A BRET-based phosphate biosensor, its construction method, and its application.

Abstract

The present invention discloses a phosphate biosensor based on BRET, a construction method and an application thereof. The biosensor is a fusion protein obtained by fusing a phosphate-binding protein Pa PBP of Pseudomonas aeruginosa that specifically responds to phosphate, a nanoluciferase NanoLuc and a yellow fluorescent protein VenusΔC10, and the amino acid sequence is shown in SEQ ID NO.2; wherein the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa, the nanoluciferase NanoLuc and the yellow fluorescent protein VenusΔC10 are all connected by a flexible peptide linker, and the amino acid sequence of the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa is shown in SEQ ID NO.1; the biosensor constructed by the present invention has extremely strong selectivity for phosphate, and can convert a phosphate concentration signal into a ratio fluorescence signal output by coupling a bioluminescence resonance energy transfer technology, thereby realizing rapid, sensitive and high-throughput detection of phosphate, and the biosensor is suitable for detecting phosphate in different environmental water bodies such as tap water, surface water and urban sewage.

Description

Technical Field

[0001] The invention belongs to the field of environmental biology, and particularly relates to a BRET-based phosphate biosensor, a construction method and an application thereof. Background Art

[0002] Phosphorus is one of the main factors leading to eutrophication of water bodies. With the increasing phosphorus discharge from industry and agriculture, the water environment has been threatened. It is urgent to establish a rapid, sensitive and high-throughput method for the phosphoric acid concentration in water bodies to improve the efficiency of environmental monitoring and more effectively prevent the hazards of eutrophication. However, the existing phosphate detection methods have many shortcomings: traditional colorimetric analysis is insensitive to low-concentration samples, time-consuming and uses toxic chemical reagents; chromatographic analysis requires expensive equipment and professional operation; electrochemical sensors are easily seriously interfered by impurities; nanoenzyme sensors are prone to introduce toxic pollutants. Therefore, it is necessary to develop a new method for phosphate detection that is highly specific, rapid, high-throughput and environmentally friendly.

[0003] In recent years, biosensors based on bioluminescence-resonance-energy transfer (BRET) have been widely used in medicine, food and other fields due to their high sensitivity and the ability to avoid photobleaching and autofluorescence. BRET biosensors are composed of sensitive (recognition) elements that specifically bind to ligands, bioluminescence donors and fluorescent receptors. Since conformational changes occur after the biosensor binds to the ligand, affecting the optical properties of the biosensor, the identification and detection of ligand compounds can be achieved by establishing a correlation between the optical properties and the ligand concentration.

[0004] In bacteria, the phosphate specific transport system (Pst) is a key system for the absorption and transport of inorganic phosphate (Pi). The Pst system is generally composed of five proteins: PstS, PstC, PstA, PstB and PhoU. Among them, the phosphate binding protein PstS is a type II periplasmic binding protein. This protein has a very high affinity for inorganic phosphate (Pi) and is responsible for sensing and binding Pi in the periplasmic space; the protein includes two similar globular domains and a phosphate binding site. Among them, the two globular domains are the basic skeleton of the PstS protein, connected by a double hinge in the middle. The phosphate binding site is the active center of the PstS protein, located in the middle (gap) of the two globular domains. The binding site consists of 10 amino acids and is connected to the N-terminus of the 1,2,6-α helix in the globular domain. It can bind to non-solvated Pi through hydrogen bonds and hydrophobic interactions. When it binds to Pi (ligand), it undergoes a large conformational change, so it can be used as a sensitive element of the phosphate biosensor. The structures of PstS proteins of different strains are roughly the same, and the main difference lies in the presence or absence of a ring structure at the N-terminus of the PstS protein. Related studies have shown that the N-terminus of the phosphate-binding protein PstS of Pseudomonas aeruginosa (PA) contains this ring structure.

[0005] NanoLuc is an engineered luciferase from deep-sea shrimp. It is a small (19 kDa), bright but stable protein that produces enhanced and sustained luminescence compared to other luciferases. Therefore, NanoLuc can be used as a bioluminescent donor for the construction of BRET biosensors. Summary of the invention

[0006] The present invention aims to provide a phosphate biosensor based on BRET, a construction method and an application thereof by designing a phosphate biosensor. By using the phosphate binding protein PstS of Pseudomonas aeruginosa as a sensitive element, nanoluciferase NanoLuc as a bioluminescence donor, and yellow fluorescent protein VenusΔC10 as a fluorescence receptor, a biosensor for detecting phosphate in water is constructed. Nanoluciferase NanoLuc can catalyze the substrate furazine to produce 460nm bioluminescence, and excite the yellow fluorescent protein VenusΔC10 to produce 528nm emission light through resonance energy transfer. When the biosensor is bound to phosphate, a conformational change occurs, resulting in a change in the BRET ratio of the nanoluciferase NanoLuc and the yellow fluorescent protein VenusΔC10, and the value of the BRET ratio change is linearly correlated with the concentration of the bound phosphate, thereby achieving accurate detection of phosphate, thereby solving the defects of the existing phosphate detection method, such as large chemical reagent pollution, long reaction time, complicated operation steps, and low sensitivity.

[0007] In order to achieve the above object, the technical solution of the present invention is as follows:

[0008] A phosphate biosensor based on BRET, wherein the biosensor is a fusion protein obtained by fusing a phosphate-binding protein Pa PBP of Pseudomonas aeruginosa that specifically responds to phosphate, a nanoluciferase NanoLuc and a yellow fluorescent protein VenusΔC10, and the amino acid sequence is shown in SEQ ID NO.2; wherein the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa, the nanoluciferase NanoLuc and the yellow fluorescent protein VenusΔC10 are all connected by a flexible peptide linker, the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa is selected from the periplasmic subunit of the Pseudomonas aeruginosa phosphate special transport system Pst, the periplasmic leader sequence is removed, and the amino acid sequence is shown in SEQ ID NO.1; and the phosphate biosensor is named VenusΔC10-Pa PBP-Nluc.

[0009] Preferably, the amino acid sequence of the flexible peptide linker is selected from (Ast C., et al., Nat Commun, 2017, 8, 431), and the sequence is GlyGlyThr-GlyGlySer, i.e. GGT-GGS.

[0010] Preferably, the yellow fluorescent protein VenusΔC10 is selected from (Dippel, AB, et al., ACS Chem Biol, 2020, 15, 904), and its amino acid sequence is shown in SEQ ID NO.3; the nanoluciferase NanoLuc is selected from (Hall, MP, et al., ACS Chem Biol, 2012, 7, 1848), and its amino acid sequence is shown in SEQ ID NO.4.

[0011] The second object of the present invention is to provide a method for constructing the above-mentioned BRET-based phosphate biosensor, comprising the following steps:

[0012] S1. Synthesis of plasmids: Full gene synthesis of yellow fluorescent protein VenusΔC10 and nanoluciferase NanoLuc connected by GGT-GGS flexible linker, using VenusΔC10-GGT-GGS-Nluc as template, PCR gene amplification, and cloning into pET-24b(+) vector to obtain recombinant plasmid pET-VenusΔC10-Nluc; full gene synthesis of Pseudomonas aeruginosa phosphate binding protein PstS, using it as a template, PCR gene amplification, and inserting it between the pET-VenusΔC10-Nluc flexible linker GGT and GGS to obtain recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc;

[0013] S2, transformation and expression: The recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc constructed in step S1 was transformed into the expression vector Escherichia coli BL21 (DE3) strain, and spread on a kanamycin-resistant LB plate for culture, and colonies were grown, each of which was a single clone. The monoclonal strain obtained by culture was selected and inoculated into a kanamycin-resistant LB liquid culture medium, and cultured until OD600 = 0.6-1.0. After IPTG induction, the expression was centrifuged to collect the bacteria;

[0014] S3, lysis and purification: The bacteria collected in step S2 are resuspended in TBS buffer, PMSF and lysozyme are added thereto, ultrasonic lysis is performed, the supernatant is collected by centrifugation, purified, and stored to obtain the biosensor protein.

[0015] Preferably, in step S2, the mass volume concentration of the kanamycin-resistant LB solid culture medium is 100 μg / mL, and the mass volume concentration of the kanamycin-resistant LB liquid culture medium is 50 μg / mL; the induction expression conditions are: 0.1-1 mM IPTG, temperature of 16-30°C, and rotation speed of 100-200 rpm; the culture conditions in the kanamycin-resistant LB liquid culture medium are: temperature of 30-37°C, and rotation speed of 100-200 rpm.

[0016] Preferably, in step S3, the pyrolysis conditions are: pyrolysis temperature is 0-4°C, power is 100-150W, and pyrolysis time is 15-30min.

[0017] The third object of the present invention is to provide the application of the BRET-based phosphate biosensor in the detection of phosphate in water.

[0018] Preferably, the method of applying phosphate biosensor to phosphate detection in water comprises the following steps:

[0019] A1. Collect water samples: Take samples of the water to be tested to obtain water samples, then add a pH adjuster to the obtained water samples to adjust the pH value to 6-8, filter, and obtain the water sample to be tested;

[0020] A2. Prepare detection buffer, furazolidone working solution, phosphate standard stock solution, phosphate standard working solution and biosensor protein solution for later use;

[0021] A3. Preparation of phosphate standard solution: pipette different volumes of phosphate standard stock solution and phosphate standard working solution into centrifuge tubes respectively, and dilute with ultrapure water to prepare phosphate series standard solutions with concentrations of 0.0001 mg / mL, 0.0005 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 5.00 mg / mL, 10.00 mg / mL, and 50.00 mg / mL;

[0022] A4. Drawing a standard curve: placing the phosphate series standard solutions prepared in step A2 in ELISA plates respectively, adding the biosensor protein solution thereto, and incubating at room temperature for 8 to 12 minutes, then adding the furazolidone working solution to obtain a bioluminescence resonance energy transfer detection system, and measuring the luminescence intensity BL in a dual-wavelength luminescence measurement mode, wherein the dual-wavelength parameters are set to 460 / 40 nm and 528 / 20 nm, respectively, and then calculating the BRET ratio according to the following formula (1), and then drawing a standard curve with the BRET ratio as the ordinate and the concentration of the phosphate standard solution as the abscissa;

[0023]

[0024] The drawn curve is subjected to nonlinear fitting to obtain the fitting equation, as shown in the following formula (2):

[0025]

[0026] Where, BRET maxis the maximum value of the BRET ratio, BRET min is the minimum value of the BRET ratio, x is the concentration of the phosphate solution, x0 is the fitting parameter, h is the Hill slope, and s is the control factor;

[0027] A5. Obtaining the phosphate concentration in the water sample to be tested: Substituting the phosphate standard solution for the water sample to be tested, repeating step A3 to obtain a bioluminescence resonance energy transfer detection system, measuring the luminescence intensity in the dual-wavelength luminescence measurement mode, and calculating the phosphate concentration in the water sample to be tested by formula (1) and formula (2).

[0028] Preferably, in step A2, the detection buffer is obtained by mixing 10-100 mM Tris and 50-250 mM NaCl, and the pH is 7.4-8.0; the working concentration of the biosensor protein solution is 1-20 nM, and the working concentration of the furazolidone solution is 10-20 μM.

[0029] In the bioluminescence resonance energy transfer detection system, the volume ratio of the phosphate standard solution, the biosensor protein solution and the furazolidone working solution is 20 μL:80 μL:20 μL.

[0030] Preferably, step A1 further comprises A1′, using an oxidant to perform microwave digestion on the water sample to be tested. This step is applicable to the determination of the total phosphorus content in the water sample. If only the inorganic phosphate content in the water sample is to be determined, this step is omitted;

[0031] Preferably, the water sample includes tap water, surface water and urban sewage.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention constructs a biosensor VenusΔC10-Pa PBP-Nluc by inserting the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa that specifically responds to phosphate between the yellow fluorescent protein VenusΔC10 and the nanoluciferase NanoLuc. The biosensor has extremely strong selectivity for phosphate. By coupling the bioluminescence resonance energy transfer technology, the phosphate concentration signal can be converted into a ratio fluorescence signal output, thereby realizing rapid and sensitive detection of phosphate in water. The detection result has a better detection limit and detection range than the ammonium molybdate spectrophotometry method, that is, the phosphate biosensor constructed by the present invention has higher accuracy in detecting phosphate in water. In addition, the optimization of the phosphate biosensor on a 96-well plate shows that the phosphate biosensor method of the present invention also has the ability to achieve high-throughput detection in a short time.

[0034] (2) In the present invention, through the spike recovery experiment, the results show that the phosphate biosensor constructed by the present invention is suitable for the detection of phosphate in different environmental water bodies such as tap water, surface water (river water) and urban sewage (simulated wastewater). BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0036] Figure 1 A schematic diagram of the recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc constructed in the present invention;

[0037] Figure 2 Schematic diagram of the SDS-PAGE verification results of the expression and purification of the biosensor VenusΔC10-Pa PBP-Nluc;

[0038] Figure 3 Schematic diagram of homology modeling and molecular docking results of the biosensor VenusΔC10-Pa PBP-Nluc;

[0039] Figure 4 Schematic diagram of the principle of the biosensor VenusΔC10-Pa PBP-Nluc constructed in the present invention;

[0040] Figure 5 is the standard curve of the biosensor VenusΔC10-Pa PBP-Nluc for phosphate;

[0041] Figure 6 A schematic diagram of the selectivity analysis results of the biosensor VenusΔC10-Pa PBP-Nluc constructed in the present invention;

[0042] Figure 7 The standard curve obtained when the biosensor VenusΔC10-Pa PBP-Nluc detects phosphate in tap water samples in Example 3;

[0043] Figure 8 The standard curve obtained when the biosensor VenusΔC10-Pa PBP-Nluc detects phosphate in river water samples in Example 4;

[0044] Fig. 9 This is the standard curve obtained when the biosensor VenusΔC10-Pa PBP-Nluc in Example 5 detects phosphate in a simulated wastewater sample. DETAILED DESCRIPTION

[0045] In the following description, for the purpose of illustration rather than limitation, specific details such as specific system structures and technologies are provided to provide a thorough understanding of the embodiments of the present invention. However, it should be clear to those skilled in the art that the present invention may also be implemented in other embodiments without these specific details.

[0046] Example 1

[0047] A phosphate sensor based on BRET is a fusion protein obtained by fusing a phosphate-specific response Pseudomonas aeruginosa phosphate-binding protein Pa PBP, a nanoluciferase NanoLuc and a yellow fluorescent protein VenusΔC10, and the amino acid sequence is shown in SEQ ID NO.2; wherein, both ends of the NC of the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa are connected to the nanoluciferase NanoLuc and the yellow fluorescent protein VenusΔC10 through a flexible peptide linker, the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa is selected from the periplasmic subunit of the Pseudomonas aeruginosa phosphate special transport system Pst, the periplasmic leader sequence is removed, and the amino acid sequence is shown in SEQ ID NO.1, the flexible peptide linker is selected from (Ast C., et al., Nat Commun, 2017, 8, 431), and its amino acid sequence is GGT-GGS; the yellow fluorescent protein VenusΔC10 is selected from (Dippel, AB, et al., ACSChem Biol, 2020, 15, 904), and its amino acid sequence is shown in SEQ ID NO.3; the nanoluciferase NanoLuc is selected from (Hall, MP, et al., ACS Chem Biol, 2012, 7, 1848), and its amino acid sequence is shown in SEQ ID NO.4; and the above phosphate biosensor is named VenusΔC10-Pa PBP-Nluc;

[0048] The method for constructing the BRET-based phosphate biosensor comprises the following steps:

[0049] S1. Plasmid synthesis: The yellow fluorescent protein VenusΔC10 and the nanoluciferase NanoLuc connected by the GGT-GGS flexible linker were fully synthesized, and VenusΔC10-GGT-GGS-Nluc was used as a template for PCR gene amplification and cloned into the pET-24b(+) vector to obtain the recombinant plasmid pET-VenusΔC10-Nluc; the sequence of the phosphate binding protein of Pseudomonas aeruginosa (Pseudomonas aeruginosa, PDB ID: 4omb) was obtained from the PDB database, and its periplasmic signal sequence was deleted. The phosphate binding protein PstS of Pseudomonas aeruginosa was fully synthesized, and it was used as a template for PCR amplification and inserted between the flexible linkers GGT and GGS of pET-VenusΔC10-Nluc to obtain the recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc. The sequence was verified by sequencing and stored in the E. coli Top10 strain to obtain the recombinant plasmid pET-VenusΔC10-Pa The map of PBP-Nluc is as follows Figure 1 As shown;

[0050] S2. Transformation and expression: The recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc constructed in step S1 was transformed into the expression vector Escherichia coli BL21 (DE3) strain, and spread on a 100 μg / mL kanamycin-resistant LB plate for culture. After colonies grow, each colony is a single clone. The monoclonal strain obtained by culture is selected and inoculated into a 50 μg / mL kanamycin-resistant LB liquid medium, and cultured at 37°C and 150 rpm until the OD 600 = 0.6-1.0, after inducing expression with 0.5 mM IPTG at 25°C, 150 rpm for 4 h, the bacteria were collected by centrifugation at 5000 g, 4°C;

[0051] S3, lysis and purification: The bacteria collected in step S2 were resuspended in TBS buffer (0.05 M, pH 7.4), 1 mM PMSF and 1 mg / mL lysozyme were added in sequence, and ultrasonic lysis was performed at a temperature of 4°C and a power of 120 W for 15 min, and then centrifuged at a speed of 10000 rpm, the supernatant was collected, and then purified by a nickel column (Ni-NTA) to obtain the phosphate biosensor protein, and the expression and purification of the biosensor protein were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as shown in FIG. Figure 2 , which is a schematic diagram of the SDS-PAGE verification results of the expression and purification of the biosensor VenusΔC10-Pa PBP-Nluc; Figure 2As a result, the molecular weight of the biosensor protein constructed in this example is about 80 kDa as expected. Finally, the phosphate biosensor protein is dialyzed into a storage buffer (25 mM Tris, 250 mM NaCl) using an ultrafiltration centrifuge tube (molecular weight cutoff 30 kDa), and the biosensor protein concentration is quantified by the NanoDrop One (Thermo Scientific) A280 method and stored at -80°C.

[0052] The structure and key amino acids of the biosensor VenusΔC10-Pa PBP-Nluc constructed above are described. Specifically, multi-template homology modeling of the biosensor VenusΔC10-Pa PBP-Nluc above is performed using the Phyre2 server, and the modeling results are verified (Ramachandran plot, Z-Score, etc.) to determine that the model meets the requirements of further research, and molecular docking is performed using Auto Dock Vina to obtain the key amino acids for interaction of the binding pocket, such as Figure 3 Shown is a schematic diagram of the homology modeling and molecular docking results of the biosensor VenusΔC10-Pa PBP-Nluc.

[0053] The above-mentioned BRET-based biosensor VenusΔC10-Pa PBP-Nluc, its principle is as follows Figure 4 As shown, since the emission spectrum of the nanoluciferase NanoLuc overlaps well with the excitation spectrum of the yellow fluorescent protein VenusΔC10, effective energy transfer can be generated. When the constructed biosensor VenusΔC10-Pa PBP-Nluc binds to phosphate, the distance between the nanoluciferase NanoLuc and the yellow fluorescent protein VenusΔC10 will be shortened or the angle will rotate, that is, the conformation will change, resulting in a change in the BRET ratio of NanoLuc and VenusΔC10, and the more phosphates are bound, the greater the change in the BRET ratio is, until saturation. Specifically, the nanoluciferase NanoLuc can catalyze the substrate furazine to produce 450nm bioluminescence, and excite the yellow fluorescent protein VenusΔC10 to produce 528nm emission light through resonance energy transfer. When the phosphate biosensor binds to phosphate, the BRET ratio of the nanoluciferase NanoLuc and the yellow fluorescent protein VenusΔC10 changes, and the value of the change in the BRET ratio is linearly related to the phosphate concentration, thereby achieving accurate detection of the phosphate concentration.

[0054] In view of this, the phosphate biosensor VenusΔC10-Pa PBP-Nluc constructed above was applied to detect phosphate in water, including the following steps:

[0055] A1. Collect water samples: Collect 100 mL of water sample in a sampling bottle (for water samples with low phosphorus content, avoid using plastic bottles for sampling, because phosphate is easily adsorbed on the wall of plastic bottles) to obtain water samples. Use 1M NaOH or HCl to adjust the pH value to 6-8, and then use a syringe filter with a pore size of 0.45 μm (water system, PES) to filter to remove suspended particles in the sample and store it at 4°C.

[0056] A2. Reagent preparation:

[0057] Assay buffer (25 mM Tris, 250 mM NaCl): weigh 7.3050 g NaCl and dissolve it in 12.50 mL Tris-HCl buffer (1 M, pH 7.4), and make up to volume in a 1000 mL volumetric flask with ultrapure water (18.2 MΩ·cm), and store at 4°C;

[0058] Furazolidone stock solution (1 mM): Weigh 0.50 mg of furazolidone, dissolve in 1.3110 mL of dimethyl sulfoxide, and store at -20°C.

[0059] Furazolidone working solution (20 μM): Take out the stored furazolidone stock solution and slowly dissolve it at room temperature. Take 24 μL of furazolidone stock solution and dilute it into 1200 μL MES buffer (100 mM, pH 6.0). This solution is prepared and used immediately.

[0060] Biosensor protein solution (5 nM): Take out the above-preserved biosensor protein solution (2 μM) and place it in a 4°C ice bath to slowly dissolve. Use the detection buffer to dilute the sensor protein 400 times. The prepared volume should be greater than 10 mL. Store it in a 4°C ice bath and use it immediately after preparation.

[0061] Phosphate standard stock solution (50 mg / L): Take an appropriate amount of potassium dihydrogen phosphate (KH2PO4) and dry it at 110℃ for 2h. After cooling in a desiccator, accurately weigh 0.2197±0.001g, dissolve it in ultrapure water and make up to volume in a 1000mL volumetric flask;

[0062] Phosphate standard working solution (50 μg / L): transfer 1.0 mL of phosphate standard stock solution to a 1000 mL volumetric flask, dilute to the mark with ultrapure water and mix well. This solution should be used immediately after preparation.

[0063] A3. Prepare phosphate standard solution:

[0064] Different volumes of phosphate standard stock solution and phosphate standard working solution were respectively pipetted into 36 10 mL centrifuge tubes, and diluted with ultrapure water to prepare phosphate series standard solutions with concentrations of 0.0001 mg / mL, 0.0005 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 5.00 mg / mL, 10.00 mg / mL, and 50.00 mg / mL (three parallel groups were set for each concentration group), as shown in Table 1 below:

[0065] Table 1 Phosphate standard solution

[0066] Tube No. 1 2 3 4 5 6 7 8 9 10 11 12 <![CDATA[Phosphate standard stock solution ( m L)]]> 0 0 0 0 0 0 0.02 0.1 0.2 1 2 10 <![CDATA[Phosphate standard stock solution ( m L)]]> 0.02 0.1 0.2 1 2 10 0 0 0 0 0 0 <![CDATA[Ultra-pure water ( m L)]]> 9.98 9.9 9.8 9 8 0 9.98 9.9 9.8 9 8 0 Phosphate standard solution concentration (mg / L) 0.0001 0.0005 0.001 0.005 0.01 0.05 0.1 0.5 1 5 10 50

[0067] A4. Draw a standard curve: Weigh 20 μL of the phosphate series standard solutions prepared in step A2 (three parallel groups for each concentration) into a 96-well all-black ELISA plate, add 80 μL of the biosensor protein solution thereto, and incubate at room temperature for 10 min, then add 20 μL of furazolidone solution to obtain a bioluminescent resonance energy transfer detection system, and use a blank solution without adding a phosphate standard solution, and use an ELISA reader to measure the luminescence intensity at 460 / 40 nm and 528 / 20 nm, and then calculate the BRET ratio according to the following formula (1), and then use the BRET ratio as the ordinate and the concentration of the phosphate standard solution as the abscissa to draw a standard curve, i.e., the standard curve of the biosensor VenusΔC10-Pa PBP-Nluc for phosphate, as shown in FIG. Figure 5 As shown;

[0068]

[0069] The drawn curve is subjected to nonlinear fitting to obtain the fitting equation, as shown in the following formula (2):

[0070]

[0071] Where, BRET max is the maximum value of the BRET ratio, BRET min is the minimum value of the BRET ratio, x is the concentration of the phosphate solution, x0 is the fitting parameter, h is the Hill slope, and s is the control factor;

[0072] ΔBRET was calculated according to the following formula (3), which represents the dynamic range of the biosensor caused by the phosphate ligand:

[0073]

[0074] Wherein, BRET0 represents the BRET ratio measured without adding phosphate (blank), BRET 100 represents the BRET ratio measured by adding a phosphate standard solution, and the ΔBRET calculated to be 25.1% indicates that the BRET ratio of the biosensor VenusΔC10-Pa PBP-Nluc constructed in the present invention changes significantly in response to an increase in phosphate concentration;

[0075] EC50 was calculated according to the following formula (4), where EC50 represents the phosphate concentration corresponding to a 50% change in BRET ratio:

[0076]

[0077] The EC50 was 355.91 nM. Since the EC50 and the apparent dissociation constant Kd were the same, the apparent dissociation constant Kd was 355.91, indicating that the biosensor VenusΔC10-Pa PBP-Nluc constructed by the present invention had a strong affinity for phosphate. Figure 5 As a result, it can be further obtained that the detection limit and detection range of the biosensor VenusΔC10-Pa PBP-Nluc for detecting phosphate are 0.0048 mg / L and 0.0096-0.3700 mg / L, respectively. Compared with the detection limit (0.01 mg / L) and the upper detection limit (0.6 mg / L) in the national standard "GB 11893-89" sodium molybdate spectrophotometry, the biosensor method of the present invention has a better detection limit and detection range for detecting phosphate. In addition, in this embodiment, a 96-well ELISA plate is used for measurement. When 3 groups of parallel water samples are set for each group, a 10-min plate reading can measure 20 water samples, and it has the ability to achieve high-throughput detection in a short time.

[0078] Example 2

[0079] The difference from Example 1 is that

[0080] Selectivity analysis of the biosensor VenusΔC10-Pa PBP-Nluc constructed in Example 1:

[0081] (I) Preparation of interference solution: Select anions that coexist with phosphate in water or are prone to interference, that is, prepare NO3 with a mass volume concentration of 10 mg / L - 、NO2 - 、SO4 2- HCOO - 、CH3COO - , Cl - 、F - and 1mg / L AsO4 3-, and prepare 10mg / L PO4 3- Solution;

[0082] (II) Determination of the dynamic range ΔBRET of the biosensor VenusΔC10-Pa PBP-Nluc: The biosensor VenusΔC10-Pa PBP-Nluc protein was diluted to 5 nM using a detection buffer (25 mM Tris, 250 mM NaCl) to obtain a biosensor protein solution. Then, 20 μM furazolidinone working solution was prepared using 0.1 M MES buffer. 80 μL of the above biosensor protein solution was added to a black 96-well plate and incubated with 20 μL of the above interfering anion solution and PO4 3- The solution was mixed and then incubated at room temperature for 10 min, then 20 μL of 20 μM furazolidinone working solution was added, and no interfering ions or PO4 3- As a blank solution, the luminescence intensity at 460 / 40 nm and 528 / 20 nm was measured using a Synergy H1 (Biotek) microplate reader (three parallel samples were set), and then the percentage of BRET ratio change, i.e., interference ΔBRET, was calculated according to formula (1) and the following formula (5). Interference ΔBRET represents the dynamic range of the biosensor response caused by interfering ions;

[0083]

[0084] Where BRET0 is the BRET ratio measured in the blank solution, BRET 100 Without adding PO4 3- BRET ratio measured in solution or interfering ion solution;

[0085] (III) Result analysis: Figure 6 As shown, the above eight interfering anions and PO4 3- Schematic diagram of the change in dynamic range of the biosensor VenusΔC10-Pa PBP-Nluc caused by Figure 6 It can be seen that due to the interfering ion AsO4 3- Biosensor VenusΔC10-Pa PBP-Nluc target molecule PO4 3- The structure is very similar, so at 10mg / L AsO4 3- The biosensor VenusΔC10-Pa PBP-Nluc produced an obvious signal response under the action of AsO4 3- The affinity is much smaller than PO4 3- Therefore, in the range of 0.001~1mg / L, AsO4 3-The presence of will not interfere with the biosensor detection of inorganic phosphate (Pi), while other anions will not interfere at all, indicating that the biosensor VenusΔC10-Pa PBP-Nluc constructed by the present invention has excellent selectivity.

[0086] Example 3

[0087] The difference from Example 2 is that:

[0088] The recovery experiment of the spiked phosphate in tap water samples detected by the biosensor VenusΔC10-Pa PBP-Nluc includes the following steps:

[0089] (I) Preparation of water samples to be tested: An appropriate amount of tap water from Nanjing was collected and filtered using a syringe filter (water system, PES) with a pore size of 0.45 μm to remove suspended particles in the sample. Phosphate (Pi) standard solutions with final concentrations of 0, 0.04 mg / L, 0.2 mg / L, and 0.6 mg / L were added to the treated water sample to obtain unspiked (blank) tap water samples and spiked series tap water samples, and three groups of parallel samples were set up;

[0090] (II) Determination of recovery rate of spiked tap water: The biosensor protein was diluted to 5 nM using detection buffer (25 mM Tris, 250 mM NaCl) to obtain a biosensor protein solution. Then, 20 μM furazolidone working solution was prepared in 0.1 M MES buffer. 80 μL of the above biosensor protein solution was added to a black 96-well plate and mixed with 20 μL of gradient concentration phosphate working solution (0.001 mg / L, 0.005 mg / L, 0.01 mg / L, 0.02 mg / L, 0.08 mg / L, 0.1 mg / L, 0.4 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, prepared with tap water, 3 groups in parallel) and unspiked (blank) tap water samples and spiked series tap water samples. After incubation at room temperature for 10 min, 20 μL of 20 μM furazolidone working solution was added and the plates were incubated using Synergy The luminescence intensity at 460 / 40 nm and 528 / 20 nm was measured by H1 (Biotek) microplate reader, and the BRET ratio was calculated according to formula (1). The standard curve was drawn with the BRET ratio as the ordinate and the concentration of the phosphate (Pi) working solution as the abscissa, as shown in the figure: Figure 7 As shown, the BRET ratio of the unspiked (blank) tap water sample and the BRET ratio of the spiked tap water sample are then calculated, and the obtained BRET ratio is substituted into the fitted Logistic5 parameter equation (see Formula 2) to obtain the concentration x0 of phosphate in the unspiked tap water sample and the concentration x of phosphate in the spiked tap water sample;

[0091] The recovery rate of phosphate in tap water detected by phosphate biosensor method was calculated according to the following formula (6):

[0092]

[0093] In the formula, x is the concentration of phosphate in the tap water sample with spikes, x0 is the concentration of phosphate in the tap water sample without spikes, and x 加标 is the concentration of the added phosphate standard solution;

[0094] (III) Experimental results analysis: Figure 7 It can be seen that by respectively measuring the unspecified (blank) tap water samples and the spiked series of tap water samples to which 0, 0.04 mg / L, 0.2 mg / L and 0.6 mg / L of phosphate standard solutions were added, the results obtained were 0.001 mg / L, 0.0542 mg / L, 0.1493 mg / L and 0.5429 mg / L, respectively, and the recovery rate was between 74.65% and 135.5%, thereby confirming that the biosensor VenusΔC10-Pa PBP-Nluc constructed by the present invention can be used for the detection of phosphate in tap water.

[0095] Example 4

[0096] The difference from Example 2 is that:

[0097] The recovery experiment of the biosensor VenusΔC10-Pa PBP-Nluc for detecting phosphate in river water samples includes the following steps:

[0098] (I) Preparation of spiked water samples: An appropriate amount of water from the Tianlai River of Nanjing University was collected and filtered using a syringe filter (water system, PES) with a pore size of 0.45 μm to remove suspended particles in the sample; phosphate standard solutions with final concentrations of 0, 0.04, 0.12, and 0.36 mg / L were added to the treated water samples to obtain unspiked (blank) river water samples and spiked series river water samples, and three sets of parallel samples were set up;

[0099] (II) Determination of recovery rate of spiked river water: This step is the same as in Example 3, and the standard curve is drawn as shown in Figure 8 As shown, the recovery rate of phosphate in river water detected by phosphate biosensor method is calculated according to formula (6);

[0100] (III) Result analysis: Figure 8It can be seen that by respectively measuring the unspecified (blank) river water samples and the spiked series of river water samples to which 0, 0.04 mg / L, 0.2 mg / L and 0.6 mg / L of phosphate standard solutions were added, the results obtained were 0.0087 mg / L, 0.057 mg / L, 0.1439 mg / L and 0.3323 mg / L, respectively, and the recovery rates were between 89.89% and 120.75%, which further confirmed that the biosensor VenusΔC10-Pa PBP-Nluc constructed by the present invention can be used for the detection of phosphate in river water samples.

[0101] Example 5

[0102] The difference from Example 2 is that:

[0103] The biosensor VenusΔC10-Pa PBP-Nluc was used to detect the spiked recovery rate of phosphate in the simulated wastewater sample of the reactor, including the following steps:

[0104] (I) Preparation of spiked water samples: Collect an appropriate amount of simulated wastewater from the reactor and filter it using a syringe filter (water system, PES) with a pore size of 0.45 μm to remove suspended particles in the sample; since the phosphate concentration in the simulated wastewater is too high, dilute it 10 times for later use; then add phosphate standard solutions with final concentrations of 0, 0.04 mg / L, 0.12 mg / L, and 0.36 mg / L to the treated inlet and outlet water samples, respectively, to obtain unspiked (blank) simulated wastewater samples and spiked series simulated wastewater samples, and set up 3 groups of parallel;

[0105] (II) Determination of recovery rate of spiked simulated wastewater: This step is the same as in Example 3, and the standard curve is drawn as shown in Fig. 9 As shown; and then the recovery rate of phosphate in simulated wastewater detected by phosphate biosensor method is calculated according to formula (6);

[0106] (III) Result analysis: Fig. 9 It can be seen that by respectively measuring the unspecified (blank) simulated wastewater samples and the spiked series of simulated wastewater samples to which 0, 0.04 mg / L, 0.12 mg / L and 0.36 mg / L phosphate standard solutions were added, the results obtained were 0.2231 mg / L, 0.2532 mg / L, 0.3923 mg / L and 0.6439 mg / L, respectively, and the recovery rates were between 75.25% and 116.89%, thereby confirming that the biosensor VenusΔC10-Pa PBP-Nluc prepared by the present invention can be used for the detection of phosphate in simulated wastewater samples.

[0107] Sequence Design Table

[0108] SEQ ID NO.1

[0109] The amino acid sequence of the phosphate-binding protein Pa PBP of Pseudomonas aeruginosa is: MKLKRLMAALTFVAAGVGAASAVAAIDPALPEYQKASGVSGNLSSVGSDTLANLMTMWAEEYKRLYPNVNIQIQAAGSSTAPPALTEGTANLGPMSRKMKDVELQAFEQKYGYKPTAVPVAVDALAIFVHKDNPIKGLTMQQVDAIFSATRLCGSKQDVKTWGDLGLTGDWAKKPVQLFGRNSVSGTYGYFKEEALCKGDFRPNVNEQPGSASVVQSVSQSLNGIGYSGIGYKTASVKTVALAKKEGAAFVEDNEQNALNGTYPLSRFLYVYVNKAPNKPLDPLEAQFLKLVLSKTGQQVVVKDGYIPLPAKVAEKAIKELGL

[0110] SEQ ID NO.2

[0111] Amino acid sequence of the biosensor VenusΔC10-Pa PBP-Nluc:

[0112] MASMTGGQQMGRGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGGGTAIDPALPEYQKASGVSGNLSSVGSDTLANLMTMWAEEYKRLYPNVNIQIQAAGSSTAPPALTEGTANLGPMSRKMKDVELQAFEQKYGYKPTAVPVAVDALAIFVHKDNPIKGLTMQQVDAIFSATRLCGSKQDVKTWGDLGLTGDWAKKPVQLFGRNSVSGTYGYFKEEALCKGDFRPNVNEQPGSASVVQSVSQSLNGIGYSGIGYKTASVKTVALAKKEGAAFVEDNEQNALNGTYPLSRFLYVYVNKAPNKPLDPLEAQFLKLVLSKTGQQVVVKDGYIPLPAKVAEKAIKELGLGGSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAKLAAALEHHHHHH*

[0113] SEQ ID NO.3

[0114] Amino acid sequence of yellow fluorescent protein VenusΔC10:

[0115] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVK FEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLLEFVTAAG

[0116] SEQ ID NO.4

[0117] Amino acid sequence of NanoLuc:

[0118] MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

[0119] The present invention is not limited to the above-mentioned specific implementation modes. Various changes made by ordinary technicians in this field based on the above-mentioned concepts without creative work are all within the protection scope of the present invention. Sequence Listing <110> Nanjing University <120> A BRET-based phosphate biosensor, construction method and application thereof <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 323 <212> PRT <213> Pseudomonas aeruginosa phosphate binding protein Pa PBP (Artificial Sequence) <400> 1 Met Lys Leu Lys Arg Leu Met Ala Ala Leu Thr Phe Val Ala Ala Gly 1 5 10 15 Val Gly Ala Ala Ser Ala Val Ala Ala Ile Asp Pro Ala Leu Pro Glu 20 25 30 Tyr Gln Lys Ala Ser Gly Val Ser Gly Asn Leu Ser Ser Val Gly Ser 35 40 45 Asp Thr Leu Ala Asn Leu Met Thr Met Trp Ala Glu Glu Tyr Lys Arg 50 55 60 Leu Tyr Pro Asn Val Asn Ile Gln Ile Gln Ala Ala Gly Ser Ser Thr 65 70 75 80 Ala Pro Pro Ala Leu Thr Glu Gly Thr Ala Asn Leu Gly Pro Met Ser 85 90 95 Arg Lys Met Lys Asp Val Glu Leu Gln Ala Phe Glu Gln Lys Tyr Gly 100 105 110 Tyr Lys Pro Thr Ala Val Pro Val Ala Val Asp Ala Leu Ala Ile Phe 115 120 125 Val His Lys Asp Asn Pro Ile Lys Gly Leu Thr Met Gln Gln Val Asp 130 135 140 Ala Ile Phe Ser Ala Thr Arg Leu Cys Gly Ser Lys Gln Asp Val Lys 145 150 155 160 Thr Trp Gly Asp Leu Gly Leu Thr Gly Asp Trp Ala Lys Lys Pro Val 165 170 175 Gln Leu Phe Gly Arg Asn Ser Val Ser Gly Thr Tyr Gly Tyr Phe Lys 180 185 190 Glu Glu Ala Leu Cys Lys Gly Asp Phe Arg Pro Asn Val Asn Glu Gln 195 200 205 Pro Gly Ser Ala Ser Val Val Gln Ser Val Ser Gln Ser Leu Asn Gly 210 215 220 Ile Gly Tyr Ser Gly Ile Gly Tyr Lys Thr Ala Ser Val Lys Thr Val 225 230 235 240 Ala Leu Ala Lys Lys Glu Gly Ala Ala Phe Val Glu Asp Asn Glu Gln 245 250 255 Asn Ala Leu Asn Gly Thr Tyr Pro Leu Ser Arg Phe Leu Tyr Val Tyr 260 265 270 Val Asn Lys Ala Pro Asn Lys Pro Leu Asp Pro Leu Glu Ala Gln Phe 275 280 285 Leu Lys Leu Val Leu Ser Lys Thr Gly Gln Gln Val Val Val Lys Asp 290 295 300 Gly Tyr Ile Pro Leu Pro Ala Lys Val Ala Glu Lys Ala Ile Lys Glu 305 310 315 320 Leu Gly Leu <210> 2 <211> 732 <212> PRT <213> Biosensor VenusΔC10-Pa PBP-Nluc (Artificial Sequence) <400> 2 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Val 1 5 10 15 Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu 20 25 30 Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly 35 40 45 Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile Cys Thr 50 55 60 Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Gly 65 70 75 80 Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys Gln His 85 90 95 Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr 100 105 110 Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys 115 120 125 Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp 130 135 140 Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr 145 150 155 160 Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile 165 170 175 Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly Val Gln 180 185 190 Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val 195 200 205 Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys 210 215 220 Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr 225 230 235 240 Ala Ala Gly Gly Gly Thr Ala Ile Asp Pro Ala Leu Pro Glu Tyr Gln 245 250 255 Lys Ala Ser Gly Val Ser Gly Asn Leu Ser Ser Val Gly Ser Asp Thr 260 265 270 Leu Ala Asn Leu Met Thr Met Trp Ala Glu Glu Tyr Lys Arg Leu Tyr 275 280 285 Pro Asn Val Asn Ile Gln Ile Gln Ala Ala Gly Ser Ser Thr Ala Pro 290 295 300 Pro Ala Leu Thr Glu Gly Thr Ala Asn Leu Gly Pro Met Ser Arg Lys 305 310 315 320 Met Lys Asp Val Glu Leu Gln Ala Phe Glu Gln Lys Tyr Gly Tyr Lys 325 330 335 Pro Thr Ala Val Pro Val Ala Val Asp Ala Leu Ala Ile Phe Val His 340 345 350 Lys Asp Asn Pro Ile Lys Gly Leu Thr Met Gln Gln Val Asp Ala Ile 355 360 365 Phe Ser Ala Thr Arg Leu Cys Gly Ser Lys Gln Asp Val Lys Thr Trp 370 375 380 Gly Asp Leu Gly Leu Thr Gly Asp Trp Ala Lys Lys Pro Val Gln Leu 385 390 395 400 Phe Gly Arg Asn Ser Val Ser Gly Thr Tyr Gly Tyr Phe Lys Glu Glu 405 410 415 Ala Leu Cys Lys Gly Asp Phe Arg Pro Asn Val Asn Glu Gln Pro Gly 420 425 430 Ser Ala Ser Val Val Gln Ser Val Ser Gln Ser Leu Asn Gly Ile Gly 435 440 445 Tyr Ser Gly Ile Gly Tyr Lys Thr Ala Ser Val Lys Thr Val Ala Leu 450 455 460 Ala Lys Lys Glu Gly Ala Ala Phe Val Glu Asp Asn Glu Gln Asn Ala 465 470 475 480 Leu Asn Gly Thr Tyr Pro Leu Ser Arg Phe Leu Tyr Val Tyr Val Asn 485 490 495 Lys Ala Pro Asn Lys Pro Leu Asp Pro Leu Glu Ala Gln Phe Leu Lys 500 505 510 Leu Val Leu Ser Lys Thr Gly Gln Gln Val Val Val Lys Asp Gly Tyr 515 520 525 Ile Pro Leu Pro Ala Lys Val Ala Glu Lys Ala Ile Lys Glu Leu Gly 530 535 540 Leu Gly Gly Ser Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp 545 550 555 560 Arg Gln Thr Ala Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly 565 570 575 Val Ser Ser Leu Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln 580 585 590 Arg Ile Val Leu Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val 595 600 605 Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu 610 615 620 Lys Ile Phe Lys Val Val Tyr Pro Val Asp Asp His His Phe Lys Val 625 630 635 640 Ile Leu His Tyr Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met 645 650 655 Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly 660 665 670 Lys Lys Ile Thr Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile 675 680 685 Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr 690 695 700 Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala Lys 705 710 715 720 Leu Ala Ala Ala Leu Glu His His His His His His 725 730 <210> 3 <211> 229 <212> PRT <213> Yellow Fluorescent Protein VenusΔC10 (Artificial Sequence) <400> 3 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Leu Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Gly Tyr Gly Leu Gln Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Gly 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Tyr Gln Ser Lys Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly 225 <210> 4 <211> 170 <212> PRT <213> NanoLuc (Artificial Sequence) of Nano Luciferase <400> 4 Met Val Phe Thr Leu Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala 1 5 10 15 Gly Tyr Asn Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30 Phe Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40 45 Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr 50 55 60 Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys 65 70 75 80 Val Val Tyr Pro Val Asp Asp His His Phe Lys Val Ile Leu His Tyr 85 90 95 Gly Thr Leu Val Ile Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe 100 105 110 Gly Arg Pro Tyr Glu Gly Ile Ala Val Phe Asp Gly Lys Lys Ile Thr 115 120 125 Val Thr Gly Thr Leu Trp Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140 Ile Asn Pro Asp Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val 145 150 155 160 Thr Gly Trp Arg Leu Cys Glu Arg Ile Leu 165 170

Claims

1. A BRET-based phosphate biosensor, characterized in that: The biosensor is a fusion protein obtained by fusing the phosphate-specific response Pseudomonas aeruginosa phosphate-binding protein Pa PBP, nanoluciferase NanoLuc and yellow fluorescent protein VenusΔC10, and the amino acid sequence is shown in SEQ ID NO.2; wherein the Pseudomonas aeruginosa phosphate-binding protein Pa PBP, nanoluciferase NanoLuc and yellow fluorescent protein VenusΔC10 are all connected by a flexible peptide linker, the Pseudomonas aeruginosa phosphate-binding protein Pa PBP is selected from the periplasmic subunit of the Pseudomonas aeruginosa phosphate special transport system Pst, and the periplasmic leader sequence is removed, and the amino acid sequence is shown in SEQ ID NO.1; The amino acid sequence of the flexible peptide linker is GGT-GGS; The amino acid sequence of the yellow fluorescent protein VenusΔC10 is shown in SEQ ID NO.3; the amino acid sequence of the nanoluciferase NanoLuc is shown in SEQ ID NO.4; The method for constructing the BRET-based phosphate biosensor comprises the following steps: S1. Synthesis of plasmids: Full gene synthesis of yellow fluorescent protein VenusΔC10 and nanoluciferase NanoLuc connected by GGT-GGS flexible linker, using VenusΔC10-GGT-GGS-Nluc as template, PCR gene amplification, and cloning into pET-24b(+) vector to obtain recombinant plasmid pET-VenusΔC10-Nluc; full gene synthesis of Pseudomonas aeruginosa phosphate binding protein PstS, using it as a template, PCR gene amplification, and inserting it between the flexible linkers GGT and GGS of pET-VenusΔC10-Nluc to obtain recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc; S2, transformation and expression: The recombinant plasmid pET-VenusΔC10-Pa PBP-Nluc constructed in step S1 was transformed into the expression vector Escherichia coli BL21 (DE3) strain, and spread on a kanamycin-resistant LB plate for culture, and colonies were grown, each of which was a single clone. The monoclonal strain obtained by culture was selected and inoculated into a kanamycin-resistant LB liquid culture medium, and cultured to OD600 = 0.6-1.

0. After IPTG induction, the expression was centrifuged to collect the bacteria; S3. Lysis and purification: The collected bacteria are resuspended in TBS buffer, PMSF and lysozyme are added thereto, the bacteria are lysed by ultrasonication, the supernatant is collected by centrifugation, purified, and preserved to obtain a biosensor protein, namely, a phosphate biosensor.

2. The phosphate biosensor according to claim 1, characterized in that In step S2, the mass volume concentration of the kanamycin-resistant LB solid culture medium is 100 μg / mL, and the mass volume concentration of the kanamycin-resistant LB liquid culture medium is 50 μg / mL; the induction expression conditions are: 0.1~1 mM IPTG, temperature is 16~30°C, and rotation speed is 100~200 rpm; the culture conditions in the kanamycin-resistant LB liquid culture medium are: temperature is 30~37°C, and rotation speed is 100~200rpm.

3. The phosphate biosensor according to claim 1, characterized in that In step S3, the pyrolysis conditions are: pyrolysis temperature is 0-4°C, power is 100-150 W, and pyrolysis time is 15-30 min.

4. Use of the phosphate biosensor according to any one of claims 1 to 3 in detecting phosphate in water.

5. The use according to claim 4, characterized in that: The method for applying phosphate biosensor to phosphate detection in water comprises the following steps: A1. Collect water samples: Take samples of the water to be tested to obtain water samples, then add pH regulator to the water samples to adjust the pH value to 6-8, filter and obtain water samples to be tested; A2. Prepare detection buffer, furazolidone working solution, phosphate standard working solution, phosphate standard stock solution and biosensor protein solution for later use; A3. Prepare phosphate standard solution: pipette different volumes of phosphate standard stock solution and phosphate standard working solution into centrifuge tubes respectively, and dilute with ultrapure water to prepare phosphate series standard solutions with concentrations of 0.0001 mg / mL, 0.0005 mg / mL, 0.001 mg / mL, 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.50 mg / mL, 1.00 mg / mL, 5.00 mg / mL, 10.00 mg / mL, and 50.00 mg / mL; A3. Drawing a standard curve: the phosphate series standard solutions prepared in step A2 are placed in ELISA plates respectively, the biosensor protein solution is added thereto, and the plates are incubated at room temperature for 8 to 12 minutes, and then the furazolidone working solution is added to obtain a bioluminescence resonance energy transfer detection system, and the luminescence intensity BL is measured in a dual-wavelength luminescence measurement mode, wherein the dual-wavelength parameters are set to 460 / 40 nm and 528 / 20 nm, respectively, and then the BRET ratio is calculated according to the following formula (1), and then the standard curve is drawn with the BRET ratio as the ordinate and the concentration of the phosphate standard solution as the abscissa; （1） The drawn curve is subjected to nonlinear fitting to obtain the fitting equation, as shown in the following formula (2): （2） Where, BRET max is the maximum value of the BRET ratio, BRET min is the minimum value of the BRET ratio, x is the concentration of the phosphate solution, x0 is the fitting parameter, h is the Hill slope, and s is the control factor; A4. Obtaining the phosphate concentration in the water sample to be tested: Substituting the phosphate standard solution with the water sample to be tested, repeating step A3 to obtain a bioluminescence resonance energy transfer detection system, measuring the luminescence intensity in the dual-wavelength luminescence measurement mode, and calculating the phosphate concentration in the water sample to be tested by equations (1) and (2).

6. The use according to claim 5, characterized in that: In step A2, the detection buffer is obtained by mixing 10~100 mM Tris and 50~250 mM NaCl, and the pH is 7.4; the working concentration of the biosensor protein solution is 1~20nM, and the working concentration of the furazolidone working solution is 10~20 μM; in the bioluminescence resonance energy transfer detection system, the volume ratio of the phosphate standard solution, the biosensor protein solution and the furazolidone working solution is 20 μL:80 μL:20 μL.

7. The use according to claim 5, characterized in that: The water samples include tap water, surface water and urban sewage.

Images