KR100539142B1 - biosensor for detecting BTEX and the manufacturing method thereof - Google Patents

Abstract

The biosensor for detecting BTEX according to the present invention comprises a first nuclear molecule comprising a sequence encoding a firefly luciferase gene having a detection report role, a BTEX degrading enzyme regulatory gene xylR , and a promoter gene for regulating the expression of the gene. Fluorescence measurement of the activity of intracellular luciferase molecules when the cells have a plasmid comprising Pr and a second nucleic acid molecule consisting of a promoter residue, Pu , which regulates BTEX degrading enzyme synthesis and when the cells are exposed to BTEX It is characterized by a kit. According to the above-described configuration, it is possible to achieve the effect of inexpensively analyzing field samples at a nano level in a short time without regard to time and place without pretreatment process for BTEX.

Description

Biosensor for detecting BTEX and its manufacturing method

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

BTEX compounds are Benzene, Toluene, Ethylbenzene, and Xylene. Along with the recent rapid growth of industrial activities, a considerable amount of harmful pollutants are released into the natural environment. BTEX compounds, a representative oil compound of organic compounds, are becoming more contaminated due to leaks and accidents in oil depots and photonic use in industry. Pollution caused by these is widely found in the public drinking water system such as surface water source, ground water source, and treated drinking water. Most VOCs are found to be toxic organic chemicals that cause carcinogenicity. Directives and legislation are on the rise.

In order to measure such BTEX, chromatography-mass spectrometry (GC-MS) and the like have been conventionally used as an instrument chemical method. However, these methods are expensive, take a long time due to complicated processing, and have no limitations in use because they are not easy to use by those without expertise and require heavy equipment. Therefore, it is necessary to develop a method for detecting BTEX easily and quickly at low cost regardless of time and place.

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 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.

The above expression regulation is possible by the interaction of a specific regulatory gene called a promoter and 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, a host factor and an 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; Holis 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, research on various biosensors measuring various chemicals and metals is underway (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. Transformable activator genes xylR and promoter Pu from Pseudomonas putida mt-2-derived TOL plasmids were transformed into E. coli cells by recombination with the reporter gene luc and induced fluorescence by benzene, toluene, etc. The search was studied. Using this biosensor, contamination of BTEX (benzene, toluene, and xylene) in water and soil was measured. Accurate concentration dependent fluorescence was observed, with 3-xylene having a K _1/2 value of 39.0 ± 3.8 μM and 3-methylbenzyl alcohol at 2,690 ± 160 μM. However, problems have been reported that fluorescence is inhibited at high salts and pH (Willardson et al., 1998). There is also a problem of high detectable threshold concentrations.

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, biosensors that can perform qualitative and quantitative analysis of BTEX compounds inexpensively and quickly are not yet developed in Korea.

Therefore, the present invention has been made to solve the above problems, an object of the present invention is to provide a biosensor for BTEX detection and its manufacturing method that can be analyzed at a short time in the field sample at a nano level without a pre-treatment process It is.

In order to achieve the above object, the biosensor for detecting BTEX 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 naphthalene degrading enzyme modulator xylR. , A gene construct comprising a second nucleic acid molecule having a promoter gene Pr for regulating expression of the gene and a promoter gene Pu for regulating naphthalene degrading enzyme synthesis, and a cell having the gene construct.

According to the biosensor for detecting BTEX having the above structure, anyone can perform BTEX analysis quickly and accurately at low cost according to the expression level of the reporter gene in the presence of BTEX.

According to a specific embodiment of the present invention, the BTEX detection biosensor is characterized in that the first nucleic acid molecule encodes the firefly luciferase gene luc .

According to the configuration, there is an advantage in that the concentration of BTEX in the analyte can be quantitatively analyzed to a level of 3 μM.

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

According to the above configuration, there is an advantage that can be inexpensively analyzed on-site samples at a nano level in a short time without pretreatment process for BTEX.

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

The means for measuring the activity of the pushperase can use any optical measuring device, a transport luminometer is preferred in the field.

According to a specific embodiment of the present invention, the cells are powdered form using a lyophilizer, and when necessary, the cells can be mixed with the sample solution to develop the presence of BTEX as a kit that can be identified in vitro. .

According to a second aspect of the present invention, a method for producing a biosensor for BTEX detection may include amplifying a naphthalene degrading enzyme regulatory gene xylR and a promoter gene Pr insertion gene for controlling expression of the gene; Cutting the amplified transgenes with restriction enzymes Kpn I and Xho I; Cutting an expression vector comprising a firefly luciferase gene with the restriction enzymes; Cloning the transgene and the expression vector and transforming the competent cells; Amplifying an insert gene comprising a promoter gene Pu that regulates BTEX degrading enzyme synthesis; Cutting the amplified transgenes with restriction enzymes Xho I and HindIII; And transforming the competent cell by cloning the insertion gene (Pu) and the expression vector.

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

Hereinafter, with reference to the accompanying drawings will be described in detail with respect to the biosensor for detecting the BTEX of the present invention and its manufacturing method.

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

At this time, the primers were prepared by determining the sequence of the evolutionarily very conserved region among the nucleotide sequences of the known BTEX degradation gene. By using the prepared primers, a polymerase chain reaction was carried out to amplify the BTEX degradation-related regulatory gene xylR and the progenitor gene Pr that regulates its expression.

After confirming this by electrophoresis, both ends were cut with restriction enzymes Kpn I and Xho I, and the fragments were purified. Then, pGL3-basic vector (Promega company), which is an expression vector cut with the same restriction enzymes as shown in FIG. Clones. Then, the expression vector containing the transgene was transformed into E. coli (DH5α).

By using the prepared primers, a polymerase chain reaction is carried out to amplify a promoter gene Pu that regulates BTEX degrading enzyme synthesis.

After confirming this by electrophoresis, both ends were cut with restriction enzymes Xho I and Hind III, and the fragments were purified. Then, the plasmid isolated from the transformed cell was used as a vector and cloned into the vector cut by the same restriction enzymes. . Then, the expression vector containing the transgene was transformed into E. coli (DH5α).

Precise cloning was confirmed by sequencing and had 99% similar bases with other degraded microorganisms.

2A and 2B are graphs showing fluorescence values (bioluminexcence) according to BTEX concentration and incubation time measured by the biosensor for detecting BTEX according to the present invention, and FIG. 2A is a graph showing fluorescence values as log values. As shown in Figures 2a and 2b, the fluorescence value increased depending on the concentration of the BTEX compound, and the fluorescence value increased with time even after the BTEX compound injection.

Hereinafter, the present invention will be described in detail based on the preferred embodiment of the present invention, but the present invention is not limited to the embodiment.

Example 1 Isolation of Genomic DNA

Pseudomonas putida KCTC1643, known to degrade BTEX, was inoculated in 3 ml of typtic 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-HCI / 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 ℃. 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 BTEX degradation regulation

The literature review confirmed that XylR, a major regulatory protein, is an important gene for recognition and degradation in BTEX degradation mechanism, and searched for xyl operon, a corresponding cluster of genes. The NCBI search site in NIH was used for this search. The nucleotide sequence of xylR and its promoter portion, Pr , and Pu , a promoter of operon regulated by XylR regulatory protein, were investigated.

Example 3 Preparation of Primer

Based on the base sequence of DNA retrieved from Genebank, the front and back of xylR operon-related genes were prepared by ordering genotech as primers.

xylR / Pr part

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

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

Pu part

Forward; 5'-CCGCTCGAGCTGCCCGGGAAAGCGCGATG-3 '(SEQ ID NO: 4)

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

{Example 4} Amplification by PCR (Polvmerase Chain Reaction)

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 mg, 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.

{Example 5} Cloning and Ligation

Restriction enzymes Kpn I and Xho I (Takara) 1U were treated for 20 hrs at 37 ° C for 1 μg of the identified PCR product xylR / Pr, and restriction enzymes Xho I and Hind III (Takara) 1U were processed for Pu lμg. Was treated at 37 ° C. for 20 hours. The PCR product was electrophoresed in 1.2% agarose gel, stained in 5 µg / ml EtBr solution for 10 minutes, and destained in distilled water for 15 minutes. XylR / 2.1 kb in UV illuminators Pr DNA fragments and 0.25kb Pu DNA fragments are identified 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 and centrifuged 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 with restriction enzyme.

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 by a CaCl2 transformation method, inoculated into 50 μl / ml ampicillin-containing LB medium, followed by shaking culture for 16 hours, and plasmid DNA was then converted to Quiaprep spin mini. Separation was performed using a prep kit (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 suspend the pellet. 250 μl of P2 buffer was added to shake the cells gently. Five times, the cells were lysed. Shake to neutralize the dissolved.

After centrifugation at room temperature for 10 minutes, the supernatant was carefully followed, transferred to a 2 ml QIAprep spin column, and then 500 µl of PB buffer was added and centrifuged for 1 minute. 750 μl of PE buffer was added to the spin column for 1 minute to wash, discard the filtered and centrifuged once more to remove the remaining buffer solution, and transfer the Qyprep spin column to 1.5 ml E-tube. 50 μl (10 mM Tris-HC1, pH8.5) was added thereto, followed by one minute, followed by centrifugation for one minute to separate the plasmid DNA. The separated DNA was electrophoresed on 0.7% agarose gel to confirm that it was cloned using Kpn I and Xho I for xylR / Pr and Xho I and HindIII for Pu.

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 Biosustems) 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. The primers used for sequencing used the following primers to identify the sequence of the gene inserted into the multiple cloning site of the pGL3 basic vector.

Meaning strand: 5'-CTAGCAAAATAGGCTGTCCC-3 '(SEQ ID NO: 6)

Reverse strand: 5'-CTTTATGTTTTTGGCGTCTTCCA-3 '(SEQ ID NO: 7)

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., 4 minutes at 60 ° C., and then at 72 ° C. for 10 minutes. 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 99% similarity with the xylR gene and Pu promoter operated by XylR. The sequence is shown in SEQ ID NO: 1.

Example 7 BTEX Compound Detection by Fluorescence

It was confirmed by fluorescence expression when the BTEX compound injection of the microorganism-derived biosensor made as described above. These compounds are the biosensor cells (OD ₆₀₀ value = 0.4 or so) after the injection is adjusted proteins XylR which specifically react with the BTEX compounds are produced when the reaction in the composite is Pu just before the fluorescence gene is luciferase gene When fluorescence was synthesized by binding to a promoter and biosynthesized by injecting a substrate, it was confirmed by measuring with a luminometer.

In the expression of fluorescence according to the incubation time of each different concentration of BTEX compound (see FIGS. 2A and 2B), the biosensor for BTEX compound detection showed that it was able to measure 3μM level of BTEX compound. It is shown as a basic experiment that can increase.

As described above, according to the biosensor for detecting the BTEX compound of the present invention, it is possible to achieve an effect that can be analyzed at a low cost in the field sample in a short time without a pretreatment process.

Therefore, the biosensor according to the present invention is to measure the compounds throughout the public drinking water system such as surface water source, ground water source, treated drinking water and the degree of contamination due to leakage and accident in oil storage, wide use of BTEX compounds in industry It can be used for the detection of BTEX compounds in the human body.

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

1 is a view showing a cloned vector map according to an embodiment of the present invention.

Figure 2a and 2b is a graph showing the bioluminescence reaction of the biosensor for BTEX detection according to the present invention.

Claims (8)

A first nucleic acid molecule encoding a firefly luciferase gene luc comprising a sequence encoding a reporter molecule having a detection report role, {Benzene, toluene, ethylbenzene, xylene (hereinafter referred to as "BTEX") Pseudomonas putida having a degrading enzyme regulator xylR , a promoter gene Pr that regulates the expression of the gene and a promoter gene Pu that regulates the synthesis of the BTEX synthase A gene construct comprising a second nucleic acid molecule derived from KCTC1643; and BTEX detection biosensor comprising a cell having the gene structure. delete The biosensor for detecting a BTEX compound according to claim 1, comprising means for measuring the activity of luciferase when the cells are exposed to the BTEX compound. delete The biosensor for detecting a BTEX compound according to claim 1 or 3, wherein the cells are in lyophilized powder form. Amplifying an insertion gene comprising {benzene, toluene, ethylbenzene, xylene (hereinafter referred to as "BTEX") degrading enzyme regulatory gene xylR , a promoter gene Pr that regulates expression of the gene; Cutting the amplified transgenes with restriction enzymes Kpn I and Xho I; Cutting an expression vector having a firefly luciferase gene with the restriction enzymes; Cloning the insertion gene and the expression vector and transforming into competent cells, the method for producing a biosensor for detecting a BTEX compound. Amplifying an insert gene comprising a promoter gene Pu that regulates {decomposition of benzene, toluene, ethylbenzene, xylene (hereinafter referred to as "BTEX") compound degrading enzyme synthesis; Cutting the amplified transgenes with restriction enzymes Xho I and HindIII; Cutting the expression vector having a firefly luciferase gene into the restriction enzymes in the transgenic cell; Cloning the insertion gene and the expression vector and transforming into competent cells, a method for producing a biosensor for detecting a BTEX compound. The method for producing a biosensor for detecting a BTEX compound according to claim 6 or 7, wherein the cells are E. coli DH5α.