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Polybrominated diphenyl ether sensory protein and whole-cell microbial sensor constructed by polybrominated diphenyl ether sensory protein

A microbial sensor, polybrominated diphenyl ether technology, applied in the biological field, can solve the problems of few biological components, low bioavailability, lack of discovery and research of biodegradation pathways, etc., and achieve the effect of improving monitoring sensitivity and broad application prospects

Active Publication Date: 2021-11-30
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the monitoring of persistent organic pollutants, the research of biosensors still faces major challenges
First of all, the strong hydrophobicity of persistent organic pollutants leads to low bioavailability, the discovery and research of biodegradation pathways are relatively lacking, and there are very few biological components that can identify persistent organic pollutants
Currently developed PAH monitoring biosensors use naphthalene or phenanthrene degradation enzyme regulatory proteins to control the bioluminescent signal of the sensor, which is not applicable to PAHs other than these degradation pathways; PCB monitoring biosensors The oxygenase regulatory protein of the biphenyl degradation pathway is used as the recognition element, so biphenyl and PCB cannot be distinguished; and at present, no specific recognition biological element of polybrominated diphenyl ether has been found at home and abroad for the research and development of biosensors

Method used

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  • Polybrominated diphenyl ether sensory protein and whole-cell microbial sensor constructed by polybrominated diphenyl ether sensory protein
  • Polybrominated diphenyl ether sensory protein and whole-cell microbial sensor constructed by polybrominated diphenyl ether sensory protein
  • Polybrominated diphenyl ether sensory protein and whole-cell microbial sensor constructed by polybrominated diphenyl ether sensory protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Acquisition of polybrominated diphenyl ether recognition gene elements

[0023] Configure inorganic salt medium, and each liter of water contains the following substances (g / L): Na 2 HPO 4 12H 2 O 2.0, KH 2 PO 4 0.7, NH 4 Cl 0.5, NaCl 0.3, MgSO 4 ·7H 2 O 0.1, CaSO 4 2H 2 O 0.05, FeCl 3 ·6H 2 O 0.2×10 -3 、NaMoO 4 0.2×10 -3 , MnCl 2 4H 2 O 0.2×10 -3 , CuCl 2 2H 2 O 0.2×10 -3 , ZnSO 4 0.2×10 -3 、H 3 BO 3 0.3×10 -3 、CoCl 2 ·6H 2 O 0.4×10 -3 , peptone 0.2, yeast extract 1.0, glucose 5.0. Inoculate S. xenophagum hydrophobic strain C1 (=CCTCC AB 2015198=KCTC 42740) and hydrophilic strain C2 (=CCTCC AB2015427=KCTC 52051) into LB liquid medium, culture in a shaker at 200rpm / min at 30°C To logarithmic growth phase, cell OD 600 The value is about 1.0. Centrifuge at 10,000×g for 10 minutes, remove the supernatant, and collect the bacteria. After washing the cells twice with inorganic salt medium, resuspend the cells with a certain volume...

Embodiment 2

[0026] Example 2: Analysis of the activity and characteristics of polybrominated diphenyl ether recognition elements

[0027] Activity analysis was performed on the four candidate PBDE recognition gene elements obtained above. By synthesizing genes at Sangon Bioengineering (Shanghai) Co., Ltd., the coding sequence of firefly luciferase small peptide HiBiT (5′-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC- 3'), synthesis includes about 500bp upstream of each candidate gene including the gene of the promoter sequence, and designs BamHI and XhoI restriction sites at both ends of the gene fragment respectively (the 5' and 3' ends of chr1_1106 synthetic gene contain EcoRI and XhoI enzymes respectively cutting point). After gene synthesis, use TaKaRa's BamHI and XhoI restriction endonucleases to treat chr1_2605, chr1_2170 and chr1_2466 synthetic gene fragments and pET24a expression vectors in K buffer at 37 °C, use TaKaRa's EcoRI and XhoI restriction endonucleases in H buffer The chr1_1106 sy...

Embodiment 3

[0031] Example 3: Construction and performance analysis of a whole-cell microbial sensor for monitoring polybrominated diphenyl ethers

[0032] Synthetic primer 2466U (5'- GGATCC GCGGCGAAGGCATCTATAT-3') and primer 2466D (5'-GCGTCTTCCATTTCGGGATAATAGCC-3'), amplify chr1_2466 gene (its nucleotide sequence is as shown in SEQ ID NO.1) and its upstream 507bp sequence (its nucleotide sequence is as shown in SEQ ID NO.1) shown in ID NO.2). Primer LucU (5′-CGGCTATTATCCCGAAATGGAAGACGCCAAAAACA-3′) and primer LucD (5′- CTCGAG TTACACGGCGATCTTTCC-3') to amplify the complete sequence of the firefly luciferase luc gene (NCBI Accession No. AB762768.1). Using 2466U (5'- GGATCC GCGGCGAAGGCATCTATAT-3′) and primer LucD (5′- CTCGAG TTACACGGCGATCTTTCC-3') performed PCR fusion on the amplified chr1_2466 and luc gene fragments to obtain the fusion fragment chr1_2466-luc containing BamHI and XhoI restriction sites at both ends respectively. Use TaKaRa's BamHI and XhoI restriction endonucleases t...

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Abstract

The invention discloses a polybrominated diphenyl ether extracellular sensing protein and a whole-cell microbial sensor constructed by the polybrominated diphenyl ether extracellular sensing protein. The amino acid sequence of the polybrominated diphenyl ether extracellular sensing protein is as shown in SEQ ID NO. 3. According to the present invention, a polybrominated diphenyl ether recognition sensing protein is cloned from Sphingobium xenophagum C1, can specifically recognize polybrominated diphenyl ether, is fused with luciferase, is transferred into the Sphingobium xenophagum C1 with high cell surface hydrophobicity, and is subjected to overexpression of the fusion protein on the bacterial cell outer membrane to prepare the hydrophobic chassis whole-cell microbial sensor, the sensor has specific response to decabromodiphenyl ether and low-brominated homologs thereof, has obvious advantages in the aspect of monitoring hydrophobic organic pollutants, can remarkably improve the monitoring sensitivity of the hydrophobic organic pollutants, and has wide application prospects in organic pollution monitoring.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a polybrominated diphenyl ether extracellular receptor protein and its application in the construction of a whole-cell microbial sensor for organic pollution monitoring. Background technique: [0002] POPs degrade slowly due to biotic / abiotic factors, persist in the environment and are a major source of environmental pollution; they are highly toxic and readily bioaccumulate, with potential negative impacts on the health of organisms. Among them, polycyclic aromatic hydrocarbons and their halogenated derivatives belong to a class of persistent organic pollutants that are particularly harmful to human health, including some dioxin-like polychlorinated biphenyls and polybrominated diphenyl ethers. Not only their presence in the environment needs to be monitored, but also their potential biotoxic effects after exposure need to be assessed. Traditionally, high performance l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/74C12N1/21G01N21/76G01N21/64G01N33/533G01N33/53C12R1/01
CPCC07K14/195C12N15/74C12N9/0069C12Y113/12007G01N21/76G01N21/6486G01N33/533G01N33/5308C07K2319/00G01N2520/00Y02A20/20
Inventor 陈杏娟许玫英姚晖宋达孙国萍
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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