fluorescence signal in FACS shifted to the right compared to the case where the sender module was not added (Fig. 2c). As a result, it was possible to verify that the phenol detection sender module detects phenol in microorganisms and regulates the expression of the LuxI gene, a communication material production gene.

Example 3: Preparation of a receiver module for microorganisms

The receiver module for microorganisms uses Vibrio fischeri- derived LuxR (GenBank Acc.No. CP000021.2) gene that detects AHL, a communication material, and a constitutive promoter (BBa_23100, BioBrick part) to construct a communication material detection site, and green fluorescence A reporter production site was constructed using a protein and a promoter ( regulating the Lux operon derived from Vibrio fischeri).

(1) Obtaining the LuxR gene for the communication material sensing site of the receiver module

The LuxR gene (GenBank: Acc. No. CP000021.2) was synthesized and obtained by Bioneer (pGENB1-LuxR). The luxR gene was obtained from a synthetic gene through a polymerase chain reaction (PCR). The primers used at this time were subjected to PCR using the forward (SEQ ID NO: 9) and reverse primers (SEQ ID NO: 10) to secure an insert containing luxR. PCR was performed in consideration of the primer binding temperature and size of each gene, and repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 1 minute at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of 810 bp, which is the expected size, was generated.

SEQ ID NO: 9;

5'-tcgtgaggatgcgtcatcgccattggtaccttaatttttaaggtatggacaattaatggc-3'

SEQ ID NO: 10,

5'-ctagctactagagattaaagaggagaaaccatgaacattaaaaatataaatgctaatgag-3'

Next, the vector containing eGFP uses pGESSv4 (pGESSv4, ACS Synth. Biol ., 3:163-171, 2014) containing the eGFP gene as a template DNA for forward (SEQ ID NO: 11) and reverse primers (SEQ ID NO: 12) was used to perform PCR to obtain a vector. PCR was carried out in consideration of the gene primer binding temperature and length, and repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 3 minutes at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of the expected size of 4,062 bp was generated.

SEQ ID NO: 11,

5'-ctcattagcatttatatttttaatgttcatggtttctcctctttaatctctagtagctag-3'

SEQ ID NO: 12,

5'-gccattaattgtccataccttaaaaattaaggtaccaatggcgatgacgcatcctcacga-3'

Using the obtained LuxR gene and 4,062bp obtained from pGESSv4 as a vector, a Gibson Assembly (Master Mix Assembly Master Mix, NEB, USA) reaction was performed according to the method provided in the kit. The recombinant plasmid was introduced into Escherichia coli DH5α by performing heat shock, spread on LB solid medium supplemented with 100 μg/ml of ampicillin as a selection marker, and cultured at 37° C. for one day.

(2) Lux box gene obtained

The promoter for regulating the expression of eGFP was prepared as follows. Vibrio fischeri- derived Lux operon-regulating region, that is, the gene sequence including the lux box and promoter (GenBank Acc. No. CP001133.1) was synthesized and secured in Bioneer (pGENB1-E.LuxBOX). A gene including a lux box and a promoter was obtained from a synthetic gene through polymerase chain reaction (PCR). As the primers used at this time, forward (SEQ ID NO: 13) and reverse primers (SEQ ID NO: 14) were used. PCR was performed in consideration of the gene primer binding temperature and size, and repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 3 minutes at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of the expected size of 199 bp was generated.

SEQ ID NO: 13;

5'-tcattagcatttatatttttaatgttcatggtttctcctctttaatctctagtagctag-3'

SEQ ID NO: 14;

5'-ggtgaacagctcctcgcccttgctcaccatatgtatatctccttttttcgactataac-3'

Next, PCR was performed using the forward (SEQ ID NO: 11) and reverse primers (SEQ ID NO: 12) using the prepared plasmid (Example 3(1)) containing the prepared LuxR and eGFP genes as template DNA. A vector was obtained. PCR was performed in consideration of the gene primer binding temperature and size, and repeated 30 times in a cycle consisting of 30 seconds at 95°C, 30 seconds at 50°C, and 3 minutes at 72°C. As a result of confirming the PCR reaction on the agarose gel, it was confirmed that the DNA of the expected size of 4,116 bp was generated.

SEQ ID NO: 15;

5'-gttatagtcgaataaaaaggagatatacatatggtgagcaagggcgaggagctgttcacc-3'

SEQ ID NO: 16,

5'-ctagctactagagattaaagaggagaaaccatgaacattaaaaatataaatgctaatga-3'

The Gibson Assembly (Master Mix Assembly Master Mix, NEB, USA) reaction was performed with the site controlling the Lux operon derived from Vibrio fischeri prepared above and the vector obtained in Example 3(1) according to the method provided in the kit. The recombinant plasmid was introduced into Escherichia coli DH5α by heat shock, spread on LB solid medium supplemented with 100 μg/ml of ampicillin as a selection marker, and cultured at 37° C. for one day (FIG. 3a).

Example 4: Operational verification of the receiver module for microorganisms

In order to verify the operation of the receiver module for microorganisms prepared in Example 3, the AHL detection receiver module plasmid prepared in Example 3 was inserted into E. coli DH5α, and then 0.1 mM AHL was added in the LB liquid medium at 30 ° C. After incubation/reaction for 24 hours, GFP expressed in single cells was analyzed by FACS, and single cell image analysis was performed using a fluorescence microscope (Cell Observer, Carl Zeiss, USA).

As a result of imaging analysis, GFP fluorescence could not be observed in the control group to which AHL was not added, but cells in which GFP fluorescence was strongly expressed could be observed when 0.1 mM AHL was added (FIG. 3b). Similar results were also confirmed in FACS analysis. Compared to the control group without addition of AHL, when the receiver module was activated by adding 0.1 mM AHL, it was observed that the fluorescence signal in FACS shifted to the right compared to the case without the addition of AHL. (Fig. 3b). As a result, it was possible to verify that the receiver module can control the expression of the reporter gene eGFP by detecting the communication material in the microorganism.

Example 5: Verification of detection of hazardous environmental substances through interaction between homogeneous and heterogeneous microorganisms using a microbial sender and receiver module

In order to verify that the sender and receiver modules built to detect harmful environmental substances present in water or soil using interactions between living things work properly in living organisms, the operation was confirmed using microorganisms. A sender module for microorganisms that detects phenolic compounds, which are harmful environmental substances, and produces AHL, a communication material, and a receiver module, a plasmid for microorganisms, which detects AHL and expresses a green fluorescent reporter protein, respectively, were introduced into Escherichia coli DH5α. After co-culture, 0.1 mM of phenol was added, incubated/reacted at 30° C. for 17 hours, and RFP and GFP expressed in the sender and receiver modules by phenol were analyzed using a microscope.

As a result, when phenol, a kind of harmful environmental substance, was present, the activated phenol-dependent transcriptional regulator of the sender module induced the expression of the communication material production gene LuxI and expressed the lower RFP, and the AHL produced by LuxI was the receiver GFP fluorescence was observed by activating the module's AHL sensing modulator, LuxR. Accordingly, the operation of the sender and receiver modules that detect harmful environmental substances using the interaction between microorganisms of the same species (E. coli-E. coli) was verified, and the effect of biological interaction between heterogeneous microorganisms was also verified. The sender module was introduced into Pseudomonas species ( P. putida KT2440ΔttgA), and as a result of culturing with E. coli loaded with a receiver module, it was confirmed that the same results observed in the same species (E. coli-E. coli) were observed ( Fig. 4).

Example 6: Construction of a receiver module for plants

The receiver module for plants consists of a communication material sensing region and a reporter production region, and broad host and binary vectors were used to operate in plant cells. The vector for a broad-spectrum host for plants in the present invention basically contains a pVS1 replication origin and a streptomycin resistance gene as a selective antibiotic marker. For the communication material detection site of the plant receiver module, the Kozac sequence and the 35S promoter of Cauliflower mosaic virus (CaMV) are fused at the N-terminus of the communication material AHL sensing gene LuxR, and RNA polymerization in eukaryotic cells and organisms such as plants at the C-terminus. Each of the VP64 sequences capable of recruiting the enzyme was fused to prepare it. For the reporter production site of the plant receiver module, eGFP was used as the reporter protein, and a plant promoter fused with lux box and the 35S promoter of Cauliflower mosaic virus (CaMV) was inserted into the N-terminus of eGFP and produced (Fig. 5a). .

(1) Obtaining the LuxR-vp64 gene, a communication material detection gene of the receiver module for plants

The gene including the LuxR-vp64 (GenBank Acc. No. DQ16010.1) was synthesized and secured by Bioneer (pGENB1-LuxR-vp64). After cutting the synthetic gene completely with restriction enzymes BamHI and XbaI, cut with the same restriction enzymes and ligated with the plant expression vector pPXRF vector, which was dephosphorylated (SAP, Roche, Switzerland), to prepare a recombinant plasmid to detect communication material for plants was produced. The recombinant plasmid was introduced into Escherichia coli DH5α by heat shock, spread on LB solid medium to which 50 μg/ml of streptomycin as a selection marker was added, and cultured at 37° C. for one day.

(2) Construction of a reporter site of a receiver module for plants

eGFP was used as the reporter protein constituting the reporter region of the receiver module for plants, and the plant-type promoter regulating the expression of this eGFP was lux box (GenBank Acc. No. CP001133.1) and CaMV minimal promoter (GenBank Acc. No. KM062057.1), and manufactured in the following way. A gene including the lux box and CaMV minimal promoter was synthesized and secured in Bioneer (pGENB1-plant lux box). The recombinant plasmid containing the synthesized gene and LuxR-vp64 prepared above was completely cut with restriction enzymes Hind III and Sac I, and then treated with SAP (Roche, Switzerland). A recombinant plasmid was prepared by ligation of a plant-type promoter (plant lux box) obtained by treatment with restriction enzymes and a plant expression vector pPXRF containing luxR-vp64 and eGFP. The recombinant plasmid was introduced into Escherichia coli DH5α by performing heat shock, spread on LB solid medium to which 50 μg/ml of streptomycin as a selection marker was added, and cultured at 37° C. for one day.

Example 7: Operational verification of receiver module for plants

In order to verify that the plant receiver module constructed in Example 6 works properly, the plant receiver module plasmid prepared in Example 6 was coated on gold particles and transformed into onion cells by bombardment method (PDS-1000/HeTM system) did After transformation, AHL (500 uM), a communication material, was treated and cultured for 24 hours in a dark room at 25° C., and fluorescence imaging analysis was performed using a fluorescence microscope (Cell Observer, Carl Zeiss). As a result of image analysis, higher GFP fluorescence was observed in onion cells treated with AHL compared to onion cells not treated with AHL ( FIG. 5b ).

In addition, in order to verify that the receiver module for plants works not only in plant cells but also in plants, the plant receiver module was transformed into Agrobacterium tumafaciens and inoculated into tobacco leaves ( Nicotiana benthamiana) by infiltration method. Two days after inoculation, 100 μM AHL, a communication substance, was treated, and 3 days later, eGFP expression was observed by activating the lux box by LuxR, which detected the communication substance AHL in the plant under a fluorescence microscope. As a negative control group, those that were not treated with the communication material AHL and those treated with tertiary distilled water were used.

As a result of analyzing the fluorescence of eGFP using a fluorescence microscope (Cell Observer system, Carl Zeiss, USA), GFP was strongly expressed in all cells in the plants treated with AHL, compared to the negative control that was not treated with water or communication material AHL. It was confirmed that (Fig. 6). These results confirm that the receiver for plants constructed in the present invention can be operated in plants by detecting the communication material AHL produced by microorganisms equipped with a sender module.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> Korea Institute of Bioscience and Biotechnology <120> Method for Detecting of Chemical Compounds from Environment Using Artificial Genetic Circuitry and Cell to Cell Communication <130> P14-B287 <160> 16 <170> KopatentIn 2.0 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 atccgccaac ctggagaagg agatatacat atgataaaaa aatcggactt tttgggcatt 60 60 <210> 2 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 aaatcttctc tcatccgcca aaacagaagc ttaatttgat acagcttttc taaactgat 59 <210> 3 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gatcagttta gaaaagctgt atcaaattaa gcttctgttt tggcggatga gagaagatt 59 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aatgcccaaa aagtccgatt tttttatcat atgtatatct ccttctccag gttggcggat 60 60 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agctgtatca aattaaaagg agatatacat atgagcgagc tgatcaagga gaacatgcac 60 60 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aaatcttctc tcatccgcca aaacagaagc ttagccatgg ctgattcgag atctgagtcc 60 60 <210> 7 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggactcagat ctcgaatcag ccatggctaa gcttctgttt tggcggatga gagaagattt 60 60 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gtgcatgttc tccttgatca gctcgctcat atgtatatct ccttttaatt tgatacagct 60 60 <210> 9 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tcgtgaggat gcgtcatcgc cattggtacc ttaattttta aggtatggac aattaatggc 60 60 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctagctacta gagattaaag aggagaaacc atgaacatta aaaatataaa tgctaatgag 60 60 <210> 11 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctcattagca tttatatttt taatgttcat ggtttctcct ctttaatctc tagtagcta 59 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gccattaatt gtccatacct taaaaattaa ggtaccaatg gcgatgacgc atcctcacga 60 60 <210> 13 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tcattagcat ttatattttt aatgttcatg gtttctcctc tttaatctct agtagctag 59 <210> 14 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ggtgaacagc tcctcgccct tgctcaccat atgtatatct cctttttatt cgactataac 60 60 <210> 15 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gttatagtcg aataaaaagg agatatacat atggtgagca agggcgagga gctgttcacc 60 60 <210> 16 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ctagctacta gagattaaag aggagaaacc atgaacatta aaaatataaa tgctaatga 59

Claims (17)

A system for detecting harmful environmental substances including sender microorganisms and receiver plants,
The sender microorganism comprises (i) a gene encoding a transcriptional regulatory protein that induces expression of a communication material production protein by recognizing harmful environmental substances and (ii) a promoter that controls the expression of the transcriptional regulatory protein of (i) Hazardous environmental substance detection area (a); and (iii) a gene encoding a communication material production protein, (iv) a promoter controlling the expression of the communication material production protein of (iii) under the control of the transcriptional regulatory protein of (i), and (v) the (iii) Containing a communication material production site (b) comprising a gene encoding a reporter protein for confirming the expression of the communication material production protein of );
The leaves of the receiver plant are (i') a gene encoding a transcriptional regulatory protein that recognizes a communication material and induces expression of a lower reporter protein, and (ii') a promoter that regulates the expression of the transcriptional regulatory protein of (i') Communication material sensing portion (a') comprising a; And (iii') a reporter region comprising a gene encoding a reporter protein and (iv') a promoter controlling the expression of the reporter protein of (iii') under the control of the transcriptional regulatory protein of (i') (b) ');contains;
The communication material is at least one selected from the group consisting of N-acyl homoserine lactone (AHL), N-butanoly-HSL, N-octanoyl-HSL and N-hexanoyl-HSL, a system for detecting hazardous environmental substances.
According to claim 1, wherein the gene encoding the transcriptional regulatory protein of (i) is phenol-sensing dmpR gene, xylSR gene, todST gene, Pseudomonas sp. A system for detecting harmful environmental substances, characterized in that it is selected from the group consisting of a stySR gene derived from stySR, a HbpR gene derived from P. azelaica , a NahR gene derived from P. fluorescens , and a tbuT gene derived from R. picketti.
The system for detecting harmful environmental substances according to claim 1, wherein the gene encoding the communication material production protein of (iii) is at least one selected from the group consisting of LuxI gene, AhyI gene, CepI gene and Cvil gene. .
The system for detecting harmful environmental substances according to claim 1, wherein the reporter protein of (v) is a fluorescent protein selected from the group consisting _{of GFP, GFP UV , sfGFP and RFP.}
According to claim 1, wherein the gene encoding the reporter protein of (v) is an antibiotic resistance gene selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene and a tetracycline resistance gene. A system for detecting environmental substances.
The method according to claim 1, wherein the transcriptional regulatory protein of (i') further comprises at least one selected from the group consisting of a Kozac sequence linked to the N-terminus and a VP64 sequence linked to the C-terminus. A system for detecting hazardous environmental substances.
The system for detecting harmful environmental substances according to claim 1, wherein the gene encoding the transcriptional regulatory protein of (i') is at least one selected from the group consisting of LuxR gene, AhyR gene, CepR gene and CviR gene.
The system for detecting harmful environmental substances according to claim 1, wherein the reporter protein of (iii') is a fluorescent protein selected from the group consisting _{of GFP, GFP UV , sfGFP and RFP.}
The method of claim 1, wherein the gene encoding the reporter protein of (iii') is an antibiotic resistance gene selected from the group consisting of an ampicillin resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, and a tetracycline resistance gene. A system for detecting hazardous environmental substances.
The system for detecting harmful environmental substances according to claim 1, wherein the promoter of (iv') is a region regulating the lux box or lux operon (luxCDABE upper sequence region).
delete delete delete delete delete delete (1) (i) a gene encoding a transcriptional regulatory protein that induces expression of a communication material production protein by recognizing harmful environmental substances Environmental substance detection area (a); and (iii) a gene encoding a communication material production protein, (iv) a promoter controlling the expression of the communication material production protein of (iii) under the control of the transcriptional regulatory protein of (i), and (v) the (iii) A step of culturing a sender microorganism containing a communication material production site (b) comprising a gene encoding a reporter protein for confirming the expression of the communication material production protein of ) in the presence of environmentally hazardous substances;
(2) the sender microorganism, (i') a gene encoding a transcriptional regulatory protein that recognizes a communication substance and induces expression of a lower reporter protein, and (ii') regulates the expression of the transcriptional regulatory protein of (i') Communication material sensing region (a') comprising a promoter; And (iii') a reporter region comprising a gene encoding a reporter protein and (iv') a promoter controlling the expression of the reporter protein of (iii') under the control of the transcriptional regulatory protein of (i') (b) '); treating the leaves of the receiver plant containing; and
(3) confirming whether the reporter protein of (iii') is expressed in the leaves of the receiver plant
including,
The communication material is at least one selected from the group consisting of N-acyl homoserine lactone (AHL), N-butanoly-HSL, N-octanoyl-HSL and N-hexanoyl-HSL, a method for detecting harmful environmental substances.

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