KR101430685B1 - Artificial biosensor for non-degradable harmful aromatic compound detection and manufacturing method thereof - Google Patents

Abstract

The present invention I relates to a decomposable harmful aromatics detect artificial biosensor and the manufacturing method thereof for, and more particularly, the footage Pseudomonas (Pseudomonas which comprises a transformant into which an expression vector containing a putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter and a marker gene is introduced into a host cell, The artificial biosensor for detection is superior to the conventional microbial biosensor in the specificity and sensitivity to the harmful aromatic compounds, and thus can be usefully used for the detection of harmful aromatic compounds.

Description

TECHNICAL FIELD The present invention relates to an artificial biosensor for detecting harmful aromatic compounds and a method for manufacturing the same,

TECHNICAL FIELD The present invention relates to an artificial biosensor for detecting harmful aromatic compounds and a method of manufacturing the same.

When a degradable aromatic compound is released into the body through a food chain or other pathway, it usually reacts with a polymer such as DNA or protein in its structural similarity and high reactivity to act as a carcinogen or an environmental hormone. For example, dioxins emitted from automobile exhaust gas and waste incineration are not only cancer-causing substances but also cause reproductive and immune system abnormalities. DDT is an environmental hormone that is produced as an insecticide and harmful to the reproductive system. . The degradable aromatic compounds introduced into nature are classified as environmental toxic substances which should be removed as priority in developed countries. In order to measure such a degradable aromatic compound, an instrumental chemical method is utilized, but it takes a long time due to a high cost due to an enormous amount of equipment and complicated treatment, and it is not easy for anyone to utilize it. to be. Also, these heavy equipment can not be used in the field. Therefore, it is important to develop technologies to detect degradation aromatic compounds more quickly, simply, and inexpensively, in terms of the national social aspect, which requires implementation of the Stockholm environmental conventions, and the economic and environmental technological aspects of solving problems at high cost and field applications , A biodegradable aromatic compound measurement biosensor development, 2004).

BTEX compounds refer to benzene, toluene, ethylbenzene, and xylene. Recently, due to the rapid growth of industrial activities, a considerable amount of harmful pollutants have been released into the natural environment. VOCs (volatile BTEX compounds, a representative oil of organic compounds, are becoming more polluted due to leaks and accidents in oil stores and widespread use in industry. Most of the VOCs have been found to be toxic organic chemicals that cause carcinogenesis. Therefore, the pollution caused by volatile organic substances in drinking water Guidelines and legislation have been strengthened. In order to measure such BTEX, conventionally, chromatographic-mass spectrometry (GC-MS) has been utilized by an instrumental chemical method. However, such a method is costly, takes a long time due to complicated processing, Is not easy to use and requires a heavy equipment and there is a limitation in usage that can not be utilized in the field (Korean Application No. 10-2003-0011554).

Therefore, it is expected that 2,4,6-trinitrotoluene (TNT) and 2,4-dinitrotoluene (DNT), which are toluene compounds derived from explosives, and BTEX compounds (benzene, toluene, ethylbenzene, Efforts to develop technologies for efficiently detecting and removing harmful aromatic compounds have been continuing.

A biosensor is a device capable of selectively sensing a substance to be analyzed by converting a biological receptor having a recognition function for a specific substance into an electric or optical signal by combining with an electric or optical transducer to convert a biological interaction and a recognition reaction into an electric or optical signal Is an analytical technique that can quickly and accurately detect substances in a variety of environments at low concentrations. Biosensors are largely divided into protein-based and cell-based methods. Cell-based whole-cell biosensors are used in terms of biological applications, selectivity, convenience, and measurement sensitivity. A lot of research is being done. The whole-cell biosensor is a genetic engineering system consisting of a reporter system such as beta-galactosidase and firefly luciferase, which can specifically react with toxicities caused by environmental pollutants. Is a state-of-the-art biomonitoring technology that can be applied to the measurement of environmental pollution of rivers, lakes, and sewage and sewage sources and soils using modified recombinant cells.

In addition, some microorganisms including Pseudomonas putida have been reported to have the ability to adapt to toxic substances such as harmful aromatic compounds and decompose them (Ramos-Gonzalez, Olson et al. 2002) . Therefore, biosensors that detect harmful aromatic compounds are mostly used. However, there is still a need for a biosensor that senses degradable aromatic compounds in high sensitivity.

Thus, the present inventors obtained todS and todT genes for translating the two proteins using the TodST 2 -phase signal transduction system composed of TodS detection protein and TodT control protein derived from Pseudomonas putida , A promoter of todX gene derived from Pseudomonas putida, and an expression vector containing a marker gene were prepared, and transformants transformed into a host cell (Escherichia coli) were prepared. In addition, a biosensor containing the biodegradable aromatic compound was prepared and the biodegradable harmful aromatic compound was detected. As a result, it was confirmed that the specificity and sensitivity were very high. Therefore, it has been found that the biosensor can be usefully used as an artificial biosensor for detecting harmful aromatic compounds, thus completing the present invention.

Simons, R. W., F. Houman, and N. Kleckner. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53: 85-96. Ramos-Gonzalez, M. I., M. Olson, A. A. Gatenby, G. Mosqueda, M. Manzanera, M. J. Campos, S. Vichez, and J. L. Ramos. 2002. Cross-regulation between a novel two-component signal transduction system and catabolism of toluene in Pseudomonas mendocina and the Todst system from Pseudomonas putida. J Bacteriol 184: 7062-7. Behzadian, F., H. Barjeste, S. Hosseinkhani, and A. R. Zarei. 2011. Construction and characterization of Escherichia coli whole-cell biosensors for toluene and related compounds. Curr Microbiol 62: 690-6. Dykxhoorn, D. M., R. St Pierre, et al. 1996. Synthesis of the beta and beta subunits of Escherichia coli RNA polymerase is autogenously regulated in vivo by both transcriptional and translational mechanisms. Mol Microbiol 19 (3): 483-493. Peters, J. E., T. E. Thate, et al. 2003. Definition of the Escherichia coli MC4100 genome by use of a DNA array. "J Bacteriol 185 (6): 2017-2021. Ramos-Gonzalez, M. I., M. Olson, et al. 2002. Cross-regulation between a novel two-component signal transduction system and catabolism of toluene in Pseudomonas mendocina and the Todst system from Pseudomonas putida. J Bacteriol 184 (24): 7062-7067. Simons, R. W., F. Houman, et al. 1987. Improved single and multicopy lac-based cloning vectors for protein and operon fusions. Gene 53 (1): 85-96.

The object of the present invention is to provide Pseudomonas sp. which comprises a transformant into which an expression vector containing a putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter and a marker gene is introduced into a host cell, A method for producing the same, and a method for detecting harmful aromatic compounds by using a biosensor.

In order to achieve the above object, the present invention provides an expression vector comprising a todS gene derived from Pseudomonas putida , a todT gene derived from Pseudomonas putida , a promoter of a todX gene derived from Pseudomonas putida and an expression vector comprising a marker gene, The present invention also provides an artificial biosensor for detecting a harmful aromatic compound.

In addition,

1) preparing an expression vector comprising a Pseudomonas putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter and a marker gene; And

2) transforming an expression vector of step 1) into a host cell to produce a transformant. The present invention also provides a method for producing an artificial biosensor for detecting a noxious degradable aromatic compound.

In addition,

1) culturing the test sample after treating the biosensor of the present invention;

2) measuring the expression level of the marker gene of the biosensor of step 1); And

3) a step of judging that a harmful aromatic compound is contained in the test sample when the level of expression of the marker gene of step 2) is increased as compared with that of the control sample.

Pseudomonas footage of the present invention is (Pseudomonas which comprises a transformant into which an expression vector containing a putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter and a marker gene is introduced into a host cell, The artificial biosensor for detection is excellent in the specificity and sensitivity compared with the conventional method for detecting harmful aromatic compounds, so that the harmful aromatic compounds can be detected more accurately and more accurately.

1 is Pseudomonas footage is (Pseudomonas putida ) A sensor protein, a regulatory protein, a promoter of a target gene whose expression is regulated by the system, and a promoter regulated by the promoter, which are used to recognize BTEX compounds such as toluene in F1 A method for producing an artificial biosensor capable of highly sensitive detection of a refractory harmful aromatic compound using a marker gene that can easily read expression.
2 is a diagram showing a sensor design of a degradable aromatic compound sensing biosensor.
3 is a diagram showing the production of an Escherichia coli strain ( E. coli MC4100 :: λΦP _todX- lacZ) into which a TodST system driving display circuit is introduced.
FIG. 4 is a graph showing the detection of harmful aromatic compounds using the biosensor manufactured in the present invention.
5 is a diagram showing a pEXT20 vector.
6 shows a vector pRS415.

Hereinafter, the present invention will be described in detail.

Pseudomonas putida ) A sensor protein, a regulatory protein, a promoter of a target gene whose expression is regulated by the system, and a promoter regulated by the promoter, which are used to recognize BTEX compounds such as toluene in F1 The present invention provides a biosensor capable of detecting highly degradable harmful aromatic compounds using a marker gene that can easily read expression (see FIG. 1).

The present invention relates to Pseudomonas sp. detection of harmful aromatic compounds including a transformant into which an expression vector containing a putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter and a marker gene is introduced into a host cell The present invention provides an artificial biosensor.

Specifically, the todS gene has a nucleotide sequence of SEQ ID NO: 5, but is not limited thereto.

Specifically, the todT gene has the nucleotide sequence of SEQ ID NO: 6, but is not limited thereto.

The promoter of the todX gene specifically includes the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.

The promoters of the todS gene, the todT gene, and the todX gene are each composed of a base sequence in which one or several bases are added, deleted, or substituted as long as they have the same activity in the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: But is not limited thereto.

The promoters of the todS gene, the todT gene and the todX gene have 80% or more homology, more specifically 90% or more homology, most specifically 95% or 96%, more preferably 90% or more homology to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: , 97%, 98%, 99% or 99.5% or more homology with the nucleotide sequence shown in SEQ ID NO.

The above-mentioned marker gene specifically includes a fluorescent protein (GFP), an alkaline phosphatase, a firefly luciferase, a bioluminescent microorganism (Vibrio, Xenorhabdus, Photorhabdus, and Photobacterium), and beta-galactosidase, but is not limited thereto. More specifically, beta-galactosidase may be selected from the group consisting of beta-galactosidase, But not limited to, beta-galactosidase.

The expression vector is specifically pEXT20-TodST, but is not limited thereto.

The host cell may be a prokaryotic microorganism or a eukaryotic microorganism but is not limited thereto. More specifically, the prokaryotic microorganism may be E. coli , and the eukaryotic microorganism may be yeast.

The refractory harmful aromatic compound is not particularly limited as long as it is a BTEX compound (Benzene, Toluene, Ethylbenzene, Xylene) and a toluene compound. More specifically, the BTEX compound Benzene and toluene, and the toluene-based compound is not limited to 2,4,6-trinitrotoluene (TNT).

To the inventors have I to produce a vector that contains the sensor of the artificial biosensor for detecting decomposable harmful aromatics, Pseudomonas footage is (Pseudomonas 2 of one TodST putida) - was used as a factor signaling pathway. The TodST 2 -phage signal transduction system consists of a TodS-sensing protein and a TodT-regulating protein. The TodS-sensing protein detects autophosphorylation using ATP when a toluene-based compound is detected. . At this time, the TodT regulatory protein is activated when it receives the phosphate group from the TodS sensing protein and attaches to the promoter of the target gene to regulate the expression of the gene (see FIG. 2). In the present invention, todS and todT genes for translating these two proteins were obtained and cloned into a pEXT20 vector (see Fig. 5), which is an expression vector for Escherichia coli capable of induction expression. As a result, a pEXT20-TodST vector in which todS and todT were inserted was obtained (see Fig. 2).

In order to detect the harmful aromatic compounds in the sample by using the recombinant strain, the present inventors obtained the promoter sequence of the Pseudomonas putida-derived todX gene whose expression is strictly controlled by the TodST system, A labeling circuit was constructed by inserting a marker gene (β-galactosidase) in front of it and inserted in a single copy on a specific site on the chromosome of E. coli. As a result, a stable labeled recombinant strain was prepared (see Fig. 3).

In addition, the inventors of the present invention found that a biosensor of the present invention, that is, E. coli MC4100 :: λΦP _todX- lacZ / pEXT20-TodST transformed with a todST expression vector, In order to confirm the degree of detection, it was confirmed that the expression of the marker gene was detected under culture conditions containing various harmful aromatic compounds, and that the corresponding aromatic compound could be detected with high sensitivity (see FIG. 4). In particular, TNT and TNF increased the expression levels of up to 4.5-fold and 7.6-fold, respectively, depending on the concentration of toluene and benzene, Expression was increased 8-fold or more (see FIG. 4). Therefore, the possibility of a heterozygous host (Escherichia coli host) biosensor capable of being sensitive to the BTEX family compound was observed.

Therefore, the biosensor of the present invention was found to be capable of detecting the corresponding aromatic compound with high sensitivity as a result of measuring the expression of the marker gene in a culture condition containing a harmful aromatic compound having various concentrations of the degradable aromatic compound. Therefore, And therefore, the present Pseudomonas putida ( Pseudomonas < RTI ID = 0.0 > a biosensor comprising a transformant into which an expression vector containing a putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter, and a marker gene is introduced into a host cell is a biodegradable, It can be used as an artificial biosensor for detection.

In addition,

1) preparing an expression vector comprising a Pseudomonas putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter and a marker gene; And

2) transforming an expression vector of step 1) into a host cell to produce a transformant. The present invention also provides a method for producing an artificial biosensor for detecting a noxious degradable aromatic compound.

In addition, the present invention provides a method of manufacturing an artificial biosensor for detecting harmful aromatic compounds, as described in more detail below.

i) securing the todS and todT genes to translate the TodS sense protein and the TodT control protein;

ii) cloning the obtained todS and todT genes of step i) into an expression vector capable of inducible expression, and introducing the todS and todT genes into a host cell;

iii) securing the promoter sequence of the todX gene whose expression is strictly controlled by the TodST system;

iv) constructing a labeling circuit by linking to the marker gene using the obtained sequence of step iii); And

v) inserting a labeling circuit of step iv) into a specific site on a recombinant strain chromosome in a single copy to prepare a stable labeled recombinant strain. The method for producing a biodegradable artificial biosensor for detecting a harmful aromatic compound is also provided.

In this method, the todS gene of step 1) specifically includes, but is not limited to, the nucleotide sequence of SEQ ID NO: 5.

In this method, the todT gene of step 1) specifically includes, but is not limited to, the nucleotide sequence of SEQ ID NO: 6.

In this method, the promoter of the todX gene of step 1) specifically includes, but is not limited to, the nucleotide sequence of SEQ ID NO: 7.

The promoters of the todS gene, the todT gene, and the todX gene are each composed of a base sequence in which one or several bases are added, deleted, or substituted as long as they have the same activity in the nucleotide sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: But is not limited thereto.

The promoters of the todS gene, the todT gene and the todX gene have 80% or more homology, more specifically 90% or more homology, most specifically 95% or 96%, more preferably 90% or more homology to the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: , 97%, 98%, 99% or 99.5% or more homology with the nucleotide sequence shown in SEQ ID NO.

In this method, the marker gene of step 1) specifically includes a fluorescent protein (GFP), an alkaline phosphatase, a firefly luciferase, a bioluminescent microorganism (Vibrio, But are not limited to, those selected from the group consisting of Xenorhabdus, Photorhabdus, and Photobacterium, and beta-galactosidase. More specifically, it is not limited to beta-galactosidase.

In the above method, the expression vector is specifically in step 1) footage Pseudomonas (Pseudomonas a todS gene derived from Pseudomonas putida , a todX gene derived from Pseudomonas putida , and a marker gene, but is not limited thereto, and more specifically, pEXT20-TodST, but is not limited thereto.

In the above method, the host cell of step 2) is not particularly limited to a prokaryotic microorganism or a eukaryotic microorganism. More specifically, the prokaryotic microorganism may be E. coli , the eukaryotic microorganism may be yeast, It is not.

In the above method, the refractory harmful aromatic compound is specifically a BTEX compound (benzene, toluene, ethylbenzene, xylene) or a toluene compound, but is not limited thereto. Specifically, the BTEX compound is not limited to benzene and toluene, and the toluene compound may be 2,4,6-trinitrotoluene (TNT).

The biosensor of the present invention was found to be capable of detecting the corresponding aromatic compound with high sensitivity as a result of measuring the expression of the marker gene in a culture condition containing a harmful aromatic compound having various concentrations of a refractory harmful aromatic compound. very specific and sensitive, since the higher, the footage of the present invention Pseudomonas (Pseudomonas a biosensor comprising a transformant into which an expression vector containing a putida- derived todS gene, a Pseudomonas putida- derived todT gene, a Pseudomonas putida- derived todX gene promoter, and a marker gene is introduced into a host cell is a biodegradable, And can be usefully used as a manufacturing method of an artificial biosensor for detection.

In addition,

1) culturing the test sample after treating the biosensor of the present invention;

2) measuring the expression level of the marker gene of the biosensor of step 1); And

3) a step of judging that a harmful aromatic compound is contained in the test sample when the expression level of the marker gene of step 2) is increased as compared with that of the control sample.

In the above method, the test sample in step 1) is specifically a substance derived from the environment, but is not limited thereto.

In this method, the marker gene of step 2) specifically includes a fluorescent protein (GFP), an alkaline phosphatase, a firefly luciferase, a bioluminescent microorganism (Vibrio, But are not limited to, those selected from the group consisting of Xenorhabdus, Photorhabdus, and Photobacterium, and beta-galactosidase. More specifically, it is not limited to beta-galactosidase.

In this method, the level of expression in step 2) is specifically determined by photomultipliers, charge coupled devices, luminometers, photometers, fiber optic cables and liquid scintillation counter scintillation counter), but the present invention is not limited thereto.

The biosensor of the present invention was found to be capable of detecting the corresponding aromatic compound with high sensitivity as a result of measuring the expression of the marker gene in a culture condition containing a harmful aromatic compound having various concentrations of a refractory harmful aromatic compound. The expression vector containing the Pseudomonas putida- derived todS gene of the present invention, the Pseudomonas putida- derived todT gene, the Pseudomonas putida- derived todX gene promoter, and the marker gene can be detected by the host A biosensor containing a transformant introduced into a cell can be usefully used in a method for detecting a harmful aromatic compound.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Manufacture of a vector with sensor of artificial biosensor for detection of harmful aromatic compounds

A pEXT20 vector (Figure 5) was used to construct a vector containing an aromatic sensor. To clone the TodS detection protein and TodT regulatory protein gene into the vector, Pseudomonas putida (&lt; RTI ID = 0.0 &gt;Pseudomonas putidaA DNA fragment of 3.77 kb was amplified by PCR using a F1 genomic DNA as a template and a primer No. 2 and an Ex-Taq enzyme (Takara, Japan) (PCR conditions: 95 ° C 30 seconds, 55 占 폚 for 30 seconds, 72 占 폚 for 3 minutes and 30 seconds, 30 cycles). PCR productsBamHI AndHind Ⅲ, and ligated using pEXT20 vector and T4 ligase (Elpis, Korea) prepared by digesting with the same restriction enzymes. The ligation sample was transformed into E. coli DH5α and a pEXT20-TodST vector with todS and todT inserted was obtained through a series of screening procedures. In order to efficiently express the cloned TodS protein in Escherichia coli Escherichia coli, an optimal ribosome-attached sequence AGGAGA for E. coli was included in the first PCR primer sequence for cloning.

As a result, pEXT20-TodST vector was constructed as shown in FIG. 2 (FIG. 2).

< Example  2> todX  Promoter promoter ) Was inserted and a vector containing the marker gene was produced

A pRS415 vector (Fig. 6) containing a beta-galactosidase (lacZ) gene lacking its own promoter was used for construction of a marker protein expression construct to visualize aromatics detection by TodST. Pseudomonas putida F1 genomic DNA as a template and a promoter sequence of the todX gene whose expression is activated by the TodST system using SEQ ID NO: 3, primer No. 4 and Ex-Taq enzyme (Takara, Japan) A DNA fragment of 0.37 kb was PCR amplified (PCR conditions: 95 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 30 cycles). The PCR products were purified by treating EcoR I restriction enzyme and treated with the purified pRS415 and T4 ligase (Elpis, Korea) treated with CIAP (Takara, Japan) to prevent self ligation after the same restriction enzyme treatment And then ligated. The ligated sample was transformed into E. coli DH5α and sequenced to obtain pRS415-ΦP _todX- lacZ vector.

< Example  3> Marker gene Expression body  chromosome( chromosome ) Introduced into Escherichia coli E. coli ) MC4100 ::? P _todX - lacZ  Production of strain

&Lt; 3-1 > Expression body  Recombination Lambda Phage ( phage )? P _todX - lacZ of making

Cells were recovered by centrifugation in 4 ml of the culture obtained by culturing the transformant obtained by introducing the pRS415-ΦP _todX- lacZ vector prepared in Example 2 into E. coli MC4100 in LB medium (Luria-Bertani medium) mM MgSO ₄ And re-suspended in 2 ml. 10 μl of lambda phage λRS45 was added to 100 μl of the cell suspension, and the mixture was allowed to stand at 37 ° C. for 20 minutes, and then re-agar (L): tryptone tryptone) 10 g, yeast extract (yeast extract) 1 g, NaCl 8 g, agar (agar) 8 g, 2 ㎖ 1 M CaCl 2, 2.5 ㎖ 50% glucose (glucose), 2 ㎖ 1 M MgSO 4, 5 ㎖ 2% X-gall (gal)) was added thereto, and the mixture was dispensed onto LB (Luria-Bertani) worm plates. The plates were incubated overnight at 37 ° C. During the above procedure, the ΦP _todx- lacZ marker gene expression vector in the vector was transplanted into lambda phage DNA by homologous recombination between the recombinant vector pRS415-ΦP _todX- lacZ and lambda phage DNA by inoculating the transformant strain with lambda phage When a recombinant phage λΦP _todx- lacZ is formed, a blue plaque is formed. Blue plaques were isolated and amplified and used in the experiments described below.

&Lt; 3-2 > Expression body  Escherichia coli inserted into the chromosome MC4100 :: λΦ P _todX - lacZ Production of strain

In order to insert the recombinant lambda phage λΦP _todx -lacZ prepared in the above Example <3-1> into a single copy at the lambda insertion site of E. coli MC4100 strain, the following experiment was conducted. First, about 4 blue plaques were added to 100 μl of E. coli MC4100 10 mM MgSO ₄ The suspension was mixed well and allowed to stand at 37 ° C for 20 minutes, after which 3 ml of LB + 10 mM MgSO ₄ was added. The mixture was shaken at 37 DEG C for 60 minutes at 100 rpm and then incubated at 30 DEG C for 4 hours. If cell lysis is observed, 100 μl of chloroform is added, vortexed strongly, centrifuged at 13000 rpm, and only the supernatant is recovered to prepare a recombinant lambda phage sample containing the marker gene expression product Respectively. 100 μl of the E. coli MC4100 10 mM MgSO ₄ was mixed with 100 μl of the recombinant phage sample and incubated at 37 ° C for 20 minutes for the preparation of the E. coli MC4100 :: λΦ P _todx -lacZ strain in which the marker gene expression vector was inserted into the chromosome. The DNA of the recombinant phage infected with the E. coli host is inserted into the lambda phage insertion site on the chromosome of the host during the above procedure. To remove the uninfected remaining phage, 1 ml of LB liquid broth was added, followed by centrifugation and washing twice to recover only the cells. Finally, 1 ml of LB liquid broth was added to suspend the cells, followed by incubation at 37 占 폚 for 30 minutes. This culture was diluted stepwise to 10 ⁶ with LB, plated on LB + X-gal (gal) plates and cultured and blue colonies were selected. Ten selected blue colonies were cultured in LB and the amount of beta-galactosidase expressed was measured and compared to obtain an E. coli MC4100 :: λΦ _P todx-lacZ strain in which the marker gene was inserted into the chromosome in a single copy.

As a result, the produced E. coli strain was confirmed as shown in FIG. 3 (FIG. 3).

< Example  4> Marker gene The expression construct  On strains containing Sensor protein  Production of a biosensor into which an expression vector is introduced

The E. coli MC4100 :: λΦP _todx- lacZ obtained in Example <3-2> The pEXT20-TodST vector, an aromatic sensor protein expression vector, was transformed into a strain to produce an aromatic compound sensing biosensor. In order to investigate the performance of the biosensor, the transformant was pre-incubated overnight in a medium containing 100 μg / ml of an antibiotic ampicillin in an LB liquid broth. The pre-cultured transformants were re-inoculated in the same medium with the medium containing 0.1 mM IPTG to induce TodS and TodT expression at a concentration of 1%. At this time, in order to evaluate the influence of each aromatic compound, it was added to the medium in the following manner. Benzene (Sigma, USA) was added at concentrations of 0, 5, 50 and 200 μM in toluene (Toluene) (Sigma, USA) at 0, 5 and 50 μM from the start of culture and incubated for 9 hours. TNT (2,4,6-trinitrotoluene) was prepared by culturing the transformant to OD ₆₀₀ 1.0, centrifuging the cells, suspending them in the same medium, adding TNT (Accustandard, USA) (Behzadian, F. and Barjeste et al., 2011). All samples were inoculated into 10 ml of medium contained in a 50 ml conical tube and incubated at 37 [deg.] C with shaking at 200 rpm.

< Example  5> tagged gene (beta-galactosidase (&bgr; 가alat 시드세제 )) analysis

The β-galactosidase assay was performed using a Z buffer (0.1 M sodium phosphate buffer, pH 7.0, 1 mM MgSO ₄ , 10 mM KCl, 50 mM mercaptoethanol ) And ONPG (Sigma, USA) was used as a substrate. First, add 30 μl of chloroform and 0.1% SDS to 1 ml of Z buffer, add 50 μl of the culture, vortex for 10 seconds, and incubate at 37 ° C for 10 minutes. 200 μl of ONPG solution (4 mg / ml) was added to initiate the reaction. When yellow color appeared at 37 ° C, 500 μl of 1 M Na ₂ CO ₃ was added to terminate the reaction. The enzyme activity was shown based on the amount of ONP produced by measuring the absorbance (OD _{420 nm} , OD _{550 nm} , OD _{600 nm} ) using a spectrophotometer (Miller unit).

As a result, as shown in FIG. 4, various concentrations of harmful aromatic compounds were contained using a marker gene expression strain [ E. coli MC4100 :: λΦP _todX- lacZ / pEXT20-TodST] transformed with a todST expression vector The expression level of reporter in the cultured condition was up to 4.5 times and 7.6 times higher than that of toluene and benzene, respectively. In addition, the sensitivity of TNT was higher than that of TNT The expression of the marker gene was increased 8-fold or more even at a concentration of 1 [mu] M (Fig. 4).

As described above, when an expression vector comprising the todS gene derived from Pseudomonas putida , the todT gene derived from Pseudomonas putida , the promoter of the todX gene derived from Pseudomonas putida , and the labeled gene of the present invention is introduced into the host cell A biosensor including a transformant can be very useful as a biodegradable harmful aromatic compound biosensor and a cell-based biosensor for the environment.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Artificial biosensor for non-degradable harmful aromatic compound          detection and manufacturing method thereof <130> 11p-10-70 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> TodST-F <400> 1 aaaggatcca ggagacatat gagctccttg gata 34 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> TodST-R <400> 2 tataagcttc tattccaggc tatcctt 27 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> todX promoter-F <400> 3 cgcgaattcc cgggatcaac gcattgagc 29 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> todX promoter-R <400> 4 cccgaattct ctccttccac atcttattt 29 <210> 5 <211> 2937 <212> DNA <213> Artificial Sequence <220> <223> todS <400> 5 atgagctcct tggatagaaa aaagcctcaa aatagatcga aaaataatta ttataatatc 60 tgcctcaagg agaaaggatc tgaagagctg acgtgtgaag aacatgcacg catcatattt 120 gatgggctct acgagtttgt gggccttctt gatgctcatg gaaatgtgct tgaagtgaac 180 caggtcgcat tggagggggg cgggattact ctggaagaaa tacgagggaa gccattctgg 240 aaggcgcgtt ggtggcaaat ttcaaaaaaa accgaggcga cccaaaagcg acttgttgaa 300 actgcatcat ccggtgaatt tgttcgctgt gatgttgaga ttcttggaaa atcaggtgga 360 agagaggtaa tagccgtcga tttttcattg ctgccaattt gcaatgaaga agggagcatt 420 gtttaccttc ttgcggaagg gcgcaatatt accgataaga agaaagccga ggccatgctg 480 gcgttgaaga accaggaatt ggagcagtcg gttgagtgta tccgaaaact cgataatgcg 540 aagagtgatt tctttgccaa ggtgagccat gagttgcgca ctccgctgtc tttgattcta 600 gggccactgg aagccgttat ggcggcagag gctgggcgtg aatcgccgta ttggaagcag 660 tttgaggtca ttcagcgtaa tgcaatgacc ctgttgaaac aggttaacac gctgcttgac 720 ttggcgaaaa tggacgcccg gcagatgggg ctttcctatc ggcgagccaa tcttagtcag 780 ctcacccgta ctattagctc gaattttgaa ggaatagccc agcaaaaatc aataacgttc 840 gatacaaaac tgcctgtaca gatggtcgct gaggtggatt gtgagaaata cgaacgcatt 900 atccttaact tgctttccaa tgcgtttaaa ttcacccctg acggggggct tatccgttgc 960 tgtcttagtt tgagtcgacc aaattatgcc ttggttactg tatctgatag cgggccgggt 1020 attcctcctg cactgcgtaa agaaatattt gaacgtttcc accagctaag ccaggaaggt 1080 caacaagcta cgcggggtac aggcttgggg ctttccattg tgaaagaatt cgttgaattg 1140 caccgtggaa caatttctgt aagtgatgcc ccgggcgggg gggcgctttt tcaggtaaag 1200 ctgccgctga atgctcctga aggtgcttat gttgcgagta acaccgcgcc gcgaagagat 1260 aatcctcagg tcgtggatac ggatgagtac cttttgctgg cgcccaatgc ggaaaatgaa 1320 gccgaggtgc ttccatttca atccgaccag cctcgggtgc taatcgttga agataaccct 1380 gatatgcgtg gttttataaa ggactgtctc agtagcgact atcaagttta tgttgcaccc 1440 gacggtgcaa aggcattgga gttgatgtca aacatgccgc cagacctgtt gattacagac 1500 ctgataatgc ctgttatgag cggcgatatg ctggttcacc aagtgcgtaa gaaaaatgaa 1560 ctttcacata tcccgatcat ggtgctgtcg gccaagtcag acgcagaact gcgtgtgaaa 1620 ttgctctccg agtcggtgca ggactttctt cttaagccat tttctgctca tgagctacga 1680 gcgcgtgtaa gcaatctggt atccatgaag gtggcaggcg atgcgttgcg taaggagctt 1740 tccgatcagg gggatgatat tgcgatactt actcaccgtc tgatcaaaag tcgccatcgt 1800 cttcagcaga gtaacatcgc attatccgcc tcggaagcgc gttggaaagc agtgtatgaa 1860 aactctgcgg ccggtattgt actgaccgac ccggaaaacc gaatactcaa cgccaatcct 1920 gcatttcaac gcattaccgg atatggggaa aaggatttgg agggactttc catggagcaa 1980 ttgactccat ctgacgaaag cccacagata aagcagcgtc tggccaattt gcttcagggt 2040 gggggagcgg aatacagtgt ggagcgctcc tatctatgca aaaatggttc tacgatttgg 2100 gccaatgcga gtgtctcgct gatgcctcaa cgtgtcggtg aatctccagt tatactgcag 2160 atcatcgatg acatcactga gaagaaacaa gcacaggaaa atcttaacca attgcagcaa 2220 caacttgtgt acgtttcccg atcagctacg atgggtgaat ttgcagccta tattgcacac 2280 gagataaacc aaccgctctc ggcgatcatg accaatgcca atgctggcac acgttggtta 2340 ggtaatgagc catctaacat cccagaggct aaagaggcac tggctcgcat tatccgagat 2400 tccgaccgcg ctgcagaaat tatccgtatg gtacgctcct tcctgaagcg tcaagaaacg 2460 gtgctgaaac cgattgatct aaaagcactg gtaactgata caagcctgat acttaaggcc 2520 cctagtcaga ataacagtgt caatttggat gttgttgcgg atgatgaact ccctgagata 2580 tggggggatg gtgtacagat ccagcagttg ataataaatc tggctatgaa cgctattgaa 2640 gcgatcagcc aagccgactg tgaaaccagg cagctaaccc tgtcattctc aggcaatgat 2700 acaggtgatg cgcttgttat ctcagtgaaa gatacaggtc caggtatttc agagaggcag 2760 atggcgcagt tgttcaacgc attctacacc acaaaaaaag aagggcttgg tatgggattg 2820 gcaatctgtc ttacaatcac ggaagtgcat aacggtaaaa tatgggttga gtgcccgccc 2880 gctgggggtg cttgtttcct ggtaagtatc cctgccagac agggctccgg cacatga 2937 <210> 6 <211> 621 <212> DNA <213> Artificial Sequence <220> <223> todT <400> 6 atgagtgatc gggcatctgt tatctatatc ctcgatgacg acaatgcagt actggaagca 60 ctgagcagct tggtgcgttc aatcggcctg agtgtcgagt gtttttcatc cgctagcgta 120 ttcctgaacg atgtcaatcg ctctgcctgt ggctgtctaa ttttggatgt ccgtatgccc 180 gagatgagcg ggttggatgt gcaacgacaa ctgaaagagc ttggcgagca aatccccatt 240 atttttatca gcggccacgg tgatattccg atggcagtca aagcgatcaa ggcgggtgcg 300 gtagacttct tcactaaacc ttttcgagaa gaggagctgc ttggcgctat tcgcgccgcg 360 ctgaagttgg cgccccagca gagatcaaac gctccccgag tcagcgagct taaagagaat 420 tacgaaagcc tcagcaaacg cgagcaacag gtgcttaagt tcgtcttgcg aggatatcta 480 aacaagcaga cggctctaga gcttgatata tcggaagcaa cagtgaaagt gcaccgccat 540 aatatcatga ggaaaatgaa agtatcttca atccaggatc tggttcgagt aactgagcgg 600 ctcaaggata gcctggaata g 621 <210> 7 <211> 366 <212> DNA <213> Artificial Sequence <220> <223> todX promoter <400> 7 ccgggatcaa cgcattgagc gccaccagca tgttcaggtc tgaggttttc atcgacatca 60 ggttatcaac ctgctcgtgg acctgaggga aactgccgtg gcggcgacgg ggcttgcgcg 120 taagcccccg ctatacgacc agcctgttcg aaagccgcaa agtgcttagg tttgggtgca 180 tatccatcag aagcggcata aaccatcgtt tatcacagtt aaactttggt tttctaagtt 240 gcgatagcca tataaaccca taagccaaaa aacaatattt cccagggcgt gattgtaata 300 ctgtgcgtgc tctaaggcgg tgtttgccta cttcacttta taaaaaaaat aagatgtgga 360 aggaga 366

Claims (15)

A todS gene derived from Pseudomonas putida consisting of the nucleotide sequence of SEQ ID NO: 5,
A todT gene derived from Pseudomonas putida consisting of the nucleotide sequence of SEQ ID NO: 6,
A promoter of the Pseudomonas putida-derived todX gene consisting of the nucleotide sequence of SEQ ID NO: 7, and
An artificial biosensor for detecting a degradable harmful aromatic compound, wherein the expression vector containing the marker gene is introduced into E. coli host cell.
delete delete delete The method according to claim 1, wherein the marker gene is selected from the group consisting of a fluorescent protein (GFP), an alkaline phosphatase, a firefly luciferase, and a beta -galactosidase Wherein the biodegradable polymer is a biodegradable biodegradable polymer.
The artificial biosensor according to claim 1, wherein the expression vector is pEXT20-TodST.
delete delete delete The harmful aromatic compound according to claim 1, wherein the refractory harmful aromatic compound is a BTEX compound (benzene, toluene, ethylbenzene, xylene) or a toluene- Artificial biosensors for detection of compounds.
The artificial biosensor according to claim 10, wherein the toluene-based compound is 2,4,6-trinitrotoluene (TNT).
1) a todS gene derived from Pseudomonas putida consisting of the nucleotide sequence of SEQ ID NO: 5,
A todT gene derived from Pseudomonas putida consisting of the nucleotide sequence of SEQ ID NO: 6,
A promoter of the Pseudomonas putida-derived todX gene consisting of the nucleotide sequence of SEQ ID NO: 7, and
Preparing an expression vector comprising the marker gene; And
2) transforming an expression vector of step 1) into a host cell Escherichia coli to produce a transformant.
13. The method of claim 12, wherein the expression vector of step 1) is pEXT20-TodST.
delete 1) treating the test sample with the biosensor of claim 1 before culturing;
2) measuring the expression level of the marker gene of the biosensor of step 1); And
3) A method for detecting a refractory harmful aromatic compound, comprising the step of judging that a harmful aromatic compound is contained in the test sample when the level of expression of the marker gene of step 2) is increased as compared with the control.

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