KR20030089115A - biosensor for detecting phenolic compounds and the manufacturing method thereof - Google Patents

Abstract

PURPOSE: A biosensor for detection of phenol compounds and a preparation method thereof are provided, thereby rapidly detecting phenol compounds without pretreatment regardless of time and place. CONSTITUTION: A biosensor for detection of phenol compounds comprises a first nucleic acid molecule containing the nucleotide sequence encoding a reporter molecule; a gene construct containing a phenol compound degrading enzyme regulating gene capR, a promoter gene Pr regulating the expression of the enzyme regulating gene capR and a second nucleic acid molecule with a promoter gene Po regulating the synthesis of the phenol compound degrading enzyme; and a cell containing the gene construct, wherein the first nucleic acid molecule encodes a firefly luciferase gene luc; the biosensor comprises a detection unit of the activity of luciferase when the cell is exposed to phenol; the second nucleic acid molecule is derived from Pseudomonas putida KCTC 1452; and the cell is a freeze-dried powder form.

Description

Biosensor for detecting phenolic compounds and manufacturing method thereof

The present invention relates to a biosensor for detecting a phenolic compound and a method for manufacturing the same, and more particularly, to a biosensor for detecting a phenolic compound using a genetic recombination technology and a method for manufacturing the same.

The phenol spill accident in the Nakdong River is raising awareness of harmful substances. Especially, aromatic organic solvents such as phenol, which are widely used industrially, are introduced into the human body through the food chain to become carcinogenic substances or environmental hormones. Due to its serious impact on living organisms, it is classified as an environmental toxic substance to be removed first in developed countries.

Instrumental chemical methods are used to measure aromatic compounds such as phenols, but the economic and technical effects are not highlighted as they are expensive and expensive due to the enormous equipment, and they are not easy to use without experts. I'm not doing it. In addition, these heavy equipment equipment is inconvenient to use in the field. In addition, these aromatic compounds also had a problem that most volatilized during the pretreatment of the analysis.

Therefore, the development of technology to detect these aromatic compounds more quickly and simply and at lower cost is economical to solve the problems such as the high cost and field application of the existing detection method in terms of national and social aspects that must comply with the global Stockholm Convention. And is very important at the environmental and technical level. In addition, in order to prevent and monitor the prior outflow of the compounds, simple and rapid analysis methods are required, and as described above, an analysis method that does not or does not require pretreatment to solve the problem of volatilization in the pretreatment process is required. .

On the other hand, it has been reported that many microorganisms, including Pseudomonas , adapt to toxic substances such as hardly degradable aromatic compounds present in the environment and have the ability to biodegrade them and use them as carbon sources (Harayama and Timmis, 1992: Kustu et. al., 1991). The properties of these microorganisms are due to their genes and related proteins, and microorganisms are evolving to have enzymes that break down these toxins for survival. These microorganisms regulate the expression of related genes to biosynthesize enzymes only when there are compounds to degrade.

Expression control is possible by the interaction of a specific regulatory gene called a promoter with a protein called an activator. The activator protein must be combined with a hardly degradable aromatic compound to induce a structural variation to initiate transcription for biosynthesis of the degrading enzyme.

This binding results in the formation of a DNA promoter complex consisting of a transcriptional activator, host factor and RNA polymerase, which expresses the downstream enzyme gene (Marques and Ramos, 1993; Perez). Martin et al., 1994). Several activator proteins, including XylR and XylS, have been discovered and reported, and the structure of the degraded genes has been elucidated. This discovery and identification made it possible to develop biosensors that detect organic pollutants.

Recombinant promoters are recombined with reporter genes, such as firefly luciferase or β -galactosidase, and reporter proteins are expressed and biochemically recognized when the promoters of these recombinant genes are activated by related degradation compounds. Attempts have been made to develop biosensors (Willardson et al., 1998; Hollis et al., 1999; Reid et al., 1998; Blouin et al., 1996; Heitzer et al., 1994).

In addition, stress genes were recombined with reporter genes (Belkin et al., 1996) to measure oxygen stress, and heat shock genes were recombined with reporter genes (Van Dyk et al., 1994). The study of the measurement of environmental toxicity by the expression of thermal shock genes induced by and the measurement of the genotoxin of the environment using the SOS lux test (Ptitsyn et al., 1997) has been reported. In addition, studies on various biosensors for measuring various compounds or metals have been conducted (Sticher et al., 1997; Peitzsch et al., 1997; Schramm et al., 1996).

The Willardson research group reported a biosensor that detects benzene, toluene and similar compounds. Transformation of the transcriptable activators xylR and promoter Pu from the Pseudomonas putida mt-2-derived TOL plasmid with the reporter gene luc was transformed into E. coli cells, followed by fluorescence by benzene, toluene, etc. Studied. Using this biosensor, contamination of BTEX (benzene, toluene, and xylene) in water and soil was measured. The exact concentration-dependent fluorescence was observed three-xylene was 39.0 ± 3.8 μ M, 3- methylbenzyl alcohol exhibited a K _1/2 value of 2,690 ± 160 μ M. However, problems have been reported that fluorescence is inhibited at high salts and pH (Willardson et al., 1998). There is also a problem of a high detectable limit concentration.

In Korea, studies on the detection of pesticides by immunoassay method using a receptor that binds pesticides and analysis of glucose by fixing glucose oxidase on the membrane have been reported. A study on the stress response of cells caused by engineering toxins has been reported. In the product development, a BOD meter measuring dissolved oxygen has been developed by bioelectronics, and a device for measuring dissolved oxygen using luminescent microorganisms has been studied.

However, a biosensor for detecting phenolic compounds has not been developed that can perform qualitative and quantitative analysis of phenolic compounds in environmental pollutants at low cost and quickly.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a biosensor for detecting phenolic compounds and a method for producing the same that can be analyzed inexpensively at a nano level in a short time without a pretreatment process. .

1 illustrates a cloned vector map according to the present invention.

2A and 2B are graphs showing bioluminescence reactions of a biosensor for detecting a phenolic compound according to the present invention.

In order to achieve the above object, the biosensor for detecting a phenolic compound of the first aspect of the present invention comprises a first nucleic acid molecule comprising a sequence encoding a reporter molecule having a detection report role, and a phenolic compound degrading enzyme regulatory gene. capR , a gene construct including a second nucleic acid molecule having a promoter gene Pr for regulating expression of the gene and a promoter gene Po for regulating phenolic compound degrading enzyme synthesis; And a cell having the gene construct.

According to the biosensor for detecting a phenolic compound having the above-described structure, there is an advantage that anyone can perform phenolic compound analysis quickly and accurately at low cost according to the expression level of the reporter gene in the presence of the phenolic compound.

According to a specific embodiment of the present invention, the biosensor for detecting a phenolic compound is characterized by expressing fluorescence as the firefly luciferase gene, luc, which is the first nucleic acid molecule is encoded by synthesizing the second nucleic acid molecule and the phenolic compound. It is done.

According to the above configuration, there is an advantage that the concentration of phenol in environmental pollutants can be quantitatively analyzed to 0.05 ppm, which corresponds to a sensitivity 100 times higher than that of a biosensor manufactured by another method (using a stress gene).

According to another specific embodiment of the present invention, the cell comprises a means for measuring the activity of intracellular luciferase when exposed to the phenolic compound.

According to the above configuration, there is an advantage that in-situ samples can be analyzed at a low cost in a short time without a pretreatment process.

The cell is not particularly limited, but E. coli is preferred.

Means for measuring the activity of the luciferase may use any photometer, and a transport luminometer is preferred in the field.

According to a specific embodiment of the present invention, the cells are powdered using a lyophilizer, and can be mixed with the sample solution wherever needed to develop a kit capable of confirming the presence of a hardly degradable aromatic compound in vitro. There is an advantage.

According to the second aspect of the present invention, a method for producing a biosensor for detecting a phenolic compound includes a phenolic compound degrading enzyme regulatory gene capR (proprietary name according to the present invention) and a promoter gene Pr for regulating expression of the gene. Amplifying an insert gene comprising a promoter gene Po for regulating degrading enzyme synthesis; Cutting the amplified transgene with restriction enzyme Kpn I; Cutting an expression vector comprising a firefly luciferase gene with the restriction enzyme; And converting the insert gene and the expression vector into competent cells.

According to the above configuration, there is an advantage that the biosensor for phenolic compound detection can be easily manufactured by a simple process, which can inexpensively analyze field samples at a nano level in a short time without a pretreatment process.

Hereinafter, a biosensor for detecting a phenolic compound of the present invention and a manufacturing method thereof will be described in detail with reference to the accompanying drawings.

First, in order to identify and obtain genes that specifically react with phenol, the genes of microorganisms having the highest resolution among phenol-degrading microorganisms were isolated and then cloned by amplifying degradation-related gene sites using polymerase chain reaction.

At this time, the primer used was produced by designating a sequence of a highly conserved region of evolutionary nucleotide sequence of a known phenol degradation gene. Phenolase- related regulatory gene capR (proprietary name according to the present invention), a promoter gene Pr for regulating its expression, and a promoter gene Po for regulating phenolase synthesis are carried out by carrying out a polymerase chain reaction using the prepared primers. Amplify.

After confirming this by electrophoresis, both ends were cut with the restriction enzyme Kpn I, and the fragment was purified, and then cloned into pGL3-basic vector (Promega company), which is an expression vector cut with the same restriction enzyme as shown in FIG. 1. Then, the expression vector containing the transgene was transformed into E. coli (DH5α). Accurate cloning was confirmed by sequencing and had 98% similar bases with other degraded microorganisms. Since the regulatory gene capR has 13 or more different amino acids from a known phenol degradation gene, it is called capR in the present invention as a unique sequence. The amino acid sequence (563) of the said CapR protein is as follows.

MPIKYKPEIQ HSDFKDLTNL IHFQSTEGKI WLGEQRMLLL QVSAMASFRR

EMVNTLGIER AKGFFLRQGY QSGLKDAELA RKLRPNASEY DMFLAGPQLH

SLKGLVKVRP TEVDIDKECG RFYAEMEWID SFEVEICQTD LGQMQDPVCW

TLLGYACAYS SAFMGREIIF KEVSCRGCGG DKCRVIGKPA EEWDDVASFK

QYFKNDPIIE ELYELQSQLV SLRTNLDKQE GQYYGIGQTP AYQTVRNMMD

KAAQGKVSVL LLGETGVGKE VIARSVHLRS KRAAEPFVAV NCAAIPPDLI

ESELFGVEKG AFTGATQSRM GRFERADKGT IFLDEVIELS PRAQASLLRV

LQEGELERVG DNRTRKIDVR VIAATHEDLA EAVKAGRFRA DLYYRLNVFP

VAIPALRERR EDIPLLVEHF LQRFHQEYGK RTLGLSDKAL EACLHYSWPG

NIRELENVIE RGIILTDPNE SISVQALFPR APEEPQTASE RVSSDGVLIQ

PGNGQGSWIS QLLSSGLSLD EIEESLMREA MQQANQNVSG AARLLGLSRP

ALAYRLKKIG IEG

2A and 2B are graphs showing fluorescence values (bioluminescence) according to phenol concentration and incubation time measured by a biosensor for detecting phenolic compounds according to the present invention, and FIG. 2A is a graph showing fluorescence values as log values. As shown in Figure 2a and 2b, the fluorescence value increased depending on the concentration from 0.05ppm (500nM) of phenol to about 3ppm, and the fluorescence value increased with time even in the hourly measurement after phenol injection.

Hereinafter, preferred embodiments of the present invention will be described.

Example 1. Isolation of Genomic DNA

Pseudomonas putida KCTC1452, known to decompose phenols from the gene bank, was inoculated in 3 ml of tryptic soy broth, shaken at 200 rpm for 18-20 hours at 25 ° C, and then for 3 minutes at 6000 rpm. Cells were collected by centrifugation.

0.567ml of 10mM Tris-HCl / 1mM EDTA (pH 8.0) was added to the collected cells and suspended. Then, 30µl of 10% SDS and 3µl of 20mg / ml proteinase K were mixed carefully and mixed for 1 hour at 37 ° C. It was left.

80 μl of 5M NaCl was added thereto and carefully mixed, 100 μl of 10% CTAB / 0.7% NaCl solution was mixed and heated at 65 ° C. for 10 minutes.

Add the same amount of chloroform / isoamyl alcohol to the solution, mix well by inverting, centrifuge, take the supernatant, transfer to a new tube, add 1/10 of 3M sodium acetate pH 5.2, and add 0.6 Pear isopropanol was added and mixed well.

When genomic DNA is precipitated by salt, white thread-shaped DNA is seen. Seal the end of the sterile Pasteur pipette well and remove it. Repeat the immersion in 1 ml of 70% ethanol to remove the salt. After lightly drying, the mixture was placed in 100 µl of 10 mM Tris-HCl / 1 mM EDTA (pH 8.0) and left for at least 10 minutes to be released.

When genomic DNA melted in the Pasteur pipette, it was placed at 4 ° C. overnight to allow the genomic DNA to spread evenly in the buffer, which was confirmed to be genomic DNA by hanging on 0.7% agarose gel.

Example 2 Search for Genes Related to Phenol Degradation Regulation

Literature research confirmed that DmpR , a major regulatory protein involved in phenol degradation, is an important gene for phenol recognition and degradation in the phenol degradation mechanism, and searched for the corresponding gene cluster, dmp operon. . The NCBI search site in NIH was used for this search. The nucleotide sequence of the dmpR and its promoter portion, Pr , and the Po portion, a promoter of the dmp operon regulated by the DmpR regulatory protein, were investigated.

Example 3. Primer Preparation

Based on the DNA sequence of the pseudoprotein found in the gene bank, primers corresponding to the beginning and end of related genes were customized to Genotech as follows.

Forward; 5'-CGGGGTACCAAGCTTATCCTAGCCTTCGATGCCGAT-3 '(SEQ ID NO: 2)

Reverse; 5'-CGGGGTACCAAGCTTTAACGAGTGAGCTGATGGAAA-3 '(SEQ ID NO: 3)

Example 4. Amplification by Polymerase Chain Reaction (PCR)

From the genomic DNA of the identified strain, the primer was used to make 50 µl of the total reaction amount. The concentration of genomic DNA to be used as template DNA was 100 ng, and the prepared primer was made to have a final concentration of 1.0 μM. The concentrations of dNTPs (dATP, dCTP, dGTP, dTTP) were set to 2.5 mM, respectively. Taq polymerase was 1.25 units and the PCR reaction was performed.

First, pre-denaturation at 95 ° C. for 10 minutes, followed by 30 cycles at 95 ° C. for 1 minute, 50 ° C. for 1 minute, and 72 ° C. for 2 minutes, followed by reaction at 72 ° C. for 10 minutes, results in DmpR-related genes. Was amplified and electrophoresed in 0.5X TAE buffer with DNA marker on 0.7% agarose gel to confirm PCR product.

Example 5. Cloning and Ligation

Restriction enzyme KpnI (Takara Co., Ltd.) 1U was treated for 2 hours at 37 ° C for the identified PCR product. The PCR product was electrophoresed in 1.2% agarose gel, stained with 5 µg / ml EtBr solution for 10 minutes and destained in distilled water for 15 minutes, and then the DNA was about 2.2 kb in UV illuminators. Check the sections using DNA molecule markers and carefully cut the sites containing them.

Weigh the cut agarose gel, add NaI solution in three times the volume, melt the gel at 65 ° C for 5 minutes, and add 5 μl glassmilk to bind the dissolved DNA. After 5 minutes in centrifugation to obtain only the pellet was added to the washing solution and centrifuged three times.

Then, the glass milk-DNA was dried at room temperature, 20 µl of TE buffer was added thereto, and the inserted DNA was extracted. In addition, pGL3 basic vector (Basic Vector, Promega) having luciferase gene was also cut to Kpn I in the same manner.

The pGL3 basic vector having the inserted gene and the luciferase gene thus obtained was added at a 5: 1 ratio of the inserted DNA: vector DNA and subjected to ligation reaction at 16 ° C. for 16 hours using a 1 U T4 DNA ligase (TAKARA). This was transformed into a DH5α competent cell (CaCl2) by a transformation method with CaCl2, and plated on an LB plate containing 50 μg / ml ampicillin, followed by incubation for 16 hours in a 37 ° C. incubator.

The colonies isolated from the plate were inoculated and cultured in LB medium containing 50 µg / ml ampicillin using a sterile toothpick for 16 hours shaking to incubate the plasmid DNA with a QIAprep Spin Miniprep kit (QIAGEN). )).

First, the cells were collected in a 1.5 ml E-tube, and then 250 μl of P1 buffer was added to float the pellet. 250 μl of P2 buffer was added to shake the cells gently, and the cells were lysed five times. Shake to neutralize the dissolved.

After centrifugation at room temperature for 10 minutes, the supernatant was carefully poured into a 2 ml QIAprep spin column, and then 500 µl of PB buffer was added and centrifuged for 1 minute. Centrifuge the spin column for 1 minute by washing with 750 μl PE buffer, discard the filtered matter, and centrifuge once more to remove the remaining buffer solution. Transfer the Quiaprep spin column to 1.5 ml E-tube, and then EB buffer. 50 μl (10 mM Tris-HCl, pH 8.5) was added thereto, followed by 1 minute, followed by centrifugation for 1 minute to separate plasmid DNA. The separated DNA was electrophoresed on 0.7% agarose gel and confirmed to have been cloned using Kpn I.

Example 6 Comparison of Sequencing and Homologs

The sequence of the cloned gene was subjected to auto sequencing using a DNA sequencing kit (ABI 377, PE Applied Biosystems) based on Sanger's dideoxy termination method.

First, 500 ng of the cloned gene, 1.6 pmole of primer and Bigdye (Bigdye) were used to proceed with the PCR reaction using 10 µl of the total reaction solution. As primers used for sequencing, the following primers were used to identify the sequence of the gene inserted into the multiple cloning site of the pGL3 basic vector.

Meaning strand: 5'-CTA GCA AAA TAG GCT GTC CC-3 '(SEQ ID NO: 4)

Reverse strand: 5'-CTT TAT GTT TTT GGC GTC TTC CA-3 '(SEQ ID NO: 5)

After 10 minutes of pre-denaturation at 95 ° C, the reaction was carried out for 25 cycles of 10 seconds at 96 ° C, 5 seconds at 50 ° C, and 4 minutes at 60 ° C, followed by 10 minutes at 72 ° C. The obtained PCR product was transferred to 1.5 ml E-tube and precipitated in ethanol to purify only amplified DNA.

Add 1 µl of 3M sodium acetate pH4.6, add 25 µl of 100% ethanol, place on ice for 25 minutes, centrifuge at 15000 rpm for 4 minutes at 4 ° C, drain the supernatant, and add 70% ethanol. After centrifugation for another 10 minutes, the supernatant is discarded, leaving only the pellets.

Then, after sufficiently drying, the loading dye (loading dye) was added, denatured at 95 ℃ for 2 minutes, and loading was carried out by following only 2μl. Sequencing by automatic sequencer and receiving only sequence as text file. This was compared with NCBI's BLAST database and found that some sequences showed about 98% similarity to the dmpR gene and the Po promoter driven by DmpR. Therefore, the regulatory gene according to the present invention is named capR , and the sequence thereof is represented by SEQ ID NO: 1 in the sequence listing.

In addition, the amino acid sequence has about 97% homology, but it is a new protein that has not been reported before. In addition to phenol, catechol, 2-chlorophenol, and 2-methylphenol ), 2,5-dimethylphenol (2,5-dimethylphenol) was also detectable.

Example 7 Phenol Detection by Fluorescence

The phenol detection capability of the microorganism-derived biosensor made as described above was confirmed by fluorescence expression upon phenol injection. When phenol is injected into a biosensor cell (OD600 = 0.4), CapR, a regulatory protein that specifically reacts with phenol, is produced and reacted, and the complex then binds to the Po promoter in front of the luciferase gene, a fluorescent gene. The biosynthesis of fluorescent expression enzymes was performed, and when the injected substrate was decomposed to fluoresce, it was confirmed by measuring with a luminometer.

At different concentrations of phenol and the expression of fluorescence at different incubation times (see FIGS. 2A and 2B), the biosensor for phenol detection was able to measure 500 nM nano-level phenol, and the sensitivity could be further increased depending on the conditions. It can be shown as a basic experiment.

According to the biosensor for detecting a phenolic compound of the present invention having the above-described configuration, it is possible to achieve an effect of analyzing a field sample at a nano level at a low cost in a short time without a pretreatment process. Therefore, the biosensor according to the present invention can be utilized to measure phenolic compounds in various fields, such as medicine and food, in addition to the environmental field.

On the other hand, according to the manufacturing method of the phenolic compound detection biosensor of the present invention, the effect that the biosensor for phenolic compound detection of the above characteristics can be manufactured by a simple process can also be achieved.

Claims (7)

A first nucleic acid molecule comprising a sequence encoding a reporter molecule having a role of detection report, a phenolic compound degrading enzyme regulatory gene capR , a promoter gene regulating expression of the gene Pr and a promoter regulating phenolic compound degrading enzyme synthesis A gene construct comprising a second nucleic acid molecule having a gene Po ; And Biosensor for detecting a phenolic compound comprising a cell having the gene structure. The biosensor for detecting a phenolic compound according to claim 1, wherein the first nucleic acid molecule encodes the firefly luciferase gene luc . The biosensor for detecting a phenolic compound according to claim 2, comprising means for measuring the activity of luciferase when the cells are exposed to phenol. The biosensor of claim 1 or 2, wherein the second nucleic acid molecule is derived from Pseudomonas putida KCTC1452. The biosensor according to claim 1 or 2, wherein the cells are in the form of lyophilized powder. Amplifying an insert gene comprising a phenolic compound degrading enzyme capR , a promoter gene Pr for regulating expression of the gene, and a promoter gene Po for regulating phenolic compound degrading enzyme synthesis; Cutting the amplified transgene with restriction enzyme Kpn I; Cutting an expression vector having a firefly luciferase gene with the restriction enzyme; Cloning the insertion gene and the expression vector to transform into a competent cell manufacturing method of a biosensor for detecting a phenol-based compound. The method of claim 6, wherein the cell is E. coli DH5α manufacturing method of the biosensor for detecting a phenol-based compound.