A stibium-selective SxArsR protein and a whole-cell microbial sensor for antimony pollution monitoring constructed therefrom

Abstract

The present invention discloses an antimony-selective SxArsR protein and a whole-cell microbial sensor for antimony pollution monitoring constructed therefrom. The amino acid sequence of the antimony-selective SxArsR protein is as shown in SEQ ID NO.3. In the present invention, an antimony-selective SxArsR protein is cloned from Sphingobium xenophagum C1, which can specifically recognize antimony trioxide. After fusing it with luciferase, it is respectively transformed into the chassis cells Sphingobium xenophagum C1 and Escherichia coli DH5α, and the fusion protein is expressed in the bacterial cytoplasm to prepare a whole-cell microbial sensor, which has a specific response to antimony trioxide, has obvious advantages in the monitoring of heavy metal antimony pollutants, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an antimony-selective SxArsR protein and its application in the construction of a whole-cell microbial sensor for heavy metal antimony pollution monitoring. Background Art

[0002] Antimony is a toxic metalloid element, located below arsenic in the periodic table, and has similar chemical and toxicological properties to arsenic. Antimony mainly exists in the form of antimony trioxide (Sb2O3) and antimony trisulfide in nature, and is the ninth most mined industrial metal in the world. Sb2O3 is the most important processed antimony commercially, accounting for 80% of the total antimony used in the manufacture of various consumer goods. Sb2O3 is widely used as a synergist for flame retardants, a catalyst for the production of polyethylene terephthalate, an additive for glassware and ceramics, a pigment in paints, a material for semiconductors and lead-acid batteries, etc. During the discharge of industrial wastewater, the treatment and recycling of plastics and electronic waste, and the dust emission during mining and smelting, a large amount of Sb2O3 is released into the environment. The extensive industrial applications and the increasingly accelerating human activities have led to a large accumulation of Sb2O3 pollutants in the environment.

[0003] Currently, the main methods for determining antimony pollution in the environment are chemical methods. Most chemical methods are reliable and have good sensitivity and reproducibility. However, they are expensive in terms of infrastructure and instruments, require trained staff and a time-consuming sample preparation process, which is unaffordable in some places. Whole-cell biosensors that use the target-responsive regulation mechanism (recognition unit) to control the expression of reporter genes (signal transducer) are an attractive option for in-situ monitoring of environmental pollutants, and can also provide more biological relevant information such as bioavailable toxicity.

[0004] Arsenic-contaminated groundwater caused by arsenite and arsenate affects the drinking water and domestic water safety of many people around the world. Although a large amount of research has been carried out on arsenite, and whole-cell biosensors based on the arsenite recognition element (ArsR protein encoded by the arsRDABC operon) have been successfully applied to field tests. However, the relevant information about the biological recognition element of Sb2O3 and its practical application in the construction of whole-cell biosensors and environmental monitoring is still very limited. ArsR derived from the Escherichia coli plasmid is a transcriptional repressor, which has a high self-regulation effect on arsenite, but has a low response ability to antimonite. Antimony pollution in the environment is mainly caused by Sb2O3. Therefore, it is imperative to develop a new type of whole-cell biosensor for accurate and efficient monitoring of Sb2O3 pollutants. Summary of the Invention

[0005] The first object of the present invention is to provide an antimony-selective ArsR family protein, named SxArsR.

[0006] The antimony-selective SxArsR protein of the present invention has an amino acid sequence as shown in SEQ ID NO.3.

[0007] The second object of the present invention is to provide the application of the above-mentioned antimony-selective SxArsR protein in specifically recognizing antimony trioxide.

[0008] Preferably, it is the application of the above-mentioned antimony-selective SxArsR protein in the preparation of a whole-cell microbial sensor for monitoring antimony pollutants, and the antimony pollutant is antimony trioxide.

[0009] Preferably, the above-mentioned antimony-selective SxArsR protein is fused with luciferase and expressed in chassis cells to obtain a whole-cell microbial sensor.

[0010] Preferably, the chassis cells are Sphingobium xenophagum C1 and Escherichia coli DH5α.

[0011] Preferably, primers 1106U: 5′- GAATTC CCGAACCCTTCAGCTTGCG-3′ and primer 1106D: 5′-ATGTTTTTGGCGTCTTCCATGGCAGCCCTTTTCT-3′ are used to amplify the sxarsR gene and its upstream sequence with the genome of Sphingobium xenophagum C1 as a template; primers LucU1: 5′-GTCAGAAAAGGGCTGCCATGGAAGACGCCAAAAACA-3′ and primer LucD1: 5′- CTCGAG TTACACGGCGATCTTTCC-3′ are used to amplify the complete sequence of the firefly luciferase luc gene with the pTAL-Luc plasmid as a template; using primers 1106U: 5′- GAATTC CCGAACCCTTCAGCTTGCG-3′ and primer LucD1: 5′- CTCGAGThe two amplified gene fragments of sxarsR and luc were subjected to PCR fusion with 5′-TTACACGGCGATCTT TCC-3′ to obtain a fusion fragment sxarsR-luc containing EcoRI and XhoI restriction sites at both ends. After digestion with the restriction enzymes EcoRI and XhoI, it was ligated to the pET24a vector, and the recombinant vector was transformed into the chassis cells Sphingobium xenophagum C1 and Escherichia coli DH5α respectively, thereby obtaining a whole-cell microbial sensor for antimony pollution monitoring.

[0012] The third object of the present invention is to provide a whole-cell microbial sensor, which is obtained by expressing the antimony-selective SxArsR protein fused with luciferase in the chassis cells.

[0013] The chassis cells are Sphingobium xenophagum C1 and Escherichia coli DH5α.

[0014] The fourth object of the present invention is to provide the application of the above-mentioned whole-cell microbial sensor in the monitoring of antimony pollutants, and the antimony pollutant is antimony trioxide.

[0015] In the present invention, an antimony-selective SxArsR protein was cloned from Sphingobium xenophagum C1, which can specifically recognize antimony trioxide. After fusing it with firefly luciferase, it was transformed into the chassis cells Sphingobium xenophagum C1 and Escherichia coli DH5α respectively, and the fusion protein was expressed in bacterial cells to prepare a whole-cell microbial sensor, which has a specific response to antimony trioxide and has obvious advantages in the monitoring of heavy metal antimony pollutants, and has broad application prospects. Description of the Drawings

[0016] Figure 1 It is the structural analysis of the ArsR element in the genome of Sphingobium xenophagum C1.

[0017] Figure 2 It is the phylogenetic analysis and conserved domain analysis of SxArsR and ArsR transcriptional repressors of other genera.

[0018] Figure 3 It is the sequence analysis of SxArsR and mutant strains and their response analysis to NaAsO2 and Sb2O3 induction.

[0019] Figure 4Performance analysis of a whole-cell microbial sensor constructed with an antimony-selective SxArsR protein. Detailed implementation mode

[0020] The following examples are further illustrations of the present invention rather than limitations thereof.

[0021] Example 1: ArsR element in the genome of Sphingobium xenophagum C1

[0022] Inoculate Sphingobium xenophagum C1 (= CCTCC AB 2015198 = KCTC 42740) into a liquid LB medium (containing 10 g of peptone, 5 g of yeast extract, and 5 g of NaCl per liter of medium, with water as the solvent; the preparation method is to dissolve each component in water and sterilize to obtain), and place it in a shaker at 200 rpm / min at 30 °C until the logarithmic growth phase, with the OD 600 value of about 1.0. Centrifuge at 10,000×g for 10 min, discard the supernatant, and collect the bacterial cells. The bacterial cells are sent to Annoroad Gene Technology (Beijing) Co., Ltd. for whole-genome extraction and sequencing analysis. The results of whole-genome sequencing analysis show that the C1 bacterial genome contains a total of 4 ars operons and 3 independent ars genes, which are distributed in its 1 replicon and 1 large plasmid gene. According to the gene annotation results, the 3 ars operons of chr1_1105-chr1_1099, chr1_1360-chr1_1358, and p4_10-p4_13 mainly encode typical ars resistance system proteins: ArsR transcriptional repressor (Chr1_1105, Chr1_1360, P4_10), pentavalent arsenic reductase ArsC (Chr1_1104, Chr1_1103, Chr1_1359, P4_11), trivalent arsenic efflux transporter ArsB (P4_12) or Acr3 (Chr1_1102, Chr1_1358) ( Figure 1A). The chr1_1106-chr1_1109 operon encodes proteins of an atypical ars resistance system: ArsR (Chr1_1106), an ArsP_1 superfamily permease (Chr1_1107), thioredoxin_3 (Chr1_1108), and an ArsO superfamily flavin monooxygenase (Chr1_1109). Alignment and analysis of the ArsR protein sequences in these four ars operons revealed significant differences between the Chr1_1106 protein and the other three ArsR proteins. The conserved cysteine sites in Chr1_1105, Chr1_1360, and P4_10 consist of three cysteine residues CCXGXXXXC located at the C-terminus. In contrast, the conserved cysteine site in Chr1_1106 consists of two cysteine residues CVC located at the N-terminus and another cysteine residue at the C-terminus ( Figure 1 B).

[0023] By the method of synthesizing genes in Sangon Biotech (Shanghai) Co., Ltd., the coding sequence of the firefly luciferase small peptide HiBiT provided by Promega (5′-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC-3′) was added to the 3′ ends of the 4 ArsR protein-coding genes chr1_1106, chr1_1105, chr1_1360, and p4_10 respectively, and fragments containing the promoter sequences of about 500 bp upstream of each gene were synthesized. Restriction enzyme cleavage sites of BamHI / XhoI or EcoRI / XhoI were designed at both ends of the gene fragments. The gene fragments and the pET24a expression vector were treated with TaKaRa's BamHI, XhoI, and EcoRI restriction endonucleases in H buffer at 37°C, and the corresponding gene fragments were ligated to the vector using the recombinase from TransGen Biotech Co., Ltd. The recombinant vectors obtained by ligation were transformed into the competent cells of C1 bacteria by electroporation, and the transformed bacterial solution was finally spread on an LB medium plate containing 50 μg / mL kanamycin and cultured statically at 30°C for 24 h. The monoclonal colonies grown on the plate were picked, and colony PCR was performed using the universal primers T7 (5′-TAATACGACTCACTATAGG-3′) and T7-TER (5′-GCTAGTTATTGCTCAGCGG-3′). After the electrophoresis of the amplified products was correct, the samples were sent for sequencing analysis. Finally, C1 bacterial sensing cells containing the correctly sequenced p24a-1106-HiBiT, p24a-1105-HiBiT, p24a-1360-HiBiT, and p24a-10-HiBiT reporter vectors were obtained. The C1 bacterial sensing cells containing different reporter vectors were respectively inoculated into an inorganic salt liquid medium containing 50 μg / mL kanamycin (containing 2.0 g of Na2HPO4·12H2O, 0.7 g of KH2PO4, 0.5 g of NH4Cl, 0.3 g of NaCl, 0.1 g of MgSO4·7H2O, 0.05 g of CaSO4·2H2O, 0.2 mg of FeCl3·6H2O, 0.2 mg of NaMoO4, 0.2 mg of MnCl2·4H2O, 0.2 mg of CuCl2·2H2O, 0.2 mg of ZnSO4, 0.3 mg of H3BO3, 0.4 mg of CoCl2·6H2O, 0.2 g of peptone, 1.0 g of yeast extract, 5.0 g of glucose per liter of the medium, and the solvent was water; the preparation method was to dissolve each component in water and sterilize it), and placed in a shaker at 200 rpm / min and cultured overnight at 30°C until the OD of the bacterial cells 600The value is approximately 1.0. The C1 sensing cell bacterial suspensions containing different reporter vectors were inoculated into the inorganic salt medium supplemented with 1 μM of different compounds at an inoculation amount of 2% by volume. Then, they were cultured in a shaker at 200 rpm / min at 30 °C for 4 h until the OD 600 value of the bacterial cells reached 0.2. 100 μL of the sensing cell samples were taken respectively for protein quantification analysis to ensure that the protein concentrations of all samples were basically the same. 100 μL of intracellular lysis buffer (containing lysis solution, large subunit of firefly luciferase LgBiT and luciferase luminescence substrate furimazine, Promega) was added to the remaining 100 μL of the sensing cell samples, and the intracellular luciferase activity analysis was carried out on a chemiluminescence instrument.

[0024] It was found that when the C1 sensing cells were exposed to different compounds, strong bioluminescence signals induced by sodium arsenite NaAsO2 and lower bioluminescence signals induced by sodium antimonite NaSbO2 were detected in the C1(p24a-1105-HiBiT), C1(p24a-1360-HiBiT) and C1(p24a-10-HiBiT) sensing cells ( Figure 1 C). Sodium arsenate Na3AsO4 could also induce bioluminescence signals in these 3 kinds of sensing cells, and the signal intensity was about one-sixth of that induced by NaAsO2, which might be because the C1 sensing cells first reduced Na3AsO4 to NaAsO2. This indicated that Chr1_1105, Chr1_1360 and P4_10 were typical ArsR proteins, which specifically recognized arsenite and had a certain response to antimonite. However, C1(p24a-1106-HiBiT) could only be induced by Sb2O3 to produce an obvious bioluminescence signal, and no other tested antimony and arsenic compounds could induce a significantly different signal compared with the non-induced control ( Figure 1 C). Due to the high toxicity of arsenic trioxide As2O3 to bacterial cells, strain C1 did not grow after incubation with As2O3, so no data on the bioluminescence signal of C1 sensing cells in response to As2O3 were collected. These results showed that the transcriptional repressor encoded by chr1_1106 was a novel ArsR family protein mediating the specific binding of Sb2O3, named SxArsR (its nucleotide sequence was shown in SEQ ID NO.1, and its amino acid sequence was shown in SEQ ID NO.3).

[0025] The SxArsR protein has the amino acid sequence shown in SEQ ID NO.3, specifically: MRIDDLLNALAEPTRLRALQIIWDGEEHCVCELMDRLDVTQSRMSRHMQRLKLVGLLTDRRDAQWVRYRRRADLSTAWMAIIDAILQALPPLPQSACQKRAA.

[0026] The coding gene sequence of the SxArsR protein is shown in SEQ ID NO.1, specifically: ATGCGAATCGATGACCTGTTAAACGCGCTTGCCGAGCCGACGCGGCTCCGCGCCCTGCAAATCATTTGGGACGGCGAAGAGCACTGCGTGTGCGAGCTGATGGACCGTCTCGACGTGACCCAATCGCGCATGTCGAGGCACATGCAGCGGTTGAAACTGGTAGGTCTCCTGACCGACCGGCGCGATGCGCAGTGGGTGCGCTATCGCAGGCGTGCCGATCTGTCGACCGCTTGGATGGCGATCATCGACGCCATCCTGCAGGCCTTACCGCCGCTGCCGCAATCGGCCTGTCAGAAAAGGGCTGCCTGA. Example 2: Comparison of SxArsR Protein with Other Types of ArsR Proteins

[0027] Phylogenetic analysis was performed on the ArsR protein sequences from 44 different bacterial sources to understand the evolutionary history of the SxArsR - like transcriptional repressors. The ArsR sequences were obtained by BLASTP search from the National Center for Biotechnology Information (NCBI) (https: / / www.ncbi.nlm.nih.gov / ) protein database and by retrieving the accession numbers listed in the references. Multiple alignments of the ArsR homologous sequences were performed using ClustalX (http: / / www.clustal.org / ) and DNAMAN (https: / / www.lynnon.com / dnaman.html). A phylogenetic tree was constructed using the maximum - likelihood method in MEGA - X (https: / / www.megasoftware.net / ) to analyze the evolutionary relationships among different types of ArsR. SmtB and CadC were added as an outgroup to construct the phylogenetic tree of ArsR. The alignment results showed that the ArsR transcriptional repressors from different sources were clustered into 5 different groups ( Figure 2A). SxArsR and similar ArsR transcriptional repressors with the conserved cysteine residue "CXC" structure are clustered in Group V and represent a novel type of ArsR transcriptional repressor not previously reported. Proteins similar to ArsR of Escherichia coli are clustered in Group I and have the conserved cysteine residue "CXCXXC" structure. Proteins similar to ArsR of Corynebacterium glutamicum are clustered in Group II and have the conserved cysteine residue "CCX(42-43)CXC" structure. Proteins similar to ArsR of Acidithiobacillus ferrooxidans are clustered in Group III and have the conserved cysteine residue "CCX(4-7)C" structure. Chr1_1105, Chr1_1360, and P4_10 in Sphingobium xenophagum C1 with the three-cysteine residue "CCXGXXXXC" structure are all clustered in Group III. Proteins similar to ArsR of Shewanella putrefaciens are clustered in Group IV and have the conserved cysteine residue "CX(17)CC" structure ( Figure 2 B). The phylogenetic relationship between SxArsR in Group V and ArsR in Group I is closer than that with the other three groups, indicating that SxArsR in Group V and ArsR in Group I share a more recent common ancestor than with ArsR in the other three groups. However, SxArsR lacks the third cysteine residue corresponding to Cys37 in ArsR of Group I Escherichia coli, suggesting that SxArsR lacks the third ligand required for high-affinity binding of arsenite. Figure 1 C also confirmed that SxArsR does not respond to the inorganic arsenic compounds NaAsO2 and Na3AsO4.

[0028] Arsenic has a high reactivity with the sulfhydryl groups of cysteine in the ArsR protein, forming a tricoordinated thioarsenite complex. The three cysteine residues in the amino acid sequence of ArsR in Group I Escherichia coli are the three ligand-binding sites for arsenite binding, consisting of Cys32, Cys34, and Cys37 at the N-terminus. The arsenite-binding sites in Group II Corynebacterium glutamicum are Cys15 and Cys16 on one subunit of ArsR and Cys55 on another subunit. The arsenite-binding site of ArsR in Group III Acidithiobacillus ferrooxidans is composed of Cys95, Cys96, and Cys102 at the C-terminus of ArsR. The ArsR in Group IV Shewanella putrefaciens has tricoordinated cysteine residues consisting of Cys83, Cys101, and Cys102, which are involved in the detoxification of methylarsenite and do not respond to arsenite. Group V SxArsR has only two conserved cysteine residues consisting of Cys29 and Cys31, and the deletion of the third conserved cysteine residue results in the loss of its high affinity for arsenite binding. The spatially distinct binding sites of SxArsR from the other four types of ArsR have evolved independently, indicating the plasticity of the regulatory sites of the ArsR family proteins and also fully demonstrating that SxArsR is a novel transcriptional repressor of the ArsR family.

[0029] Example 3: Analysis of the metal-binding site of the SxArsR protein and its high affinity for Sb2O3

[0030] Different from Group I ArsR, Group V SxArsR lacks the third cysteine residue Cys34 at its metal-binding site, which is replaced by a methionine residue Met34. In addition, before the first conserved cysteine residue Cys29 in the SxArsR protein is a histidine residue His28 instead of a leucine residue in the ArsR of Escherichia coli ( Figure 2C). The two amino acid residues His28 and Met34 likely play important roles in the high affinity of SxArsR for Sb2O3. However, there is another cysteine residue Cys97 at the C-terminus of SxArsR, which is not conserved in most group V SxArsR-like ArsRs, and its contribution to SxArsR binding to Sb2O3 is also unclear. To explore the mechanism of specific binding of SxArsR to Sb2O3, the codons of the amino acid residues His28, Met34, and Cys97 were mutated to Leu28, Cys34, and Ala97, respectively, generating 7 different SxArsR mutants, namely Chr1_1106-1 to Chr1_1106-7( Figure 3 A).

[0031] By synthesizing the gene at Sangon Biotech (Shanghai) Co., Ltd., the coding sequence (5′-GTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC-3′) of the firefly luciferase small peptide HiBiT provided by Promega was added to the 3′ end of the sxarsR mutant gene, and a fragment including the promoter sequence of about 500 bp upstream of each gene was synthesized. EcoRI and XhoI restriction enzyme sites were designed at both ends of the gene fragment. Each gene fragment and the pET24a expression vector were treated with TaKaRa's EcoRI and XhoI restriction enzymes in H buffer at 37 °C, and the corresponding gene fragment and the vector were ligated using the recombinase of TransGen Biotech Co., Ltd. in Beijing. The obtained recombinant vector was transformed into the competent cells of C1 bacteria by electroporation, and the transformed bacterial solution was finally spread on an LB medium plate containing 50 μg / mL kanamycin and cultured statically at 30 °C for 24 h. Monoclonal colonies grown on the plate were picked, and colony PCR was performed using the universal primers T7 (5′-TAATACGACTCACTATAGG-3′) and T7-TER (5′-GCTAGTTATTGCTCAGCGG-3′). After the electrophoresis of the amplified product was correct, the sample was sent for sequencing analysis. Finally, C1 bacterial sensing cells containing the p24a-1106-1-HiBiT to p24a-1106-7-HiBiT reporter vectors with correct sequences were obtained. The C1 bacterial sensing cells containing different reporter vectors were respectively inoculated into an inorganic salt medium containing 50 μg / mL kanamycin and cultured overnight at 30 °C in a shaker at a rotation speed of 200 rpm / min until the cell density OD 600The value is approximately 1.0. Different C1 sensing cell suspensions were inoculated into inorganic salt media supplemented with 0.5 μM of Sb2O3 and NaAsO2 respectively at an inoculation amount of 2% by volume. Then, they were cultured in a shaker at 200 rpm / min at 30 °C for 4 h until the OD 600 value reached 0.2. 100 μL of the sensing cell samples were taken for protein quantification analysis to ensure that the protein concentrations of all samples were basically the same. The remaining 100 μL of the sensing cell samples were added with 100 μL of intracellular lysis buffer (containing lysis solution, large subunit of firefly luciferase LgBiT, and luciferase luminescence substrate furimazine, Promega), and intracellular luciferase activity analysis was carried out on a chemiluminescence instrument.

[0032] As Figure 3As shown in Figure B, the bioluminescence signal induced by Sb2O3 was only detected in Chr1_1106 and its H28 mutants Chr1_1106-1, -2, and -5. Compared with the control without Sb2O3 induction, there was no significant difference in the bioluminescence signal of all H28L mutants, indicating that the His28 residue of SxArsR is necessary for its specific response to Sb2O3. Neither the H28L / M34C / C97 mutant Chr1_1106-6 nor the H28L / M34C / C97A mutant Chr1_1106-7 could produce bioluminescence signals in cells induced by NaAsO2 and Sb2O3. This shows that the binding site "CXCXXC" of Group I ArsR cannot bind to arsenic or antimony on SxArsR without the His28 residue. Compared with the H28 / M34 / C97A mutant Chr1_1106-1, the H28 / M34C / C97A mutant Chr1_1106-5 and the H28 / M34 / C97 wild-type Chr1_1106 both showed lower Sb2O3-induced bioluminescence signals, indicating that neither Met34 nor Cys97 was involved in the binding of SxArsR to Sb2O3. However, Cys34 and Cys97 could enhance the binding of the H28 / M34C / C97 mutant Chr1_1106-2 to Sb2O3 and NaAsO2. SxArsR lacks the third ligand-binding site Cys34 required for high-affinity binding of arsenite, but shows a high response to Sb2O3. The Met34 and Cys97 residues of SxArsR are not necessary for its binding to Sb2O3, while His28 located before Cys29 plays a key role in forming the "HCXC" binding site for specific response to Sb2O3. The His and Cys conserved amino acid residues crucially determine the specific binding affinity of SxArsR to Sb2O3. The "HCXC" domain in Group V SxArsR is a conserved binding site for Sb2O3, which is significantly different from the "CXCXXC" domain in the ArsR group of Group I.

[0033] Example 4: Construction and Performance Analysis of a Whole-Cell Microbial Sensor for Antimony Pollution Monitoring

[0034] Synthesize primer 1106U (5′- GAATTCCCGAACCCTTCAGCTTGCG-3′) and primer 1106D (5′-ATGTTTTTGGCGTCTTCCATGGCAGCCCTTTTCT-3′), using the genome of Sphingobium xenophagum C1 as a template, amplified the sxarsR gene (whose nucleotide sequence is shown in SEQ ID NO.1) and its upstream 506bp sequence (whose nucleotide sequence is shown in SEQ ID NO.2). Synthesized primer LucU1 (5′-GTCAGAAAAGGGCTGCCATGGAAGACGCCAAAAACA-3′) and primer LucD1 (5′- CTCGAG TTACACGGCGATCTTTCC-3′), using the pTAL-Luc plasmid (Clontech, catalog number 631909) as a template, amplified the complete sequence of the firefly luciferase luc gene (NCBI accession number No. AB909458.1). Used 1106U (5′-GAATTCCCGAACCCTTCAGCTTGCG-3′) and primer LucD1 (5′-CTCGAGTTACACGGCGATCTTTCC-3′) to perform PCR fusion on the amplified sxarsR and luc gene fragments, obtaining a fusion fragment sxarsR-luc containing EcoRI and XhoI restriction sites at both ends. Treated the fusion fragment sxarsR-luc and the pET24a expression vector with TaKaRa's EcoRI and XhoI restriction endonucleases in H buffer at 37°C, and used the recombinase of TransGen Biotech Co., Ltd. in Beijing to ligate the digested fragments with the vector. The obtained recombinant vector was transformed into the competent cells of Sphingobium xenophagum C1 and Escherichia coli DH5α by electroporation, and the transformed bacterial solution was finally spread on an LB medium plate containing 50 μg / mL kanamycin and cultured statically at 30°C for 24 h. Picked the monoclonal colonies grown on the plate and performed colony PCR using the universal primers T7 (5′-TAATACGACTCACTATAGG-3′) and T7-TER (5′-GCTAGTTATTGCTCAGCGG-3′). After the electrophoresis of the amplified product was correct, the sample was sent for sequencing analysis. Finally, the Sphingobium xenophagum C1 (p24a-SxArsR-Luc) and Escherichia coli DH5α (p24a-SxArsR-Luc) whole-cell microbial sensors with the correct sequence and containing the reporter vector were obtained.

[0035] The 506 bp sequence upstream of the sxarsR gene is shown in SEQ ID NO.2, specifically: CCGAACCCTTCAGCTTGCGGTTTCACAGCGAAGCCCCGCCATGAGGTCGGAGCACATTTCCGGCGCGCCCTGGCAGCAATCCTGCATCAGGAAGGTGAGCAGGCGGCGCATTGCGGCATAGTCGACGCGGTAATGGATCAGGCGGCTCTCGCGCTCGGACTGAACGAGGCCAGCCCGCTCTAGCGTCGCCAGATGGTGCGACATGGTCGAAGGCGGCACGCCAGCGCGTTCGGCAATGGCGCCCGCGACATGACCTTCGGGACCGGCTATCACCAGCATCCGAAACACGCTGAGCCGCGTCTCATGCGCAAGCGCGCCAAGTGCATCGACCGCCCAGACCTGTGTGGTCAGTTCATCCATGATTCGTCGATCCTTCCAGTTTCGTCGAATTATGGATCCCGGGACTTCGCGTCAATCGATGATTCGAGAATTATCGAACTAATGACTTTCGGTTGACCTGCGTCAAAGCGCATATGCGGGCAGGCGCATACGCAAGGTGCATGGAA。

[0036] The sensing cells C1 (p24a-SxArsR-Luc) and DH5α (p24a-SxArsR-Luc) were respectively inoculated into the inorganic salt medium and LB medium containing 50 μg / mL kanamycin, and cultured overnight at 30 °C in a shaker at a rotation speed of 200 rpm / min until the OD 600 value was approximately 1.0. According to the inoculation amount of 2% by volume, the bacterial suspensions of the sensing cells C1 (p24a-SxArsR-Luc) and DH5α (p24a-SxArsR-Luc) were respectively inoculated into the inorganic salt medium and LB medium supplemented with different concentrations of Sb2O3, and cultured at 30 °C in a shaker at a rotation speed of 200 rpm / min for 4 h and 1 h respectively until the OD 600 value of the bacterial cells reached 0.2. 100 μL of the sensing cell samples were respectively taken for protein quantitative analysis to ensure that the protein concentrations of all samples were basically the same. The remaining 100 μL of the sensing cell samples were added with 100 μL of intracellular lysis buffer (containing lysis solution, large subunit of firefly luciferase LgBiT and luciferase luminescence substrate furimazine, Promega), and the luciferase activity analysis was carried out on a chemiluminescence analyzer.

[0037] The sensitivity analysis results of the whole-cell microbial sensor showed that among the tested concentrations of Sb2O3, 6.0 μM of Sb2O3 induced the maximum fluorescence signal ( Figure 4 A, 4B). As the concentration of Sb2O3 increased, the fluorescence signal decreased at first, indicating that high concentrations of Sb2O3 were toxic to the sensing cells. The lowest detection limits of both the C1(p24a-SxArsR-Luc) and DH5α(p24a-SxArsR-Luc) whole-cell microbial sensors were 0.01 μM, which was approximately 2.44 μg / L of antimony. By performing a correlation analysis on the Sb2O3 concentration and the fluorescence signal between 0.01 and 6.0 μM, it was found that the correlation index R 2 was 0.9925 in C1(p24a-SxArsR-Luc) ( Figure 4 A), and 0.9948 in DH5α(p24a-SxArsR-Luc) ( Figure 4 B). SxArsR is a novel antimony-selective ArsR family transcriptional repressor. The C1(p24a-SxArsR-Luc) and DH5α(p24a-SxArsR-Luc) whole-cell microbial sensors constructed based on SxArsR showed specific and sensitive responses to Sb2O3. The linear range, detection limit, cultivation time, etc. of the whole-cell microbial sensor based on SxArsR were comparable to existing analytical methods. For example, the detection limit of the whole-cell biosensor based on Escherichia coli ArsR for arsenite was 1 μg / L - 10 mg / L, and the typical detection time was 30 minutes to several hours. The response time of the whole-cell microbial sensor based on Aspergillus niger AcrA was significantly slower, being 12 hours, and the detection limit was 20 μg / L of arsenite. Generally speaking, the SxArsR whole-cell microbial sensor has the advantages of strong specificity, fast reaction, and good detection limit, laying a foundation for the future application of ArsR biosensors in the monitoring of arsenic and antimony dual signals.

[0038] The above are only the preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be defined by the scope of the claims. For those of ordinary skill in the art of this technology, without departing from the spirit and scope of the present invention, several improvements and refinements can also be made, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. Use of the antimony-selective SxArsR protein in specifically recognizing antimony trioxide, wherein the amino acid sequence of the antimony-selective SxArsR protein is as shown in SEQ ID NO.

3.

2. The application according to claim 1, wherein Use of the antimony-selective SxArsR protein in preparing a whole-cell microbial sensor for monitoring antimony pollutants, wherein the antimony pollutant is antimony trioxide.

3. The application according to claim 2, wherein It is to express the antimony-selective SxArsR protein in chassis cells to obtain a whole-cell microbial sensor.

4. The application according to claim 3, wherein It is to fuse the antimony-selective SxArsR protein with luciferase and then express it in chassis cells to obtain a whole-cell microbial sensor.

5. The application according to claim 2, 3 or 4, characterized in that, The whole cells are Sphingobium xenophagum C1 and Escherichia coli DH5α.

6. The application according to claim 2, wherein Using primer 1106U: 5′-GAATTCCCGAACCCTTCAGCTTGCG-3′ and primer 1106D: 5′-ATGTTTTTGGCGTCTTCCATGGCAGCCCTTTTCT-3′, the sxarsR gene and its upstream sequence were amplified using the genome of Sphingobium xenophagum C1 as a template; using primer LucU1: 5′-GTCAGAAAAGGGCTGCCATGGAAGACGCCAAAAACA-3′ and primer LucD1: 5′- CTCGAG TTACACGGCGATCTTTCC-3′, the complete sequence of the firefly luciferase luc gene was amplified using the pTAL-Luc plasmid as a template; the two amplified gene fragments of sxarsR and luc were PCR fused using primer 1106U: 5′-GAATTCCCGAACCCTTCAGCTTGCG-3′ and primer LucD1: 5′-CTCGAGTTACACGGCGATCTTTCC-3′ to obtain a fusion fragment sxarsR-luc containing EcoRI and XhoI restriction sites at both ends. After digestion with the restriction enzymes EcoRI and XhoI, it was ligated to the pET24a vector. The recombinant vector was transformed into Sphingobium xenophagum C1 and Escherichia coli DH5α respectively, thereby obtaining a whole-cell microbial sensor for antimony pollutant monitoring.

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