Genetically-mutated bacterial strain for detecting estrogenic compound and method for detecting estrogenic compound using the same

Patent metadata

Field	Value
Publication number	US-2019127774-A1
Application number	US-201816146677-A
Country	US
Kind code	A1
Filing date	Sep 28, 2018
Priority date	Mar 30, 2016
Publication date	May 2, 2019
Grant date	—

Abstract

The present invention can provide genetically mutated bacteria for detecting an estrogenic compound and a method for detecting an estrogenic compound by using same since the bacteria are based on estrogen receptor protein originated from the human body, and thus are environmentally friendly, and the detection of the bacteria can be performed in a very short time with low cost and labor by virtue of a relatively simple process.

First claim

1 . A bacterial strain having an ability of detecting an estrogenic compound, the strain being transformed by:
plasmid A having a base sequence in which a gene encoding a coregulatory factor interacting with an estrogen receptor ligand-binding domain (ER LBD) is conjugated with a gene encoding a λCI protein; and plasmid B in which a gene encoding ER LBD is conjugated with a gene encoding an αNTD protein.

2 . The bacterial strain according to claim 1 , wherein the coregulatory factor interacting with the ER LBD is any one selected from the group comprising a RIP140 protein, a TIF2 protein, a TIF1 protein and a SRC1 protein.

3 . The bacterial strain according to claim 1 , wherein the gene encoding the coregulatory factor interacting with the ER LBD or the gene encoding the ER LBD is obtained by transcribing mRNA from human genomic DNA, preparing intron-deleted mRNA through splicing with respect to the mRNA, and amplifying the intron-deleted mRNA by PCR using cDNA synthesized by reverse transcription as a template.

4 . The bacterial strain according to claim 2 , wherein a FLAG sequence for confirming the expression of the coregulatory factor interacting with the ER LBD and the λCI protein is conjugated to the 3′-end of the gene encoding the coregulatory factor interacting with the ER LBD.

5 . The bacterial strain according to claim 1 , wherein the plasma A has a nucleic acid sequence of SEQ ID NO: 1 or 3.

6 . The bacterial strain according to claim 1 , wherein a FLAG sequence for confirming the expression of the ER LBD and the αNTD protein is conjugated to the 3′-end of the gene encoding the ER LBD.

7 . The bacterial strain according to claim 1 , wherein the plasmid B has a nucleic acid sequence of SEQ ID NO: 2.

8 . The bacterial strain according to claim 1 , wherein the bacterial strain is any one strain selected from the group comprising Escherichia coli, Bacillus subtilis, Bacillus licheniformis and lactic acid bacteria.

9 . The bacterial strain according to claim 1 , wherein the estrogenic compound is selected from the group comprising norethynodrel, 5α-androstane, nonylphenol, dodecylphenol, octylphenol, bisphenol A, bisphenol S, bisphenol F, 2-ethylhexyl-4-hydroxybenzoate, 4,4′-dihyroxybenzophenone, 2,4-dihydroxybenzophenone, dihydroxymethoxychlorolefin, o,p′-DDT, dihydroxymethoxychlor (HPTE), 2′,3′,4′,5′-tetrachloro-4-biphenylol, nordihydroguaiaretic acid, aurin, phenolphthalein, phenol red, and a mixture thereof.

10 . A method for detecting an estrogenic compound, comprising:
preparing the bacterial strain having an ability of detecting an estrogenic compound of claim 1 ; culturing the bacterial strain to which a specimen containing an estrogenic compound is added; and lysing the culture bacterial strain and analyzing a degree of the expression of a reporter protein.

11 . The method according to claim 10 , wherein the reporter protein is a β-galactosidase, a fluorescent protein or an antibiotic resistance-imparting protein.

12 . The method according to claim 11 , wherein the fluorescent protein is a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), a red fluorescent protein (RFP) or a luciferase.

13 . The method according to claim 10 , wherein the degree of the expression of a reporter protein is analyzed using a UV-VIS spectrophotometer.

14 . The method according to claim 10 , wherein the degree of the expression of the β-galactosidase is measured by adding O-nitrophenyl-β-D-galactopyranoside (ONPG) as a colorimetric reagent after the lysis, and analyzing the expression degree.

Assignees

• Univ Hanyang Ind Univ Coop Found

Inventors

• Lee Jin-Won
• Gye Myung Chan
• Ryu Su-Hyun
• Kim Young-Pil

Classifications

• C07K14/245

  Escherichia (G) · CPC title

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title

• C07K14/4702

  Regulators; Modulating activity · CPC title

• C07K14/721

  Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor · CPC title

• C12N15/74 Primary

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title