阅读说明：本技术 一种多溴联苯醚感受蛋白及其构建的全细胞微生物传感器 (Polybrominated diphenyl ether sensing protein and whole-cell microbial sensor constructed by same ) 是由 陈杏娟 许玫英 姚晖 宋达 孙国萍 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种多溴联苯醚胞外感受蛋白及其构建的全细胞微生物传感器。所述的多溴联苯醚胞外感受蛋白,其氨基酸序列如SEQ ID NO.3所示。本发明从Sphingobium xenophagum C1克隆出一个多溴联苯醚的识别感受蛋白,其能特异性识别多溴联苯醚,将其融合荧光素酶后转入高细胞表面疏水性的Sphingobium xenophagum C1,在细菌细胞外膜超表达融合蛋白,制成疏水底盘全细胞微生物传感器,其对十溴联苯醚及其低溴代同系物都具有特异性的响应,在疏水性有机污染物的监测方面具有明显的优势,并且可以显著提高疏水性有机污染物的监测灵敏性,在有机污染监测中具有广阔的应用前景。(The invention discloses a polybrominated diphenyl ether extracellular receptive protein and a whole-cell microbial sensor constructed by the same. The amino acid sequence of the polybrominated diphenyl ether extracellular sensing protein is shown in SEQ ID NO. 3. The invention clones a polybrominated diphenyl ether recognition receptive protein from Sphingobium xenophagum C1, which can specifically recognize polybrominated diphenyl ether, transfers the polybrominated diphenyl ether into Sphingobium xenophagum C1 with high cell surface hydrophobicity after being fused with luciferase, and overexpresses the fusion protein on the outer membrane of bacterial cells to prepare the hydrophobic chassis whole-cell microbial sensor, which has specific response to decabrominated diphenyl ether and low brominated homologues thereof, has obvious advantages in the aspect of monitoring hydrophobic organic pollutants, can obviously improve the monitoring sensitivity of the hydrophobic organic pollutants, and has wide application prospect in organic pollution monitoring.)

1. The polybrominated diphenyl ether extracellular sensing protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 3.

2. Use of the extracellular sensory protein of polybrominated diphenyl ethers according to claim 1 for specific biorecognition of polybrominated diphenyl ethers.

3. Use according to claim 2, wherein the use of polybrominated diphenyl ether extracellular receptive proteins for the preparation of hydrophobic underpan whole cell microbial sensors for monitoring organic contaminants, said organic contaminants being polybrominated diphenyl ethers.

4. The use according to claim 3, wherein the hydrophobic bottom plate whole cell microbial sensor is obtained by over-expressing polybrominated diphenyl ether extracellular receptor protein in a hydrophobic bottom plate whole cell.

5. The use according to claim 4, wherein the hydrophobic bottom plate whole cell microbial sensor is obtained by co-overexpression of the PBDEs extracellular receptor protein and the luciferase in the hydrophobic bottom plate whole cells.

6. The use of claim 3, 4 or 5, wherein the whole hydrophobic underplate cell is Sphingobium xenophagum C1.

7. The use of claim 2, wherein the primer is 2466U: 5' -GGATCCGCGGCGAAGGCATCTATAT-3' and primer 2466D: 5'-GCGTCTTCCATTTCGGGATAATAGCC-3', using the genome of Sphing obium xenophagum C1 as a template, amplifying to obtain chr1_2466 gene and an upstream sequence thereof, and using a primer LucU: 5'-CGGCTATTATCCCGAAATGGAAGACGCCAAAAACA-3' and primer LucD: 5' -CTCGAGTTACACGGCGATCTTTCC-3', amplifying the complete sequence of the luciferase luc gene of the firefly; with 2466U: 5' -GGATCCGCGGCGAAGGCATCTATAT-3' and primer LucD: 5' -CTCGAGTTACACGGCGATCTTTCC-3' PCR fusion of the two amplified gene fragments chr1_2466 and luc to obtain a fused fragment chr1_2466-luc containing BamHI and XhoI cleavage sites at both ends, which is digested with restriction enzymes BamHI and XhoI, ligated into pET24a vector to transform Sphingobium xenophagum C1, thereby obtaining a hydrophobic bottom plate whole cell microbial sensor.

8. A whole-cell biosensor, wherein the polybrominated diphenyl ether extracellular receptor protein fusion luciferase according to claim 1 is overexpressed in whole cells on a hydrophobic base plate to obtain the whole-cell biosensor.

9. The whole-cell microbial sensor of claim 8, wherein the whole-cell of the hydrophobic bottom plate is Sphingobium xenophagum C1.

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a polybrominated diphenyl ether extracellular receptive protein and application thereof in construction of a whole-cell microbial sensor for monitoring organic pollution.

Background art:

persistent organic pollutants are slowly degraded due to biological/non-biological factors, are persistently existed in the environment and are the main sources of environmental pollution; they are highly toxic and prone to bioaccumulation, with potential negative effects on the health of the organism. The polycyclic aromatic hydrocarbons and halogenated derivatives thereof belong to a class of persistent organic pollutants which are particularly harmful to human health, and particularly comprise some dioxin-like polychlorinated biphenyls and polybrominated diphenyl ethers. Not only are there a need to monitor their occurrence in the environment, but there is also a need to evaluate their potential biotoxic effects after exposure. Traditionally, the persistent organic pollutants are mainly determined by a high performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry combined method. The analysis methods have high measurement sensitivity and strong recognition specificity, so the analysis methods are widely applied. However, these methods require specialized equipment and training, are complicated to operate, are time-consuming and expensive, and more importantly, cannot measure bioavailability and evaluate biotoxicity effects, so that monitoring of persistent organic pollutants urgently requires development of new monitoring techniques and instrumentation. Biosensors, as an analytical device that integrates a biological recognition element with a physical sensing element to produce a measurable signal proportional to the concentration of a target analyte, is a promising option. It has the advantages of rapidness, convenience, low cost, high efficiency, sensitivity and the like; compared with the traditional analysis method, the biosensor also has the characteristics of in-situ on-line monitoring, biological toxicity effect early warning and the like.

At present, the research of the biosensor is developed significantly, and the biosensor is widely applied to the fields of environmental monitoring, clinical diagnosis, food safety, geological exploration and the like. But the development of biosensors also faces significant challenges in the monitoring of persistent organic pollutants. First, the strong hydrophobicity of persistent organic pollutants results in low bioavailability, the discovery and study of biodegradation pathways are relatively lacking, and few biological elements capable of recognizing persistent organic pollutants. The currently developed polycyclic aromatic hydrocarbon monitoring biosensors control bioluminescent signals of the sensors by using naphthalene or phenanthrene degrading enzyme regulating and controlling proteins, which are not suitable for polycyclic aromatic hydrocarbons outside the degradation pathways; the polychlorinated biphenyl monitoring biosensor utilizes oxygenase regulatory protein in a biphenyl degradation pathway as an identification element, so that biphenyl and polychlorinated biphenyl cannot be distinguished; at present, no specific identification biological element of polybrominated diphenyl ethers is found at home and abroad to be used for the research and development of biosensors. Therefore, finding specific biorecognition elements that respond to persistent organic pollutants is a major challenge in developing new biosensors for organic pollution monitoring. Secondly, most of the current environmental monitoring biosensors use bacteria as the underpan cells. How to improve the availability of hydrophobic organic contaminants to the Chassis bacteria is another challenge in developing new biosensors for organic contamination monitoring. Compared with biological recognition elements such as enzymes, antibodies and nucleic acids, the bacterial cells have more economic advantages and can be used for evaluating the toxicity of compounds on living systems. More importantly, the bacteria can change their own cell surface hydrophobicity to better adapt to environmental changes. The availability of the bacteria with high cell surface hydrophobicity to the hydrophobic persistent organic pollutants is higher, the concentration of the hydrophobic organic matters adsorbed on the surfaces of the bacteria is higher, and the monitoring sensitivity is higher.

A new bacterial strain with stable high cell surface hydrophobicity, namely Sphingobium xenophagum (named Sphingobium hydrophibicum) C1, is the generalized sphingomonas with the highest cell surface hydrophobicity reported at present, and can tolerate the toxicity of polybrominated diphenyl ethers with high concentration; hydrophilic mutants of the hydrophobic sphingosine bacteria were obtained in serial passage experiments. The method provides resource guarantee for searching the specific biological recognition element of the polybrominated diphenyl ethers; the hydrophobic sphingosine bacteria has good adsorption capacity on polybrominated diphenyl ethers, and provides a new direction for developing a novel whole-cell microbial sensor for monitoring hydrophobic organic pollutants as hydrophobic chassis cells.

The invention content is as follows:

the first purpose of the invention is to provide a polybrominated diphenyl ether extracellular sensing protein.

The amino acid sequence of the polybrominated diphenyl ether extracellular sensing protein is shown in SEQ ID NO. 3.

The second purpose of the invention is to provide the application of the polybrominated diphenyl ether extracellular sensing protein in specific biological recognition of polybrominated diphenyl ethers.

Preferably, the polybrominated diphenyl ether extracellular receptive protein is applied to the preparation of a hydrophobic chassis whole-cell microbial sensor for monitoring organic pollutants, wherein the organic pollutants are polybrominated diphenyl ethers.

Preferably, the PBDEs extracellular receptor protein is overexpressed in the whole hydrophobic chassis cells to obtain the hydrophobic chassis whole cell microbial sensor.

Preferably, the PBDEs extracellular receptor protein and luciferase are co-overexpressed in whole cells of the hydrophobic chassis to obtain the hydrophobic chassis whole cell microbial sensor.

Preferably, the whole hydrophobic underplate cell is Sphingobium xenophagum C1.

Preferably, the primer 2466U: 5' -GGATCCGCGGCGAAGGCATCTATAT-3' and primer 2466D: 5'-GCGTCTTCCATTTCGGGATAATAGCC-3', using Sphingobium xenophagum C1 genome as template, obtaining chr1_2466 gene and its upstream sequence by amplification, using primer LucU: 5'-CGGCTATTATCCCGAAATGGAAGACGCCAAAAACA-3' and primersLucD：5′-CTCGAGTTACACGGCGATCTTTCC-3', amplifying the complete sequence of the luciferase luc gene of the firefly; with 2466U: 5' -GGATCCGCGGCGAAGGCATCTATAT-3' and primer LucD: 5' -CTCGAGTTACACGGCGATCTTTCC-3' PCR fusion of the two amplified gene fragments chr1_2466 and luc to obtain a fused fragment chr1_2466-luc containing BamHI and XhoI cleavage sites at both ends, which is digested with restriction enzymes BamHI and XhoI, ligated into pET24a vector to transform Sphingobium xenophagum C1, thereby obtaining a hydrophobic bottom plate whole cell microbial sensor.

The third purpose of the invention is to provide a whole-cell microbial sensor, which is obtained by carrying out overexpression on the polybrominated diphenyl ether extracellular receptor protein fusion luciferase in whole cells of a hydrophobic chassis.

The whole hydrophobic underplate cell is Sphingobium xenophagum C1.

The invention clones a polybrominated diphenyl ether recognition receptive protein from Sphingobium xenophagum C1, which can specifically recognize polybrominated diphenyl ether, transfers the polybrominated diphenyl ether into Sphingobium xenophagum C1 with high cell surface hydrophobicity after being fused with luciferase, and overexpresses the fusion protein on the outer membrane of bacterial cells to prepare the hydrophobic chassis whole-cell microbial sensor, which has specific response to decabrominated diphenyl ether and low brominated homologues thereof, has obvious advantages in the aspect of monitoring hydrophobic organic pollutants, can obviously improve the monitoring sensitivity of the hydrophobic organic pollutants, and has wide application prospect in organic pollution monitoring.

Drawings

FIG. 1: differential genetic analysis of response of decabromodiphenyl ether-induced sphingosine bacteria C1 and C2, wherein C1 and C2 represent controls, and B-C1 and B-C2 represent decabromodiphenyl ether-induced sphingosine bacteria C1 and C2.

FIG. 2: response activity analysis of four candidate recognition elements to decabromodiphenyl oxide

FIG. 3: gel blocking assay Chr1_2466 was analyzed for recognition and binding properties of a receptive protein with polybrominated diphenyl ethers, where M represents a protein molecular weight marker, + represents decabromodiphenyl ether-induced conditions, -represents control conditions without decabromodiphenyl ether induction.

FIG. 4: sensitivity and specificity analysis of whole-cell microbial sensor constructed by Chr1_2466 receptive protein

FIG. 5: comparison of sensing Performance of hydrophobic Chassis cells on different cell surfaces

The specific implementation mode is as follows:

the following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1: obtaining of polybrominated diphenyl ether recognition gene element

Preparing an inorganic salt culture medium, wherein each liter of water respectively contains the following substances (g/L): na (Na)₂HPO₄·12H₂O 2.0、KH₂PO₄ 0.7、NH₄Cl 0.5、NaCl 0.3、MgSO₄·7H₂O 0.1、CaSO₄·2H₂O 0.05、FeCl₃·6H₂O 0.2×10^-3、NaMoO₄ 0.2×10^-3、MnCl₂·4H₂O 0.2×10^-3、CuCl₂·2H₂O 0.2×10^-3、ZnSO₄ 0.2×10^-3、H₃BO₃ 0.3×10^-3、CoCl₂·6H₂O 0.4×10^-3Peptone 0.2, yeast extract 1.0, and glucose 5.0. Inoculating hydrophobic strain C1 (CCTCC AB 2015198 ═ KCTC 42740) and hydrophilic strain C2 (CCTCC AB 2015427 ═ KCTC 52051) to LB liquid culture medium, culturing at 30 deg.C in shaker at 200rpm/min until logarithmic growth phase, and culturing to obtain thallus OD₆₀₀The value is about 1.0. The cells were centrifuged at 10,000 Xg for 10min, and the supernatant was removed to collect the cells. After the cells were washed twice with the inorganic salt medium, the cells were resuspended in a volume of the inorganic salt medium to OD₆₀₀Reaching about 1.0. To a 1L volume Erlenmeyer flask were added 0 (control) and 5mL of the mother solution of decabromodiphenyl ether BDE-209 (500. mu.M in dichloromethane), respectively, and after the solvent was evaporated in the dark, 500mL of inorganic salt medium was added to bring the final concentration of BDE-209 to 0 and 5. mu.M, respectively. Respectively inoculating 10mL of hydrophobic strain C1 and hydrophilic strain C2 bacterial suspensions into a culture medium according to the inoculation amount of 2 percent of volume fraction, and placing the culture medium at a rotating speed of 200Culturing at 30 deg.C for 8h in a shaker at rpm/min. The cells were centrifuged at 10,000 Xg for 10min, and the supernatant was removed to collect the cells. Total RNAs of the cells were extracted using RNA extraction Kit (Takara RNAioso Plus Kit), and rRNAs were removed using Epicentre Ribo-zeroTM Magnetic Kit. Then, mRNA is subjected to library establishment according to the operation method of the TruSeq RNA Sample Prep kit for Illumina kit, and finally, mRNA expression quantity is subjected to sequencing analysis by utilizing an Illumina Hiseq 2000 platform of Yongzhou Diao Biotechnology Co., Ltd.

According to the RPKM (Reads Per kb Per Million reads) value of gene expression, FDR (false discovery rate) was used<0.05 and | log₂FC|>1 to screen for differentially expressed genes. The reference genomic information was the s.xenophagum C1 genome (NCBI accession nos. cp022745, CP022746, CP022747, CP022748, CP022749, CP022750, and CP 022751). The results showed that under BDE-209 exposure conditions, a total of 101 genes were significantly changed among 4428 genes in the whole genome of C1 and C2, with 29 genes significantly up-regulated and 72 genes significantly down-regulated. Of the 29 genes that were significantly up-regulated, 17 genes were up-regulated in both C1 and C2 bacteria, with the same expression pattern. The other 12 genes were up-regulated in C1 strain only, and were not significantly changed in C2 strain. Gene annotation indicates that 12 genes specifically up-regulated in C1 bacteria are mainly involved in heavy metal resistance (chr1_1106-chr1_1109), NO metabolism (p2_0159-p2_0162), porphyrin metabolism (chr1_2498-chr1_2500) and small molecule recognition (chr1_ 2466); 17 genes up-regulated in both C1 and C2 bacteria were mainly involved in styrene degradation (chr1_1483), xenobiotic response (chr1_2605), heavy metal response (chr1_2170), and unknown function (chr2_0630-chr2_0636) (fig. 1A).

The biological recognition element (sensor protein) commonly used in biosensors mainly includes transcription regulatory protein, two-component sensing protein, methyl receptor sensing protein and periplasmic binding protein. Annotation analysis of 29 genes with a significant BDE _209 response upregulation according to these classifications revealed that 4 gene elements belong to these recognition element classes, chr1_2605 encoding xenobiotic response transcription factors (NCBI accession No. asy45241.1), chr1_2170 encoding the UrcA family transcriptional regulator (NCBI accession No. asy44883.1), chr1_1106 encoding the metalloregulator ArsR/SmtB family transcription factors (NCBI accession No. asy43965.1), and chr1_2466 encoding the Cache domain receptive protein (NCBI accession No. asy45126.1). Wherein the genes chr1_2605 and chr1_2170 are respectively up-regulated by 2.28 and 2.88 times during the BDE-209 response process of the C1 strain; it was up-regulated by 2.01 and 2.43 fold during the response of C2 strain to BDE-209, respectively (FIG. 1B). While the gene elements chr1_1106 and chr1_2466 only up-regulate the expression in the process that the C1 strain responds to BDE-209, and the up-regulation is 3.25 and 2.03 times respectively; without significant change in the response of the C2 strain to BDE-209 (fig. 1C). The 4 gene elements are used as candidate polybrominated diphenyl ether recognition gene elements for further analysis.

Example 2: activity and characteristic analysis of polybrominated diphenyl ether recognition element

The activity analysis was performed on the 4 candidate polybrominated diphenyl ether recognition gene elements obtained above. A gene including a promoter sequence of about 500bp upstream of each candidate gene was synthesized by adding a coding sequence of firefly luciferase small peptide HiBiT (5'-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC-3') provided by Promega to the C-terminus of 4 candidate recognition element genes by a method of synthesizing genes by Biochemical engineering (Shanghai) Ltd, and BamHI and XhoI cleavage sites were designed at both ends of the gene fragment, respectively (EcoRI and XhoI cleavage sites were contained at 5 'and 3' ends of the synthesized gene chr 1-1106, respectively). After gene synthesis, the gene fragment and pET24a expression vector were synthesized by treating chr1_2605, chr1_2170 and chr1_2466 with BamHI and XhoI restriction enzymes of TaKaRa at 37 ℃ in K buffer, by treating chr1_1106 and pET24a expression vector with EcoRI and XhoI restriction enzymes of TaKaRa at 37 ℃ in H buffer, and by ligating the corresponding gene fragment and vector with the recombinase of Beijing Quanyu gold Biotech (TransGen Biotech) Co., Ltd. The pET24a vector connected with the recombinant fragment is transformed into competent cells of the C1 strain by an electric shock transformation method, and the transformed strain liquid is finally spread on an LB medium plate containing 50 ug/mL kanamycin and is statically cultured for 24h at 30 ℃. Single clones growing on the plate were picked, colony PCR was performed using the universal primers T7 (5'-TAATACGACTCACTATAGG-3') and T7-TER (5'-GCTAGTTATTGCTCAGCGG-3'), and the amplified products were run correctly and then sampled for sequencing analysis. Finally, C1 strain underplate cells respectively containing pET-1106-HiBiT, pET-2170-HiBiT, pET-2466-HiBiT and pET-2605-HiBiT report vectors with correct sequences are obtained.

Inoculating the Chassis cells of C1 bacteria containing 4 kinds of reporter vectors into LB liquid medium containing 50. mu.g/mL kanamycin, and culturing overnight at 30 deg.C to OD of bacteria in a shaker at 200rpm/min₆₀₀The value is about 1.0. The cells were centrifuged at 10,000 Xg for 10min, and the supernatant was removed to collect the cells. After the cells were washed twice with the inorganic salt medium, the cells were resuspended in a volume of the inorganic salt medium to OD₆₀₀Reaching about 1.0. To a 5mL volume brown glass bottle were added 0, 5, 10 and 20. mu.L of BDE-209 mother liquor (50. mu.M in dichloromethane), respectively, and after evaporation of the solvent in the dark, 200. mu.L of mineral salts medium was added to bring the final concentration of BDE _209 to 0, 1.25, 2.5 and 5. mu.M, respectively. Respectively inoculating 4 μ L of Chassis cell bacterial suspension to 200 μ L of inorganic salt culture medium pre-added with BDE-209 with different concentrations according to the inoculation amount of 2%, and culturing in a shaker at the rotation speed of 200rpm/min at 30 deg.C for 8h to obtain thallus cell OD₆₀₀The value reached 0.4. 100 mul of the bottom plate cell samples are respectively taken for protein quantitative analysis to ensure that the protein concentration of each sample is basically consistent. The other 100. mu.L of the Chassis cell samples were added with 100. mu.L of extracellular non-lysis buffer (containing firefly luciferase large subunit LgBiT and luciferase luminescent substrate furimazine, Promega corporation) or intracellular lysis buffer (containing lysis buffer, firefly luciferase large subunit LgBiT and luciferase luminescent substrate furimazine, Promega corporation), and extracellular or intracellular luciferase activity analysis was performed on a chemiluminescence apparatus.

The analysis result shows that the C1 bacterial underpan cells of the 4 reporter vectors can detect the intracellular luciferase activity, but the extracellular luciferase activity is detected only in C1(pET-2170-HiBiT) and C1(pET-2466-HiBiT), which indicates that the proteins encoded by the gene elements of chr1_2170 and chr1_2466 are positioned outside the cell membrane, and the proteins encoded by the gene elements of chr1_1106 and chr1_2605 are positioned in cytoplasm. The luciferase activity induced by BDE-209 at different concentrations is analyzed, and the intracellular luciferase activity of C1(pET-1106-HiBiT) and C1(pET-2605-HiBiT) is not in a linear relation with the concentration of BDE-209, and the extracellular and intracellular luciferase activity of C1(pET-2170-HiBiT) is also in a linear relation with the concentration of BDE-209; while C1(pET-2466-HiBiT) alone, whether extracellular luciferase activity or intracellular activity, showed a significant linear relationship with BDE _209 concentration (FIG. 2), suggesting that Chr1_2466 is likely to be a novel recognition receptor for polybrominated diphenyl ethers.

Bioinformatics analysis shows that the protein Chr1_2466 encoded by the gene element Chr1_2466(NCBI accession No. ASY45126.1) is an extracellular sensing protein of a Cache domain, is 154 amino acids long (the amino acid sequence of the protein is shown as SEQ ID NO. 3), and contains a signal peptide of 24 amino acids (MFPFSRTLTAMVCALALSAAPALA) at the N end, which indicates that Chr1_2466 is a cell membrane protein. The isoelectric point of the protein is 5.59, and the molecular weight is 16.5 KD. The Cache domain is reported in the literature to be an extracellular domain protein and has an important role in the recognition of small molecules. Therefore, the experimental design verifies the identification and binding characteristics of Chr1_2466 and polybrominated diphenyl ethers through a gel blocking experiment. The cells of C1(pET-2466-HiBiT) cultured for 8 hours in BDE-209 inorganic salt medium of 0 and 5 mu M were centrifuged at 10,000 Xg for 10min, and the supernatant was removed to collect the cells. After the cells were washed twice with an inorganic salt medium, the cells were resuspended in a volume of lysis buffer (50mM Tris-HCl, pH 8.0,150mM NaCl,1mM EDTA, 0.25% Triton X-100) to make the OD of the cells₆₀₀Reaching about 1.0. The bacterial suspension was treated with freshly prepared lytic enzyme to a final concentration of 0.5mg/mL, protease inhibitor from Merck was added to inhibit protein degradation, and the suspension was then treated with slow shaking at 25 ℃ for 20 min. The lysate was centrifuged at 10,000 Xg for 5min at 4 ℃ to remove the precipitate, and the supernatant was subjected to protein electrophoresis in 12% polyacrylamide non-denaturing gel at 4 ℃ for 1h at 100V. The protein gel was then transferred to a nitrocellulose membrane in a transfer bath at 180mA for 2h, and the nitrocellulose membrane was incubated in TBST buffer (20mM Tris-HCl, pH 7.5,150mM NaCl, 0.1% Tween 20) with slow shaking at room temperature for 30 min. Nitrocellulose was transferred to blotting buffer from Promega, 100nM luciferase large subunit LgBiT protein was added and incubated at room temperature for 1h with slow shaking. Finally adding 25 mu M luciferase substrate furimazine,the cells were incubated at room temperature for 5min and the nitrocellulose membrane was placed on a ChemiDoc XRS + imaging system from Bio-Rad for photographic analysis. The experimental results show that under non-denaturing conditions, Chr1_2466 is a tetrameric protein with a molecular weight of about 66 kD. Since the water solubility of BDE-209 is very low, very little part of BDE-209 dissolved in water is combined with Chr1_2466, which causes the lag and diffusion of the protein band of Chr1_2466 on the electrophoresis gel. In addition, it can be seen from the electropherogram that BDE-209 can significantly induce the up-regulated expression of the protein encoded by the Chr1_2466 gene (FIG. 3). These results confirmed that Chr1_2466 is a novel recognition receptive protein for polybrominated diphenyl ethers.

Example 3: construction and performance analysis of polybrominated diphenyl ether monitoring whole-cell microbial sensor

Synthesis of primer 2466U (5' -GGATCCGCGGCGAAGGCATCTATAT-3 ') and a primer 2466D (5'-GCGTCTTCCATTTCGGGATAATAGCC-3'), amplifying the chr1_2466 gene (the nucleotide sequence of which is shown in SEQ ID NO. 1) and the 507bp upstream sequence (the nucleotide sequence of which is shown in SEQ ID NO. 2). Synthesis of the primer LucU (5'-CGGCTATTATCCCGAAATGGAAGACGCCAAAAACA-3') and the primer LucD (5-CTCGAGTTACACGGCGATCTTTCC-3'), amplifying the complete sequence of the firefly luciferase luc gene (NCBI accession No. AB762768.1). Using 2466U (5')GGATCCGCGGCGAAGGCATCTATAT-3') and the primer LucD (5-CTCGAGTTACACGGCGATCTTTCC-3') carrying out PCR fusion on the two amplified gene fragments of chr1_2466 and luc to obtain a fused fragment chr1_2466-luc with BamHI and XhoI enzyme cutting sites at two ends. The fused fragment chr 1-2466-luc and the pET24a expression vector were treated with BamHI and XhoI restriction enzymes of TaKaRa at 37 ℃ in K buffer, and the ligation of the digested fragment and the vector was performed by using recombinase of Beijing Quanyu gold Biotechnology (TransGen Biotech) Co.Ltd. The pET24a vector connected with the recombinant fragment is respectively transformed into competent cells of C1 bacteria and C2 bacteria by an electric shock transformation method, and the transformed bacteria liquid is finally coated on an LB culture medium plate containing 50 ug/mL kanamycin and is statically cultured for 24h at 30 ℃. Selecting single clone grown on plate, performing colony PCR with universal primer T7 (5'-TAATACGACTCACTATAGG-3') and T7-TER (5'-GCTAGTTATTGCTCAGCGG-3'), and electrophoresing amplified productAnd (5) after the accuracy is achieved, sending a sample for sequencing analysis. Finally, the microbial sensors of C1 underpan cells and C2 underpan cells containing pET-2466-Luc reporter vectors with correct sequences are obtained.

Inoculating C1(pET-2466-Luc) basal disc cells into LB liquid medium containing 50. mu.g/mL kanamycin, placing the cells in a shaker with the rotating speed of 200rpm/min for overnight culture at 30 ℃ until the thallus OD₆₀₀The value is about 1.0. The cells were centrifuged at 10,000 Xg for 10min, and the supernatant was removed to collect the cells. After the cells were washed twice with the inorganic salt medium, the cells were resuspended in a volume of the inorganic salt medium to OD₆₀₀Reaching about 1.0. Adding 0, 5, 10, 20, 24, 25, 30, 35 and 40 μ L of BDE-209 mother liquor (50 μ M dissolved in dichloromethane) into a brown glass bottle with a volume of 5mL, volatilizing the solvent in dark, and adding 200 μ L of inorganic salt medium to make the final concentration of BDE _209 reach 0, 1.25, 2.5, 5.0, 6.0, 6.25, 7.50, 8.75 and 10.0 μ M; adding 0, 0.4, 2, 5, 10, 20 and 50 μ L of BDE-209 mother liquor (5 μ M dissolved in dichloromethane) into a brown glass bottle with a volume of 5mL respectively, volatilizing the solvent in dark, and adding 200 μ L of inorganic salt culture medium to make the final concentration of BDE _209 reach 0, 0.01, 0.05, 0.125, 0.5 and 1.25 μ M respectively; 1 mu M of PCB-209, diphenyl ether, phenol, BDE-1, BDE-47, BDE-99, BDE-153, BDE-183, BDE-209, potassium chloride, sodium chloride, magnesium chloride, zinc chloride, calcium chloride, mercuric chloride, antimony trichloride, antimony trioxide, antimony potassium tartrate, manganese sulfate, nickel sulfate, chromium sulfate, cobalt chloride, cadmium sulfate, copper nitrate, sodium arsenate, sodium arsenite, lead nitrate, ferric trichloride and aluminum nitrate are respectively added into a brown glass bottle with the volume of 5mL, and 200 mu L of inorganic salt culture medium is added. Respectively inoculating 4 μ L of C1(pET-2466-Luc) Chassis cell bacterial suspension into 200 μ L of inorganic salt culture medium added with substrate with different concentrations according to the inoculation amount of 2%, and culturing in a shaker at the rotation speed of 200rpm/min at 30 deg.C for 8h to obtain thallus cell OD₆₀₀The value reached 0.4. 100 mul of the bottom plate cell samples are respectively taken for protein quantitative analysis to ensure that the protein concentration of each sample is basically consistent. The remaining 100. mu.L of the bottom plate cell samples were added with the luciferase luminescent substrate furimazine and analyzed for extracellular luciferase activity on a chemiluminescence apparatus.

The sensitivity analysis of the C1(pET-2466-Luc) whole cell microbial sensor shows that in the BDE-209(0-10 mu M) of the tested concentration, the BDE-209 of 6 mu M induces the maximum fluorescence signal. As the concentration of BDE-209 is increased to be more than 6.25 mu M, the fluorescence signal is sharply reduced, which indicates that BDE-209 with high concentration generates toxicity to the underpan cells. On the other hand, the C1(pET-2466-Luc) whole cell microbial sensor can be used for monitoring the toxicity of BDE-209, and the cytotoxicity threshold is 6.25 mu M. The lowest detection limit of the C1(pET-2466-Luc) whole cell microbial sensor was 0.01. mu.M, about 9.59. mu.g/L BDE-209. Through the correlation analysis of the BDE-209 concentration between 0.05 and 6.0 mu M and the fluorescence signal, the correlation index R of the BDE-209 concentration and the fluorescence signal²Is 0.98. The results of the specificity analysis of the whole-cell microbial sensor show that C1(pET-2466-Luc) has no obvious response to polychlorinated biphenyl, diphenyl ether, phenol, metal ions and inorganic salts, and can distinguish the polybrominated diphenyl ether from the diphenyl ether. Among representative polybrominated diphenyl ethers, C1(pET-2466-Luc) had the highest affinity for BDE-209, and the affinity decreased with decreasing degree of bromination of polybrominated diphenyl ethers, and the response to monobrominated diphenyl ethers was the lowest (FIG. 4). This also suggests that bromide is likely one of the key ions for Chr1_2466 sensory protein binding to polybrominated diphenyl ethers.

Example 4: comparison of sensing Performance of hydrophobic Chassis cells on different cell surfaces

Whole cell biosensors C1(pET-2466-Luc) and C2(pET-2466-Luc) constructed from underpan cells with different cell surface hydrophobicity, which were constructed in example 3, were inoculated into LB liquid medium containing 50. mu.g/mL kanamycin, and cultured overnight at 30 ℃ in a shaker at 200rpm/min until the OD of the cells reached₆₀₀The value is about 1.0. The cells were centrifuged at 10,000 Xg for 10min, and the supernatant was removed to collect the cells. After the cells were washed twice with the inorganic salt medium, the cells were resuspended in a volume of the inorganic salt medium to OD₆₀₀Reaching about 1.0. To a 5 mL-volume brown glass bottle were added 0. mu.M and 2.5. mu.M of BDE-1, BDE-47, BDE-99, BDE-153, BDE-183, and BDE-209, respectively, and after the solvent was evaporated in the dark, 200. mu.L of the inorganic salt medium was added. Inoculating 4 μ L of the mixture according to the inoculum size of 2%The suspension of the Chassis cells of C1(pET-2466-Luc) and C2(pET-2466-Luc) is added into 200 mu L of inorganic salt culture medium which is pre-added with polybrominated diphenyl ethers with different concentrations, and the mixture is put into a shaking table with the rotating speed of 200rpm/min for culturing for 8 hours at the temperature of 30 ℃ until the OD of the somatic cells is up to₆₀₀The value reached 0.4. 100 mul of the bottom plate cell samples are respectively taken for protein quantitative analysis to ensure that the protein concentration of each sample is basically consistent. The remaining 100. mu.L of the bottom plate cell samples were added with the luciferase luminescent substrate furimazine and analyzed for extracellular luciferase activity on a chemiluminescence apparatus.

Analysis of the luciferase activity induced by different concentrations of polybrominated diphenyl ethers revealed that none of the whole-cell biosensor C2(pET-2466-Luc) constructed from hydrophilic underpant cells responded significantly to different concentrations of polybrominated diphenyl ethers, whereas the C1(pET-2466-Luc) whole-cell biosensor constructed from hydrophobic underpant cells responded specifically to BDE-209 and its low-brominated homologues (FIG. 5). Since hydrophilic C2(pET-2466-Luc) does not have significant adsorption to polybrominated diphenyl ethers, its low cell surface hydrophobicity is not conducive to underplate cell contact and perception of hydrophobic polybrominated diphenyl ethers. The result shows that the hydrophobic underpan cells have obvious advantages in the aspect of monitoring hydrophobic organic pollutants, and the sensitivity of monitoring the hydrophobic organic pollutants can be obviously improved.

Sequence listing

<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)

<120> polybrominated diphenyl ether sensing protein and whole-cell microbial sensor constructed by same

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 465

<212> DNA

<213> Sphingobium xenophagum C1

<400> 1

atgttcccat tttcgcgcac cctcacggct atggtctgcg ccctcgcgct ttccgccgcc 60

cccgccctgg ccgccccgca tgccgatgca gcacaggcgc aggccatgct cgaaaaagcc 120

gtggcgacca tcaagacggc cggtgccggg cctgcctttg ctgcgttcaa tcaaaaggac 180

ggggcattca acaccgggga actctatgtg ttcgttttcg acctgaacgg ggtctacgaa 240

gcctatggcg cgcatcccgg cttggtcggc cgcgatgtca gcgatctgac cgatgcggaa 300

ggcaaaccga tcgtgcgcga catgatagag attgcccgca ccagcggcca tggaaagatc 360

aattatgtct ggctcaaccg cgctgacaat cgggtcgaac gcaagatgtc gctgatcgaa 420

ctggtcgaca atcatgtcgt gggtgtcggc tattatcccg aatag 465

<210> 2

<211> 507

<212> DNA

<213> Sphingobium xenophagum C1

<400> 2

gcggcgaagg catctatatc gatcatgtcg ccatggacca ggactggcgc acgcatcatg 60

tccccctttt ttacggcttt caaagctatg tgtcgctgcc tgtcattctg cttgatggca 120

gcttctacgg cacgctgtgc gccattgatc ccaacccccg ttcggtcagc gcgccggcca 180

tcgtcgccgc catgcaggac tatgcccgat cgatcggtgc gatattgtcg gcgaaatgat 240

gctgttgttc agtcgatagt gccgggactt cgcctgactc gtttgcgtcc agcggggcca 300

aggtagattt ccgaccttga gcggtgcggc cgcgacaggc ctctccatgc atatggcctc 360

ataccaaagg tcgatttgat ctgcgtctaa atgcgcgggc gcggatcggg ttaattacgc 420

tctgtcatcg gcaatcggat cgggaccgaa ggtccgcctg catccggctt cagcgtttgt 480

tgtcgatgat cagaggagat ttggctc 507

<210> 3

<211> 154

<212> PRT

<213> Sphingobium xenophagum C1

<400> 3

Met Phe Pro Phe Ser Arg Thr Leu Thr Ala Met Val Cys Ala Leu Ala

1 5 10 15

Leu Ser Ala Ala Pro Ala Leu Ala Ala Pro His Ala Asp Ala Ala Gln

20 25 30

Ala Gln Ala Met Leu Glu Lys Ala Val Ala Thr Ile Lys Thr Ala Gly

35 40 45

Ala Gly Pro Ala Phe Ala Ala Phe Asn Gln Lys Asp Gly Ala Phe Asn

50 55 60

Thr Gly Glu Leu Tyr Val Phe Val Phe Asp Leu Asn Gly Val Tyr Glu

65 70 75 80

Ala Tyr Gly Ala His Pro Gly Leu Val Gly Arg Asp Val Ser Asp Leu

85 90 95

Thr Asp Ala Glu Gly Lys Pro Ile Val Arg Asp Met Ile Glu Ile Ala

100 105 110

Arg Thr Ser Gly His Gly Lys Ile Asn Tyr Val Trp Leu Asn Arg Ala

115 120 125

Asp Asn Arg Val Glu Arg Lys Met Ser Leu Ile Glu Leu Val Asp Asn

130 135 140

His Val Val Gly Val Gly Tyr Tyr Pro Glu

145 150