KR102083729B1 - Adenylate cyclase-based bacterial strain for the detection of estrogenic compounds and a method for detecting estrogenic compounds using the same - Google Patents

Abstract

The present invention relates to an adenylate cyclase-based bacterial strain having a detectability of an estrogen compound and a method for detecting an estrogen compound using the same. The present invention relates to an estrogen receptor protein derived from the human body and a co-regulator thereof. The bacterial strain transformed by conjugating the TIF protein with an adenylate cycle has low sensitivity to noise signals other than the estrogen compound and excellent detection performance on the estrogen compound. In addition, the bacterial strain of the present invention can be used to detect the estrogenous compound in an environmentally friendly manner.

Description

Adenylate cyclase-based bacterial strain for the detection of estrogenic compounds and a method for detecting estrogenic compounds using the same}

The present invention relates to an adenylate cyclase-based bacterial strain having a detectability of an estrogen compound and a method for detecting an estrogen compound using the same.

Endocrine Disrupting Compounds (EDC) refer to a group of compounds that exhibit adverse effects on humans and wildlife by mimicking, blocking, and disturbing the physiological action of hormones. The American Endocrinology Association has reported that endocrine disruptors can affect male and female reproduction, breast development and cancer, prostate cancer, neuroendocrine, thyroid activity, metabolism, obesity, and cardiovascular endocrine. It warns of serious health concerns.

Hormones interact with receptors on target cells to perform a variety of reactions, such as growth, development, behavior and reproduction, and trigger normal biological functions. However, substances that interfere with hormone activity, such as EDC described above, can lead to a variety of reversible or irreversible biological damage, including dwarf growth, short-term memory damage, fallopian pregnancy, low sperm counts, reproductive disorders, and immune system damage. .

In general, EDCs can be divided into three main groups: androgen (compounds that mimic or block natural testosterone), thyroid (compounds that have a direct or indirect effect on the thyroid), and estrogens (imitate natural estrogens). Compound or a blocking compound). In particular, estrogen compounds (EC) are known to be closely related to sexual development and disorders of children and men, as well as to the development of cancer, as well as adult men and women, and the severity is very serious because they are found in thousands of products used in everyday life Do.

Endocrine disrupting compounds containing estrogens are widely reported to be present in very low concentrations in the environment, but they have relatively high fat solubility that accumulate in the fats of organisms and animals at high positions in the food chain, and even at relatively low concentrations. Will result. It is also found in industrial chemicals such as insecticides, herbicides, fungicides, plasticizers, plastics, resins and detergents.

Analytical methods for detecting estrogens and estrogen-like compounds to date include solid phase extraction (SPE), high performance liquid chromatography (HPLC), liquid chromatography / weight spectrophotometer (LC / MS) or gas chromatography / weight spectrophotometer (GC). / MS) and the like are known. However, these methods are disadvantageous in that the apparatus to be used is expensive and the analysis process is complicated and requires a lot of analysis cost and time.

Meanwhile, the inventor has invented a detection sensor based on a bacterial protein hybrid method using RNA polymerase αNTD and λCI protein (Korean Patent Publication No. 10-2017-0112017), but the detection sensor is a reporter expressed by a noise signal. Since the lacZ gene, which is a gene, is colored by X-gal even if it is not treated with estrogen or environmental hormone, it is not suitable for use in a screening method with high noise signal on a plate.

Thus, in the present invention to solve the problems of the prior art by conjugating the estrogen receptor protein derived from the human body and its co-regulatory TIF protein with adenylate cycle, bacteria using the existing RNA polymerase αNTD and λCI protein Compared to a protein hybrid based sensor, the sensitivity to noise is significantly lower than that of an adenylate cyclase-based bacterial strain capable of immediately detecting an estrogen compound even with a small sample amount, and a method for detecting an estrogen compound using the same. I would like to.

According to the present invention, a gene encoding a ligand binding site (hERα LBD) of human estrogen receptor alpha and a plasmid comprising a nucleotide sequence conjugated with a gene encoding a TIF2 protein is encoded by a gene encoding a T18 region of an adenylate cycle. Provided is an adenylate cyclase-based bacterial strain transformed by a plasmid comprising a nucleotide sequence conjugated with a gene encoding a T25 site of nylate cyclase, having the detectability of an estrogenous compound.

According to one embodiment of the present invention, a FLAG sequence for confirming expression of the TIF2 protein and the T18 site may be conjugated to the 3 'end of the gene encoding the TIF2 protein.

According to another embodiment of the present invention, the FLAG sequence for confirming the expression of the hERα LBD and the T25 site may be conjugated to the 3 'end of the gene encoding the hERα LBD.

According to another embodiment of the present invention, the plasmid comprising the nucleotide sequence conjugated with the gene encoding the TIF2 protein and the gene encoding the T18 site of the adenylate cycle may have a nucleotide sequence of SEQ ID NO: 1 have.

According to another embodiment of the present invention, the plasmid conjugated with the gene encoding the hERα LBD and the gene encoding the T25 site of the adenylate cycle may have a nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention provides a gene encoding a ligand binding site (hERα LBD) of human estrogen receptor alpha and a plasmid comprising a nucleotide sequence conjugated with a gene encoding a TIF2 protein and a gene encoding a T25 region of an adenylate cycle. Provides an adenylate cyclase based bacterial strain transformed by a plasmid comprising a nucleotide sequence conjugated with a gene encoding a T18 site of an adenylate cyclase, having an detectability of an estrogenous compound. do.

According to one embodiment of the invention, the 3 'end of the gene encoding the TIF2 protein may be a conjugated FLAG sequence for confirming the expression of the TIF2 protein and the T25 site.

According to another embodiment of the present invention, a FLAG sequence for confirming the expression of the hERα LBD and the T18 site may be conjugated to the 3 'end of the gene encoding the hERα LBD.

According to another embodiment of the present invention, the plasmid comprising the nucleotide sequence conjugated with the gene encoding the TIF2 protein and the gene encoding the T25 site of the adenylate cycle may have a nucleotide sequence of SEQ ID NO: 3 have.

According to another embodiment of the present invention, the plasmid conjugated with the gene encoding the hERα LBD and the gene encoding the T18 site of the adenylate cycle may have a nucleotide sequence of SEQ ID NO.

According to another embodiment of the present invention, the gene encoding the TIF2 protein or the gene encoding the hERα LBD, transcription of mRNA from human genomic DNA, introns are removed through the splicing process for the mRNA After preparing the mRNA, it may be PCR amplified by using the cDNA synthesized through the reverse transcription process for the mRNA from which the intron is removed as a template.

According to another embodiment of the present invention, the bacterial strain may be any one strain selected from the group consisting of Escherichia coli strain, Bacillus subtilis , Bacillus licheniformis and Lactobacillus. .

According to another embodiment of the present invention, the estrogen compound is noretil nodrel, 5α-androstane, dodecylphenol, octylphenol, bisphenol A, bisphenol S, bisphenol F, 2-ethylhexyl-4-hydroxy Benzoate, 4,4'-dihydroxybenzophenone, 2,4-dihydroxybenzophenone, dihydroxymethoxycycloolefin, o, p'-DDT, dihydroxymethoxycyclo, 2 ', 3 It may be selected from the group consisting of ', 4', 5'-tetrachloro-4-biphenylol, nordihydroguai aretic acid, aurine, phenolphthalein and phenol red.

In another embodiment, the present invention provides a method for preparing an adenylate cyclase-based bacterial strain having a detectability of an estrogen compound, adding and culturing a sample containing an estrogen compound to the bacterial strain. And lysing the cultured bacterial strain, and then analyzing the expression level of the reporter protein.

According to one embodiment of the invention, the reporter protein may be beta-galactosidase, fluorescent light emitting protein or antibiotic resistance imparting protein.

According to another embodiment of the present invention, the fluorescent protein may be green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) or luciferase.

According to another embodiment of the present invention, the expression level of the reporter protein may be performed by a UV-VIS spectrophotometer.

According to another embodiment of the present invention, the expression level of the beta-galactosidase is, after the lysis, O-nitrophenyl-β-D-galactopyranoside (O-nitrophenyl-β- D-galactopyranoside (ONPG) can be added and assayed for expression.

According to the present invention, a bacterial strain transformed by conjugating an estrogen receptor protein derived from the human body and its co-regulatory TIF protein with an adenylate cycle, has low sensitivity to noise signals other than the estrogen compound and Excellent detection performance. In addition, by using the adenylate cyclase-based bacterial strain of the present invention, it is possible to detect the estrogenous compound simply and environmentally.

1 schematically illustrates the expression of a reporter gene in the presence of an estrogen compound by an estrogen compound detection sensor comprising an adenylate cyclase based bacterial strain according to one embodiment of the invention.
FIG. 2 shows that an estrogen compound detection sensor comprising an adenylate cyclase-based bacterial strain according to the present invention is estrogen by ligand dependent specific binding between a ligand binding site (hERα LBD) of human estrogen receptor alpha and a TIF2 protein. It is the result of the comparative experiment that detects the sex compound.
3 is a comparative experiment of the sensitivity of the noise signal of the estrogen compound detection sensor based on the conventional λCI protein and αNTD protein and the estrogen compound detection sensor comprising the adenylate cyclase-based bacterial strain according to the present invention One result.
Figure 4 is a test result of the detection performance and noise sensitivity of the estrogen compound using an estrogen compound detection sensor comprising an adenylate cycle race based bacterial strain according to the present invention.
Figure 5 is confirmed through the beta galactosidase experiments the detection performance of the estradiol (E2) of the estrogen compound sensor comprising an adenylate cyclase-based bacterial strain according to the present invention.
Figure 6 confirms the detection performance of the bisphenol A (BPA) of the estrogen compound sensor including the adenylate cyclase-based bacterial strain according to the present invention through beta galactosidase experiments.

Hereinafter, the present invention will be described in detail.

The present invention provides bacterial strains based on adenylate cycles with detectability of estrogenous compounds.

In the present invention, "Adenylate cyclase" (Adenylate cyclase) is also called adenylate cyclase, is a cell membrane-type enzyme that catalyzes the ATPAMP + PP _i reaction. This enzymatic activity is controlled by various extracellular signals, and the produced cAMP acts as a second messenger in the cell. Regardless of prokaryotic and eukaryotic cells, they are generally present in living organisms. Especially, they are known as plasma membrane binding enzymes in animal tissues.

In the present invention, it was intended to use the principle that the reporter gene is expressed as follows. As shown in Fig. 1, the ligand binding site (hERα LBD) of human estrogen receptor alpha and the binding site protein of TIF2, its co-regulator, are respectively bound to the T25 site or T18 site of the adenylate cycle. When no ligand is present, no interaction occurs between hERα LBD and TIF2 protein and no adenylate cyclase enzyme effects by T18 and T25. However, when the ligand is present, an interaction occurs between hERα LBD and TIF2 protein, and T18 and T25 bound to them are sufficiently close to have the ability of the adenylate cyclase enzyme. As a result, ATP can be converted to cAMP, and cAMP can bind to the CAP protein to express the reporter gene lacZ gene. That is, when estrogen compounds are present in the sample to be analyzed, the estrogen compounds bind to the hERα LBD, and the binding causes interaction between the hERα LBD and the TIF2 protein. The reporter gene is expressed by the action. In addition, the expressed reporter gene causes a color reaction by a color developing reagent, and by quantifying the degree of the color reaction, it is possible to accurately quantify the concentration of the estrogen compound in the sample.

According to the present invention, an adenylate cyclase-based bacterial strain includes a plasmid in which a gene encoding a TIF2 protein comprises a nucleotide sequence conjugated with any one of a T18 region and a T25 region of an adenylate cycle; And a plasmid comprising a nucleotide sequence conjugated with a gene encoding a ligand binding site (hERα LBD) of human estrogen receptor alpha with a gene encoding one of the T18 and T25 sites that is not conjugated with a TIF2 protein. Adenylate cyclase based bacterial strains with detectability of transformed estrogenous compounds.

In one specific embodiment, an adenylate cyclase-based bacterial strain according to the present invention comprises a plasmid comprising a nucleotide sequence conjugated with a gene encoding a TIF2 protein and a gene encoding a T18 site of an adenylate cycle; And a plasmid comprising a nucleotide sequence in which a gene encoding a ligand binding site (hERα LBD) of human estrogen receptor alpha is conjugated to a gene encoding a T25 site of an adenylate cycle. Adenylate cyclase based bacterial strains with detectability of compounds.

In another specific embodiment, the bacterial strain according to the present invention comprises a plasmid comprising a nucleotide sequence in which a gene encoding a TIF2 protein is conjugated with a gene encoding a T25 region of an adenylate cycle; And a plasmid comprising a nucleotide sequence in which a gene encoding a ligand binding site (hERα LBD) of human estrogen receptor alpha is conjugated with a gene encoding a T18 site of an adenylate cycle. Adenylate cyclase based bacterial strains with detectability of sex compounds.

The adenylate cyclase based bacterial strain according to the present invention is transformed by the plasmid, which encodes a set of proteins involved in the expression of the reporter gene.

In the present invention, a FLAG sequence may be conjugated to the 3 'end of the gene encoding the TIF2 protein to confirm the expression of the TIF2 protein and the T18 or T25 region bound thereto. Likewise, the 3 'end of the gene encoding the hERα LBD can be conjugated to the FLAG sequence for confirming the expression of the hERα LBD and the T25 site or T18 site bound thereto. By conjugation of such FLAG sequences, it becomes possible to confirm using a method such as western blotting in which only the expressed protein is recognized as a specific antibody.

In the present invention, the gene encoding the TIF2 protein or the gene encoding the hERα LBD, the mRNA is transcribed from human genomic DNA, and the intron is removed through a splicing process for the mRNA to prepare an mRNA , PCR may be amplified by using a cDNA synthesized through the reverse transcription process for the mRNA from which the intron is removed as a template.

More specifically, the nucleotide sequence conjugated with the gene encoding the TIF2 protein and the gene encoding the T18 region of the adenylate cycle may be the nucleotide sequence of SEQ ID NO: 1. The sequence shown in SEQ ID NO: 1 comprises the gene encoding the T18 site of the adenylate cycle (pUT18C), the linker amino acid sequence, the gene encoding the TIF2 protein and the FLAG sequence, in the 5 'to 3' direction.

The base sequence conjugated with the gene encoding the hERα LBD and the gene encoding the T25 region of the adenylate cycle may be the nucleotide sequence of SEQ ID NO: 2. The sequence shown in SEQ ID NO: 2 comprises a gene encoding the T25 site of the adenylate cycle (pKT25), a linker amino acid sequence, a gene encoding hERα LBD and a FLAG sequence, in the 5 'to 3' direction.

The base sequence of the gene encoding the TIF2 protein conjugated with the gene encoding the T25 region of the adenylate cycle may be the nucleotide sequence of SEQ ID NO: 3. The sequence shown in SEQ ID NO: 3 includes the gene encoding the T25 site of the adenylate cycle (pKT25), the linker amino acid sequence, the gene encoding the TIF2 protein, and the FLAG sequence, in the 5 'to 3' direction.

The base sequence conjugated with the gene encoding the hERα LBD and the gene encoding the T18 region of the adenylate cycle may be the nucleotide sequence of SEQ ID NO: 4. The sequence shown in SEQ ID NO: 4 includes the gene encoding the T18 site of the adenylate cycle (pUT18C), the linker amino acid sequence, the gene encoding the hERα LBD and the FLAG sequence, in the 5 'to 3' direction.

In the following example, as a cell transformed by the plasmid containing the nucleotide sequence according to the present invention, in the following example was described as E. coli strains, the target strain is not necessarily limited to E. coli, Bacillus subtilis, Bacillus All bacterial strains that can be genetically modified, such as licheniformis, lactic acid bacteria and the like, can be used as the target strain.

In addition, the subject compounds detectable by the bacterium according to the present invention include the detection of all types of hormones and analogs capable of binding to estrogen receptors, including human estrogen hormones. For example, but not limited to, various steroid hormones (Norethylnodrel, 5α-Androstane, etc.), alkylphenol compounds (Dodecylphenol, Octylphenol, etc.), bisphenol compounds (Bisphenol A, Bisphenol S, Bisphenol F, etc.), preservatives Commonly used paraben compounds (2-Ethylhexyl 4-hydroxybenzoate, Heptyl 4-hydroxybenzoatea, etc.), benzophenone compounds (4,4'-Dihydroxybenzophenone, 2,4-Dihydroxybenzophenone, etc.) Organochlorine substances in pesticides (Dihydroxymethoxychlor olefin, o, p'-DDT, Dihydroxymethoxychlor (HPTE), 2 ', 3', 4 ', 5'-Tetrachloro-4-biphenylol, etc.) Compounds such as Nordihydroguaiaretic acid, Aurin, Phenolphthalein and Phenol red, which are widely used as acid base indicators, can be detected by the present invention.

In one embodiment of the present invention, an experiment for detecting an estrogenous compound on a plate enables the adenylate cyclase-based bacterial strain according to the present invention to specifically detect only an estrogenic compound without being sensitive to noise. It was confirmed that there was (Example 3).

In another embodiment of the present invention, the adenylate cyclase-based bacterial strain according to the present invention based on the bacterial protein hybridization method using the existing RNA polymerase αNTD and λCI protein through a comparative experiment for detecting estrogen compounds on the plate It was confirmed that the detection ability of the estrogen compound was lower than that of the detection sensor, and the noise sensitivity was lower (Example 4).

In another embodiment of the present invention, β-galactosidase was used to test the detection ability of the adenylate cyclase-based bacterial strains according to the present invention for estrogenous compounds, and for estradiol and bisphenol A. It was confirmed that the detection capability was very excellent (Example 5).

From the results of the above examples, the adenylate cyclase-based bacterial strains according to the present invention show that the detection ability for the estrogen compound is very good and the noise sensitivity is low, so that it can be usefully applied to the sensor for detecting the estrogen compound. Can be.

The present invention thus also provides a sensor for detecting estrogenous compounds comprising adenylate cycle-based bacterial strains according to the present invention.

The present invention also provides a method for preparing an adenylate cyclase-based bacterial strain; Adding and culturing a sample containing an estrogen compound to the bacterial strain; After lysing the cultured bacterial strain, the present invention provides a method for detecting an estrogenous compound using an adenylate cycle race-based bacterial strain comprising analyzing the expression level of a reporter protein.

In the detection method according to the present invention, the adenylate cyclase-based bacterial strain according to the present invention is prepared, incubated with the sample to be analyzed, and then lysed (bacteria) by culturing the cultured bacterial strain, The reporter protein expressed is analyzed. At this time, the reporter protein is produced by the reporter gene only when the estrogen compound is present in the sample, and the estrogen compound in the sample can be detected by quantitatively analyzing the produced reporter protein.

That is, the method according to the present invention is to analyze the estrogen compounds in the sample using the property of producing a specific protein only when the estrogen compound present in the sample acts as a ligand and binds to hERα LBD.

According to the present invention, the method of analysis may vary depending on the type of reporter protein to be expressed. For example, the reporter protein may be a beta-galactosidase, a fluorescent protein, or an antibiotic resistance conferring protein, The protein can be green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) or luciferase. The color or fluorescent protein can be quantitatively analyzed using equipment such as a UV-VIS spectrophotometer.

In addition, in the case of using β-galactosidase as a reporter protein, after the above lysis, O-nitrophenyl-β-D-galactopyranoside (O-nitrophenyl-β-D-galactopyranoside) is used as a color developing reagent. , ONPG) can be added and assayed for expression. For example, when O-nitrophenyl-β-D-galactopyranoside (ONPG) is added as a coloring reagent, the added ONPG is added to β-galactosidase. And orthonitrophenol is produced which produces a strong yellow luminescence as a degradation product. At this time, the yellow light emission degree depends on the concentration of the estrogen compound present in the sample. Therefore, when the degree of color development by ONPG is measured using a UV-VIS spectrophotometer or the like, the concentration of the estrogen compound present in the sample is determined. Quantitative analysis becomes possible.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Preparation of Plasmid

The human estrogen receptor alpha ligand binding domain (hERα LBD (N ₃₀₄ to T ₅₅₃ : from the 304th amino acid asparagine to the 553th amino acid threonine)) is the cloned plasmid pJL2397 (pDG1730 :: PxylA :: αNTD-hERα LBD- FLAG) was used as a template and amplified using a PCR (Polymerase Chain Reaction) method. Primers used for PCR include Bam HI and Eco RI sites at 5 'and 3', respectively, and DNA including FLAG sequence and terminator to identify protein expression as specific antibodies. The part was amplified.

The TIF2 binding domain (TIF2 BD (Q ₆₂₄ to T ₈₆₉ : 624th amino acid glutamic acid to 869th amino acid threonine) is the cloned plasmid pJL2398 (pDG1730 :: PxylA :: λCI-TIF2 BD-FLAG) as a template And amplified using the PCR method. Primers used in PCR contained Bam HI and Eco RI sites at 5 'and 3', respectively, and amplified DNA parts including the flag sequence and the terminator to confirm protein expression as specific antibodies.

The amplified hERα LBD-FLAG and TIF2 BD-FLAG genes were digested with restriction enzymes Bam HI and Eco RI. A plasmid containing the nucleotide sequence of pUT18C :: TIF2 BD-FLAG (SEQ ID NO: 1) using a ligase at pUT18C and pKT25 sites digested with Bam HI and Eco RI, pKT25 :: hERα LBD-FLAG A plasmid comprising the nucleotide sequence of (SEQ ID NO: 2), a plasmid comprising the nucleotide sequence of pKT25 :: TIF2 BD-FLAG (SEQ ID NO: 3), and a nucleotide sequence of pUT18C :: hERα LBD-FLAG (SEQ ID NO: 4) The plasmid was cloned.

Example 2. E. coli transformation

Receptor cells of E. coli BTH101 (Euromedex), an E. coli strain, in which the adenylate cyclase gene was deleted, were prepared. Cells cultured _to OD ₆₀₀ (optical density at ₆₀₀ nm light _{) to 0.4} were obtained. Then 100 mM CaCl ₂ for about 4 hours. The treated cells were obtained again and then used after releasing the cells with a volume of 100 mM CaCl ₂ corresponding to 1/50 of the volume of the medium for the first culture.

50 μL of the prepared recipient cells were added 1 μL (about 100 ng) of each of the cloned pUT18C :: TIF2 BD-FLAG and pKT25 :: hERα LBD-FLAG and pUT18C :: hERα LBD-FLAG and pKT25 :: TIF2 BD-FLAG And then stored at 0 ° C. for about 30 minutes. Then, the heat of 42 ° C. was applied for 1 minute 40 seconds, and then stored at 0 ° C. for 5 minutes. Subsequently, 1 mL of LB medium was added and incubated at 37 ° C. for 1 hour, and then plated on a medium containing kanamycin (pKT25 selection) and ampicillin (pUT18C selection), which are selection markers.

Example 3 Determination of Estrogenic Compound Detecting Performance of Adenylate Cyclone-Based Bacteria Strains According to the Present Invention

In order to determine whether the adenylate cyclase-based bacterial strain according to the present invention specifically reacts only with estrogen compounds, the bacterial strain transformed with plasmids having different sequence numbers and some nucleotide sequences according to the present invention. To prepare a On-plate test (spotting) experiment as follows.

A medium (-X-gal medium) containing 100 μg / mL empicillin, 50 μg / mL kanamycin, 1 mM IPTG was prepared. Medium (+ X-gal medium) containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 1 mM IPTG, 200 μM X-gal was prepared. And prepared an LB-agar plate (1.5%) (+ X-gal + Estradiol medium) containing 100 μg / mL ampicillin, 50 μg / mL kanamycin, 1 mM IPTG, 200 μM X-gal, 10 μM estradiol It was. Each strain cultured _to OD _{600 ~ 1} was spotted in each of the medium by 5 μL, and then dried for about 30 minutes and then incubated at 37 ° C. overnight. The color change was observed by placing each strain on a medium containing X-gal, which is a blue substance, and estradiol, an estrogen, by expression of the reporter gene, lacZ gene.

As a result, as shown in Figure 2, according to the present invention, a bacterial strain (pUT18C :: TIF2 cloned with a ligand binding site (hERα LBD) and TIF2 protein of human estrogen receptor alpha at adenylate cycle T18 and T25 sites, respectively) Only estrogen compound detection sensors using BD-FLAG and pKT25 :: hERα LBD-FLAG or bacterial strains transformed with pUT18C :: hERα LBD-FLAG and pKT25 :: TIF2 BD-FLAG, respectively, are estrogen compounds. Only when estradiol was present, it interacted specifically to confirm that the lacZ gene was expressed and appeared blue.

On the other hand, bacterial strains transformed with pUT18C-zip and pKT25-zip showed blue color even without estradiol. This is because, unlike the bacterial strain according to the present invention, the bacterial strain transformed by pUT18C-zip and pKT25-zip expresses the lacZ gene even when no estrogen compound is present, thereby making the strain sensitive to noise (X-gal). It can be seen that. In the adenylate cyclase-based bacterial strain according to the present invention and bacterial strains transformed by other sequences except for bacterial strains transformed with pUT18C-zip and pKT25-zip, no visually observed reaction was observed.

Accordingly, it can be seen that the adenylate cyclase-based bacterial strains according to the present invention can specifically detect only estrogen compounds without being sensitive to noise.

Example 4 Measurement of Noise Sensitivity and Estrogen Compound Detection Performance of a Sensor Using Bacterial Strains According to the Present Invention

Example 4-1. Comparative experiment with existing sensor

A protein hybrid based sensor using a conventional RNA polymerase αNTD and λCI protein using on-plate disk diffusion (control: E. coli FW102 OL2-62 pBRαNTD :: hERα LBD-FLAG pACλCI :: TIF2 BD -FLAG strain) and the sensitivity to the noise of the estrogen detection sensor using the adenylate cycle-based bacterial strain according to the present invention was compared.

A bottom agar plate (1.5%, 15 mL) containing 100 μg / mL empicillin, 50 μg / mL kanamycin, 1 mM IPTG (20 μM for FW102 strain) was prepared (-X-gal disc).

A bottom agar plate (1.5%, 15 mL) containing 100 μg / mL empicillin, 50 μg / mL kanamycin, 1 mM IPTG (20 μM for FW102 strain) and 200 μM X-gal was prepared (+ X-gal disk).

E. coli FW102 OL2-62 pBRαNTD :: hERα LBD-FLAG pACλCI :: TIF2 BD-FLAG strain (control) and E. coli BTH101 pUT18C :: hERα LBD − incubated overnight on each bottom agar plate prepared as above. FLAG pKT25 :: TIF2 BD-FLAG strain was inoculated to the top agar (0.8%, 8 mL) of the same composition as the bottom agar to OD ₆₀₀ 0.1 and poured on the bottom agar and dried for 30 minutes.

Thereafter, the paper disk was placed on a plate, and 10 μL of ethanol, 1 mM estradiol, 1 mM bisphenol A, 1 mM bisphenol S, 1 mM bisphenol F, and 1 mM nonylphenol were respectively treated on the disk. All plates were incubated overnight at 37 ° C.

As a result, as shown in Figure 3, the control plate, which is a conventional sensor strain in the + X-gal disk was shown that the entire plate is blue because it is sensitive to the noise signal. On the other hand, the sensor using the bacterial strain according to the present invention, when treated with estradiol, bisphenol enol A, bispheol S, bisphenol F, it was observed that the blue color only specifically in the treated part.

Example 4-2. Determination of Detecting Estrogen Compounds Using Real Samples

An experiment was conducted to determine whether the sensor using the bacterial strain according to the present invention can detect estrogen compounds from the actual sample.

Receipt, blue ink, eco-friendly receipt (BPA-free), eco-friendly receipt (Phenol-free), disposable that is a paper sample on the X-gal disk and + X-gal disk prepared in Example 4-1. The paper cup and A4 paper were all cut to a width of 0.5 cm and placed on the top agar.

As a result, as shown in Figure 4, as a result of measuring by the sensor using the bacterial strain according to the present invention was able to visually confirm the color change of the blue color in the actual sample receipt and eco-friendly receipt (BPA-free), the present invention The estrogen compound detection sensor using a bacterial strain according to the present invention can easily detect the estrogen substance which is an environmental hormone just by placing the actual sample on the sensor.

Example 5. β-galactosidase assay

The detection ability of estradiol, bisphenol A, nonylphenol of the estrogen compound detection sensor using the bacterial strain according to the present invention was confirmed.

E. coli BTH101 pUT18C :: hERα LBD after the -FLAG and pKT25 TIF2 BD :: FLAG-strain inoculated, by the addition of 1mM IPTG and incubated until growth phase point to OD _{600 ~ 0.4.} Thereafter, estradiol, bisphenol A, and nonylphenol and EDCs corresponding to each concentration were added to 800 μL of cells, and further incubated for 45 minutes. Next, cells were collected and freed with 800 μL of Z-buffer (Na ₂ HPO ₄ 60 mM, NaH ₂ PO ₄ 40 mM, KCl 10 mM, MgSO ₄ 7H ₂ O 1 mM, Dithiothreitol 400 μM). Then OD ₆₀₀ (cell content) was measured. Thereafter, the cells were lysed through sonication and then treated with 160 μL of 4 mg / mL ONPG to confirm the time when the color of the sample turned yellow. When the sample turned yellow, the reaction was stopped with 400 μL of 1 M Na ₂ CO ₃ . Subsequently, OD ₅₅₀ (cell residue) and OD ₄₂₀ (yellow) were measured, and then the expression of lacZ gene was measured in Miller unit formula (1000 * (((OD _420- (1.75 * OD ₅₅₀ )) / (t * v * OD ₆₀₀ )) to determine the activity level.

As a result, as shown in Figures 5 and 6, the estrogen compound detection sensor using the bacterial strain according to the present invention confirmed that the estradiol at 1 μM level, bisphenol A can be detected at the 1 mM level.

It can be seen from the above examples that the bacterial strain and the estrogen-based compound detection sensor using the same can easily screen the estrogen-like compound. In addition, by improving the high noise signal which was a disadvantage of the conventional detection system, it can be effectively applied to the device that can directly see the signal of the sensor.

The description of the present invention set forth above is for illustrative purposes, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> INDUSTRY-UNIVERSITY COOPERATION FOUNDATION HANYANG UNIVERSITY <120> Adenylate cyclase-based bacterial strain for the detection of          estrogenic compounds and a method for detecting estrogenic          compounds using the same <130> PD18-034-P1 <150> KR 1020180043810 <151> 2018-04-16 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 3821 <212> DNA <213> Artificial Sequence <220> <223> pUT18C :: TIF2 BD-FLAG sequence <400> 1 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 60 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 120 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 180 caagcttagc cgccagcgag gccacgggcg gcctggatcg cgaacgcatc gacttgttgt 240 ggaaaatcgc tcgcgccggc gcccgttccg cagtgggcac cgaggcgcgt cgccagttcc 300 gctacgacgg cgacatgaat atcggcgtga tcaccgattt cgagctggaa gtgcgcaatg 360 cgctgaacag gcgggcgcac gccgtcggcg cgcaggacgt ggtccagcat ggcactgagc 420 agaacaatcc tttcccggag gcagatgaga agattttcgt cgtatcggcc accggtgaaa 480 gccagatgct cacgcgcggg caactgaagg aatacattgg ccagcagcgc ggcgagggct 540 atgtcttcta cgagaaccgt gcatacggcg tggcggggaa aagcctgttc gacgatgggc 600 tgggagccgc gcccggcgtg ccgagcggac gttcgaagtt ctcgccggat gtactggaaa 660 cggtgccggc gtcacccgga ttgcggcggc cgtcgctggg cgcagtggaa cgccactgca 720 ggtcgactct agaggatcca gagagagctg acgggcagag cagactgcat gacagcaaag 780 ggcagaccaa actcctgcag ctgctgacca ccaaatctga tcagatggag ccctcgccct 840 tagccagctc tttgtcggat acaaacaaag actccacagg tagcttgcct ggttctgggt 900 ctacacatgg aacctcgctc aaggagaagc ataaaatttt gcacagactc ttgcaggaca 960 gcagttcccc tgtggacttg gccaagttaa cagcagaagc cacaggcaaa gacctgagcc 1020 aggagtccag cagcacagct cctggatcag aagtgactat taaacaagag ccggtgagcc 1080 ccaagaagaa agagaatgca ctacttcgct atttgctaga taaagatgat actaaagata 1140 ttggtttacc agaaataacc cccaaacttg agagactgga cagtaagaca gatcctgcca 1200 gtaacacaaa attaatagca atgaaaactg agaaggagga gatgagcttt gagcctggtg 1260 accagcctgg cagtgagctg gacaacttgg aggagatttt ggatgatttg cagaatagtc 1320 aattaccaca gcttttccca gacacgaggc caggcgcccc tgctggatca gttgacaagc 1380 aagccatcat caatgacctc atgcaactca cagctgaaaa cagccctgtc acacctgttg 1440 gagcccagaa aacagcactg cgaatttcac agagcactcc atgggattat aaagatgatg 1500 atgataaata ataatctagt gcagcccgcc taatgagcgg gcttttttcc atgcaagcga 1560 attcatcgat ataactaagt aatatggtgc actctcagta caatctgctc tgatgccgca 1620 tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 1680 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 1740 ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 1800 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 1860 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 1920 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 1980 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 2040 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 2100 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 2160 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 2220 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 2280 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 2340 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 2400 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 2460 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 2520 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 2580 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 2640 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 2700 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 2760 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 2820 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 2880 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 2940 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 3000 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 3060 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 3120 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 3180 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 3240 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3300 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3360 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 3420 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 3480 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 3540 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 3600 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 3660 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 3720 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 3780 cgcaaaccgc ctctccccgc gcgttggccg attcattaat g 3821 <210> 2 <211> 4253 <212> DNA <213> Artificial Sequence <220> <223> pKT25 :: hERa LBD-FLAG sequence <400> 2 aagctttaat gcggtagttt atcacagtta aattgctaac gcagtcaggc accgtgtatg 60 aaatctaaca atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc tgtaggcata 120 ggcttggtta tgccggtact gccgggcctc ttgcgggatc tggcacgaca ggtttcccga 180 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 240 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 300 atttcacaca ggaaacagct atgaccatgc agcaatcgca tcaggctggt tacgcaaacg 360 ccgccgaccg ggagtctggc atccccgcag ccgtactcga tggcatcaag gccgtggcga 420 aggaaaaaaa cgccacattg atgttccgcc tggtcaaccc ccattccacc agcctgattg 480 ccgaaggggt ggccaccaaa ggattgggcg tgcacgccaa gtcgtccgat tgggggttgc 540 aggcgggcta cattcccgtc aacccgaatc tttccaaact gttcggccgt gcgcccgagg 600 tgatcgcgcg ggccgacaac gacgtcaaca gcagcctggc gcatggccat accgcggtcg 660 acctgacgct gtcgaaagag cggcttgact atctgcggca agcgggcctg gtcaccggca 720 tggccgatgg cgtggtcgcg agcaaccacg caggctacga gcagttcgag tttcgcgtga 780 aggaaacctc ggacgggcgc tatgccgtgc agtatcgccg caagggcggc gacgatttcg 840 aggcggtcaa ggtgatcggc aatgccgccg gtattccact gacggcggat atcgacatgt 900 tcgccattat gccgcatctg tccaacttcc gcgactcggc gcgcagttcg gtgaccagcg 960 gcgattcggt gaccgattac ctggcgcgca cgcggcgggc tgcagggtcg actctagagg 1020 atccaaacag cctggccttg tccctgacgg ccgaccagat ggtcagtgcc ttgttggatg 1080 ctgagccccc catactctat tccgagtatg atcctaccag acccttcagt gaagcttcga 1140 tgatgggctt actgaccaac ctggcagaca gggagctggt tcacatgatc aactgggcga 1200 agagggtgcc aggctttgtg gatttgaccc tccatgatca ggtccacctt ctagaatgtg 1260 cctggctaga gatcctgatg attggtctcg tctggcgctc catggagcac ccagggaagc 1320 tactgtttgc tcctaacttg ctcttggaca ggaaccaggg aaaatgtgta gagggcatgg 1380 tggagatctt cgacatgctg ctggctacat catctcggtt ccgcatgatg aatctgcagg 1440 gagaggagtt tgtgtgcctc aaatctatta ttttgcttaa ttctggagtg tacacatttc 1500 tgtccagcac cctgaagtct ctggaagaga aggaccatat ccaccgagtc ctggacaaga 1560 tcacagacac tttgatccac ctgatggcca aggcaggcct gaccctgcag cagcagcacc 1620 agcggctggc ccagctcctc ctcatcctct cccacatcag gcacatgagt aacaaaggca 1680 tggagcatct gtacagcatg aagtgcaaga acgtggtgcc cctctatgac ctgctgctgg 1740 agatgctgga cgcccaccgc ctacatgcgc ccactcggcc ggattataaa gatgatgatg 1800 ataaataata atctagtgca gcccgcctaa tgagcgggct tttttccatg caagcgaatt 1860 cggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc 1920 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc 1980 cttcccaaca gttgcgcagc ctgaatggcg aatggcgctg atgtccggcg gtgcttttgc 2040 cgttacgcac caccccgtca gtagctgaac aggagggaca gggggtgggc gaagaactcc 2100 agcatgagat ccccgcgctg gaggatcatc cagccggcgt cccggaaaac gattccgaag 2160 cccaaccttt catagaaggc ggcggtggaa tcgaaatctc gtgatggcag gttgggcgtc 2220 gcttggtcgg tcatttcgaa ccccagagtc ccgctcagaa gaactcgtca agaaggcgat 2280 agaaggcgat gcgctgcgaa tcgggagcgg cgataccgta aagcacgagg aagcggtcag 2340 cccattcgcc gccaagctct tcagcaatat cacgggtagc caacgctatg tcctgatagc 2400 ggtccgccac acccagccgg ccacagtcga tgaatccaga aaagcggcca ttttccacca 2460 tgatattcgg caagcaggca tcgccatggg tcacgacgag atcctcgccg tcgggcatcc 2520 gcgccttgag cctggcgaac agttcggctg gcgcgagccc ctgatgctct tcgtccagat 2580 catcctgatc gacaagaccg gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg 2640 cttggtggtc gaatgggcag gtagccggat caagcgtatg cagccgccgc attgcatcag 2700 ccatgatgga tactttctcg gcaggagcaa ggtgagatga caggagatcc tgccccggca 2760 cttcgcccaa tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc 2820 aaggaacgcc cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgg agttcattca 2880 gggcaccgga caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga 2940 acacggcggc atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct 3000 ccacccaagc ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc 3060 ctcatcctgt ctcttgatca gatcttgatc ccctgcgcca tcagatcctt ggcggcaaga 3120 aagccatcca gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca 3180 attccggttc gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac 3240 tgcaagctac ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc 3300 tgacattcat ccggggtcag caccgtttct gcggactggc tttctacgtg ttccgcttcc 3360 tttagcagcc cttgcgccct gagtgcttgc ggcagcgtga agctgtgcct agaaatattt 3420 tatctgatta ataagatgat cttcttgaga tcgttttggt ctgcgcgtaa tctcttgctc 3480 tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag gttctctgag ctaccaactc 3540 tttgaaccga ggtaactggc ttggaggagc gcagtcacca aaacttgtcc tttcagttta 3600 gccttaaccg gcgcatgact tcaagactaa ctcctctaaa tcaattacca gtggctgctg 3660 ccagtggtgc ttttgcatgt ctttccgggt tggactcaag acgatagtta ccggataagg 3720 cgcagcggtc ggactgaacg gggggttcgt gcatacagtc cagcttggag cgaactgcct 3780 acccggaact gagtgtcagg cgtggaatga gacaaacgcg gccataacag cggaatgaca 3840 ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg agccgccagg ggaaacgcct 3900 ggtatcttta tagtcctgtc gggtttcgcc accactgatt tgagcgtcag atttcgtgat 3960 gcttgtcagg ggggcggagc ctatggaaaa acggctttgc cgcggccctc tcacttccct 4020 gttaagtatc ttcctggcat cttccaggaa atctccgccc cgttcgtaag ccatttccgc 4080 tcgccgcagt cgaacgaccg agcgtagcga gtcagtgagc gaggaagcgg aatatatcct 4140 gtatcacata ttctgctgac gcaccggtgc agcctttttt ctcctgccac atgaagcact 4200 tcactgacac cctcatcagt gccaacatag taagccagta tatacactcc gct 4253 <210> 3 <211> 4241 <212> DNA <213> Artificial Sequence <220> <223> pKT25 :: TIF2 BD-FLAG sequence <400> 3 aagctttaat gcggtagttt atcacagtta aattgctaac gcagtcaggc accgtgtatg 60 aaatctaaca atgcgctcat cgtcatcctc ggcaccgtca ccctggatgc tgtaggcata 120 ggcttggtta tgccggtact gccgggcctc ttgcgggatc tggcacgaca ggtttcccga 180 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc 240 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca 300 atttcacaca ggaaacagct atgaccatgc agcaatcgca tcaggctggt tacgcaaacg 360 ccgccgaccg ggagtctggc atccccgcag ccgtactcga tggcatcaag gccgtggcga 420 aggaaaaaaa cgccacattg atgttccgcc tggtcaaccc ccattccacc agcctgattg 480 ccgaaggggt ggccaccaaa ggattgggcg tgcacgccaa gtcgtccgat tgggggttgc 540 aggcgggcta cattcccgtc aacccgaatc tttccaaact gttcggccgt gcgcccgagg 600 tgatcgcgcg ggccgacaac gacgtcaaca gcagcctggc gcatggccat accgcggtcg 660 acctgacgct gtcgaaagag cggcttgact atctgcggca agcgggcctg gtcaccggca 720 tggccgatgg cgtggtcgcg agcaaccacg caggctacga gcagttcgag tttcgcgtga 780 aggaaacctc ggacgggcgc tatgccgtgc agtatcgccg caagggcggc gacgatttcg 840 aggcggtcaa ggtgatcggc aatgccgccg gtattccact gacggcggat atcgacatgt 900 tcgccattat gccgcatctg tccaacttcc gcgactcggc gcgcagttcg gtgaccagcg 960 gcgattcggt gaccgattac ctggcgcgca cgcggcgggc tgcagggtcg actctagagg 1020 atccagagag agctgacggg cagagcagac tgcatgacag caaagggcag accaaactcc 1080 tgcagctgct gaccaccaaa tctgatcaga tggagccctc gcccttagcc agctctttgt 1140 cggatacaaa caaagactcc acaggtagct tgcctggttc tgggtctaca catggaacct 1200 cgctcaagga gaagcataaa attttgcaca gactcttgca ggacagcagt tcccctgtgg 1260 acttggccaa gttaacagca gaagccacag gcaaagacct gagccaggag tccagcagca 1320 cagctcctgg atcagaagtg actattaaac aagagccggt gagccccaag aagaaagaga 1380 atgcactact tcgctatttg ctagataaag atgatactaa agatattggt ttaccagaaa 1440 taacccccaa acttgagaga ctggacagta agacagatcc tgccagtaac acaaaattaa 1500 tagcaatgaa aactgagaag gaggagatga gctttgagcc tggtgaccag cctggcagtg 1560 agctggacaa cttggaggag attttggatg atttgcagaa tagtcaatta ccacagcttt 1620 tcccagacac gaggccaggc gcccctgctg gatcagttga caagcaagcc atcatcaatg 1680 acctcatgca actcacagct gaaaacagcc ctgtcacacc tgttggagcc cagaaaacag 1740 cactgcgaat ttcacagagc actccatggg attataaaga tgatgatgat aaataataat 1800 ctagtgcagc ccgcctaatg agcgggcttt tttccatgca agcgaattcg gccgtcgttt 1860 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc 1920 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt 1980 tgcgcagcct gaatggcgaa tggcgctgat gtccggcggt gcttttgccg ttacgcacca 2040 ccccgtcagt agctgaacag gagggacagg gggtgggcga agaactccag catgagatcc 2100 ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga ttccgaagcc caacctttca 2160 tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc 2220 atttcgaacc ccagagtccc gctcagaaga actcgtcaag aaggcgatag aaggcgatgc 2280 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc 2340 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac 2400 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca 2460 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatccgc gccttgagcc 2520 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga 2580 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga 2640 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata 2700 ctttctcggc aggagcaagg tgagatgaca ggagatcctg ccccggcact tcgcccaata 2760 gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg 2820 tcgtggccag ccacgatagc cgcgctgcct cgtcttggag ttcattcagg gcaccggaca 2880 ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga cagccggaac acggcggcat 2940 cagagcagcc gattgtctgt tgtgcccagt catagccgaa tagcctctcc acccaagcgg 3000 ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg aaacgatcct catcctgtct 3060 cttgatcaga tcttgatccc ctgcgccatc agatccttgg cggcaagaaa gccatccagt 3120 ttactttgca gggcttccca accttaccag agggcgcccc agctggcaat tccggttcgc 3180 ttgctgtcca taaaaccgcc cagtctagct atcgccatgt aagcccactg caagctacct 3240 gctttctctt tgcgcttgcg ttttcccttg tccagatagc ccagtagctg acattcatcc 3300 ggggtcagca ccgtttctgc ggactggctt tctacgtgtt ccgcttcctt tagcagccct 3360 tgcgccctga gtgcttgcgg cagcgtgaag ctgtgcctag aaatatttta tctgattaat 3420 aagatgatct tcttgagatc gttttggtct gcgcgtaatc tcttgctctg aaaacgaaaa 3480 aaccgccttg cagggcggtt tttcgaaggt tctctgagct accaactctt tgaaccgagg 3540 taactggctt ggaggagcgc agtcaccaaa acttgtcctt tcagtttagc cttaaccggc 3600 gcatgacttc aagactaact cctctaaatc aattaccagt ggctgctgcc agtggtgctt 3660 ttgcatgtct ttccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 3720 actgaacggg gggttcgtgc atacagtcca gcttggagcg aactgcctac ccggaactga 3780 gtgtcaggcg tggaatgaga caaacgcggc cataacagcg gaatgacacc ggtaaaccga 3840 aaggcaggaa caggagagcg cacgagggag ccgccagggg aaacgcctgg tatctttata 3900 gtcctgtcgg gtttcgccac cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg 3960 ggcggagcct atggaaaaac ggctttgccg cggccctctc acttccctgt taagtatctt 4020 cctggcatct tccaggaaat ctccgccccg ttcgtaagcc atttccgctc gccgcagtcg 4080 aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa tatatcctgt atcacatatt 4140 ctgctgacgc accggtgcag ccttttttct cctgccacat gaagcacttc actgacaccc 4200 tcatcagtgc caacatagta agccagtata tacactccgc t 4241 <210> 4 <211> 3833 <212> DNA <213> Artificial Sequence <220> <223> pUT18C :: hERa LBD-FLAG sequence <400> 4 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 60 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 120 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 180 caagcttagc cgccagcgag gccacgggcg gcctggatcg cgaacgcatc gacttgttgt 240 ggaaaatcgc tcgcgccggc gcccgttccg cagtgggcac cgaggcgcgt cgccagttcc 300 gctacgacgg cgacatgaat atcggcgtga tcaccgattt cgagctggaa gtgcgcaatg 360 cgctgaacag gcgggcgcac gccgtcggcg cgcaggacgt ggtccagcat ggcactgagc 420 agaacaatcc tttcccggag gcagatgaga agattttcgt cgtatcggcc accggtgaaa 480 gccagatgct cacgcgcggg caactgaagg aatacattgg ccagcagcgc ggcgagggct 540 atgtcttcta cgagaaccgt gcatacggcg tggcggggaa aagcctgttc gacgatgggc 600 tgggagccgc gcccggcgtg ccgagcggac gttcgaagtt ctcgccggat gtactggaaa 660 cggtgccggc gtcacccgga ttgcggcggc cgtcgctggg cgcagtggaa cgccactgca 720 ggtcgactct agaggatcca aacagcctgg ccttgtccct gacggccgac cagatggtca 780 gtgccttgtt ggatgctgag ccccccatac tctattccga gtatgatcct accagaccct 840 tcagtgaagc ttcgatgatg ggcttactga ccaacctggc agacagggag ctggttcaca 900 tgatcaactg ggcgaagagg gtgccaggct ttgtggattt gaccctccat gatcaggtcc 960 accttctaga atgtgcctgg ctagagatcc tgatgattgg tctcgtctgg cgctccatgg 1020 agcacccagg gaagctactg tttgctccta acttgctctt ggacaggaac cagggaaaat 1080 gtgtagaggg catggtggag atcttcgaca tgctgctggc tacatcatct cggttccgca 1140 tgatgaatct gcagggagag gagtttgtgt gcctcaaatc tattattttg cttaattctg 1200 gagtgtacac atttctgtcc agcaccctga agtctctgga agagaaggac catatccacc 1260 gagtcctgga caagatcaca gacactttga tccacctgat ggccaaggca ggcctgaccc 1320 tgcagcagca gcaccagcgg ctggcccagc tcctcctcat cctctcccac atcaggcaca 1380 tgagtaacaa aggcatggag catctgtaca gcatgaagtg caagaacgtg gtgcccctct 1440 atgacctgct gctggagatg ctggacgccc accgcctaca tgcgcccact cggccggatt 1500 ataaagatga tgatgataaa taataatcta gtgcagcccg cctaatgagc gggctttttt 1560 ccatgcaagc gaattcatcg atataactaa gtaatatggt gcactctcag tacaatctgc 1620 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 1680 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 1740 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 1800 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact 1860 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 1920 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 1980 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 2040 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 2100 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 2160 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 2220 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 2280 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 2340 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 2400 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 2460 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 2520 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 2580 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 2640 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 2700 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 2760 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 2820 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 2880 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 2940 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 3000 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 3060 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 3120 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 3180 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 3240 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 3300 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 3360 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 3420 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 3480 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 3540 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 3600 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 3660 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 3720 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 3780 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atg 3833

Claims (18)

A plasmid in which a gene encoding a TIF2 protein comprises a nucleotide sequence conjugated with a gene encoding a T18 region of an adenylate cycle; And
A plasmid in which a gene encoding a ligand binding site (hERα LBD) of human estrogen receptor alpha comprises a nucleotide sequence conjugated with a gene encoding a T25 site of an adenylate cycle;
An adenylate cyclase-based bacterial strain transformed by means of having an detectability of an estrogenous compound. The method of claim 1,
The 3 'end of the gene encoding the TIF2 protein is adenylate cyclase-based bacteria having a detectability of the estrogen compound, characterized in that the FLAG sequence for confirming the expression of the TIF2 protein and the T18 site is conjugated Strain. The method of claim 1,
The 3 'end of the gene encoding the hERα LBD protein is based on the adenylate cycle having a detectability of the estrogen compound, characterized in that the FLAG sequence for confirming the expression of the hERα LBD and the T25 site is conjugated Bacterial strains. The method of claim 1,
The plasmid comprising the nucleotide sequence conjugated with the gene encoding the TIF2 protein and the gene encoding the T18 region of the adenylate cycle has a detectable estrogen compound, characterized in that it has the nucleotide sequence of SEQ ID NO: 1. Adenylate cyclase based bacterial strains. The method of claim 1,
The plasmid conjugated with the gene encoding the T25 region of the adenylate cycle, wherein the gene encoding the hERα LBD protein has a nucleotide sequence of SEQ ID NO: 2, has an adenylate cycle having detectability of an estrogen compound. Race based bacterial strains. A plasmid in which a gene encoding a TIF2 protein comprises a nucleotide sequence conjugated with a gene encoding a T25 site of an adenylate cycle; And
A plasmid in which the gene encoding the ligand binding site (hERα LBD) of human estrogen receptor alpha comprises a nucleotide sequence conjugated with a gene encoding the T18 site of adenylate cycle;
An adenylate cyclase-based bacterial strain transformed by means of having an detectability of an estrogenous compound. The method of claim 6,
The 3 'end of the gene encoding the TIF2 protein is adenylate cyclase-based bacteria having a detectability of the estrogen compound, characterized in that the FLAG sequence for confirming the expression of the TIF2 protein and the T25 site is conjugated Strain. The method of claim 6,
The 3 'end of the gene encoding the hERα LBD is adenylate cycle-based based on the detection ability of the estrogen compound, characterized in that the FLAG sequence for confirming the expression of the hERα LBD protein and the T18 site is conjugated Bacterial strains. The method of claim 6,
The plasmid containing the nucleotide sequence conjugated with the gene encoding the TIF2 protein and the gene encoding the T25 region of the adenylate cycle has a detectable estrogen compound, characterized in that it has the nucleotide sequence of SEQ ID NO: 3 Adenylate cyclase based bacterial strains. The method of claim 6,
The plasmid conjugated with the gene encoding the hERα LBD and the gene encoding the T18 region of the adenylate cycle has an adenylate cycle having a detectability of an estrogen compound, characterized in that it has the nucleotide sequence of SEQ ID NO: 4. Based bacterial strains. The method according to claim 1 or 6,
The gene encoding the TIF2 protein or the gene encoding the hERα LBD is used to transcribe mRNA from human genomic DNA, to prepare mRNA from which introns are removed by splicing the mRNA, and then the intron is removed. Adenylate cyclase-based bacterial strain having a detection ability of the estrogen compound, characterized in that PCR amplified by the cDNA synthesized through a reverse transcription process for the mRNA. The method according to claim 1 or 6,
The bacterial strain has a detectability of an estrogen compound, characterized in that any one strain selected from the group consisting of Escherichia coli strain, Bacillus subtilis , Bacillus licheniformis and lactic acid bacteria. Adenylate cyclase based bacterial strains. The method according to claim 1 or 6,
The estrogenic compounds include noretynodrel, 5α-androstane, dodecylphenol, octylphenol, bisphenol A, bisphenol S, bisphenol F, 2-ethylhexyl-4-hydroxybenzoate, 4,4'-dihydrate Hydroxybenzophenone, 2,4-dihydroxybenzophenone, dihydroxymethoxycycloolefin, o, p'-DDT, dihydroxymethoxycyclo, 2 ', 3', 4 ', 5'-tetrachloro Adenylate cyclase-based bacterium having detectability of estrogenic compound, characterized in that any one or more selected from the group consisting of -4-biphenylol, nordihydroguai aretic acid, aurine, phenolphthalein and phenol red Strain. Preparing an adenylate cycle race based bacterial strain having a detectability of the estrogen compound according to claim 1;
Adding and culturing a sample containing an estrogen compound to the bacterial strain; And
Lysing the cultured bacterial strain and analyzing the expression level of the reporter protein;
Method for detecting an estrogen compound comprising a. The method of claim 14,
The reporter protein is a detection method for an estrogen compound, characterized in that β-galactosidase, fluorescent light-emitting protein or antibiotic resistance imparting protein. The method of claim 15,
The fluorescent light emitting protein is a green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP) or luciferase detection method characterized in that the estrogen compound. The method of claim 14,
The expression level of the reporter protein is a detection method of the estrogen compound, characterized in that carried out by UV-VIS spectrophotometer. The method of claim 15,
The expression level of the beta-galactosidase was added after O-nitrophenyl-β-D-galactopyranoside (ONPG) as a coloring reagent after the lysis. And detecting the estrogen compound by analyzing the degree of expression.

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