A detection method based on a two-component biosensor and a smart terminal cooperation

Abstract

The application discloses a detection method based on a two-component biosensor and a smart terminal, and belongs to the technical field of biosensing. The application takes Escherichia coli as a host cell, constructs a biosensor based on a NarX-NarL two-component signal transduction system, and is used for identifying nitrate in water. The method has the characteristics of clear structure, simple detection process and suitability for on-site application, and can be used for rapid detection of nitrate in environmental water samples.

Description

Technical Field

[0001] This invention relates to a detection method based on the collaboration of a two-component biosensor and a smart terminal, belonging to the field of biosensing technology. Background Technology

[0002] Nitrate is a common dissolved compound in natural water bodies, agricultural irrigation water, aquaculture water, and domestic sewage. Its concentration changes are closely related to eutrophication, ecological imbalance, and drinking water safety. Especially in scenarios such as agricultural fertilization, industrial discharge, and domestic sewage discharge, abnormal accumulation of nitrate can easily lead to environmental risks. Therefore, it is of great significance to establish rapid, accurate, low-cost detection methods suitable for field applications.

[0003] Currently, methods for nitrate detection mainly include spectrophotometry, ion chromatography, electrochemical analysis, and mass spectrometry. While these methods offer high accuracy, they typically rely on large instruments and specialized operators, involve complex sample pretreatment, have long detection cycles, and struggle to meet the demands of rapid on-site detection, distributed monitoring, and immediate results. Especially in scenarios such as grassroots environmental monitoring, agricultural non-point source pollution monitoring, and portable on-site detection, traditional methods suffer from significant limitations in terms of equipment size, cost, ease of operation, and real-time performance.

[0004] In recent years, the development of synthetic biology has driven the application of whole-cell biosensors in environmental monitoring. Whole-cell biosensors utilize the cell's own signal recognition and transduction capabilities to sense target substances and output detectable signals through reporter genes, offering advantages such as flexible construction, low cost, and biological amplification. Among them, two-component biosensors sense extracellular signals through transmembrane histidine kinases, and then downstream promoter activity is regulated by response regulatory proteins, thereby driving reporter gene expression. This makes them particularly suitable for detecting soluble small molecules in water. For nitrates, sensor cells constructed based on the NarX-NarL two-component signal transduction system have shown good recognition potential.

[0005] However, the application of existing two-component biosensors remains primarily confined to laboratory conditions, typically relying on benchtop devices such as ELISA readers and fluorescence spectrophotometers to read fluorescence signals. While these detection methods can perform quantitative analysis, the equipment is expensive, bulky, and lacks portability, hindering on-site deployment and immediate application. Furthermore, the detection results often depend on laboratory software for subsequent processing, lacking an integrated solution for data acquisition, transmission, analysis, and result output that combines with mobile terminals, making it difficult to form a complete technological chain suitable for rapid on-site monitoring.

[0006] Furthermore, the output signal of whole-cell biosensors is easily affected by factors such as cell state, culture conditions, sample matrix, and detection procedure. Without a unified detection procedure, standard curve conversion method, and signal normalization strategy, the stability and comparability of detection results will be limited. Therefore, it remains necessary to provide a detection method and platform based on a two-component biosensor, enabling rapid, portable, and quantitative detection of nitrates using fluorescence detection devices and smart terminals, while retaining the high specificity and biological amplification advantages of two-component signal transduction systems. Summary of the Invention To address the aforementioned issues, this invention first constructs a nitrate-responsive histidine kinase NarX mutant and a whole-cell biosensor, and then provides a detection method based on the synergy of a two-component biosensor and a smart terminal.

[0007] The first objective of this invention is to provide a nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase NarX mutant has one or more mutations at position 507 or 562 based on the amino acid sequence such as SEQ ID NO.1.

[0008] In one embodiment, the nitrate-responsive histidine kinase NarX mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has leucine at position 507 mutated to phenylalanine and serine at position 562 mutated to arginine, i.e., NarX. L507F / S562R .

[0009] This invention also provides another nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has leucine at position 507 mutated to serine, arginine at position 446 mutated to histidine, and asparagine at position 439 mutated to lysine, i.e., NarX. L507F / R446H / N439K .

[0010] This invention also provides another nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has its serine at position 562 mutated to arginine, its leucine at position 490 mutated to methionine, and its methionine at position 418 mutated to isoleucine, i.e., NarX. S562R / L490M / M418I .

[0011] This invention also provides another nitrate-responsive histidine kinase NarX mutant, wherein the nitrate-responsive histidine kinase mutant, based on the amino acid sequence as shown in SEQ ID NO.1, has the following mutations: leucine at position 507 is mutated to phenylalanine; serine at position 562 is mutated to arginine; proline at position 492 is mutated to alanine; valine at position 541 is mutated to alanine; leucine at position 490 is mutated to methionine; methionine at position 418 is mutated to isoleucine; arginine at position 446 is mutated to histidine; and asparagine at position 439 is mutated to lysine, i.e., NarX. L507F / S562R / P492A / V541A / L490M / M418I / R446H / N439K .

[0012] A second objective of this invention is to provide a polynucleotide encoding the aforementioned nitrate-responsive histidine kinase NarX mutant.

[0013] A third objective of this invention is to provide cells expressing the above-mentioned nitrate-responsive histidine kinase NarX mutant, characterized in that the cells comprise Escherichia coli, Bacillus subtilis, and Corynebacterium glutamicum.

[0014] A fourth objective of this invention is to provide a genetically engineered Escherichia coli strain that expresses any of the above-mentioned nitrate-responsive histidine kinase NarX mutants.

[0015] In one embodiment, the response regulatory protein NarL and the fluorescent protein sfGFP are also expressed; The amino acid sequence of the response regulatory protein NarL is shown in SEQ ID NO.2, and the amino acid sequence of the fluorescent protein sfGFP is shown in SEQ ID NO.3.

[0016] In one implementation, promoter P is used. narG365 It expresses fluorescent proteins; promoter J23109 expresses the nitrate-responsive histidine kinase NarX mutant; promoter J23101 expresses the responsive regulatory protein NarL. Among them, promoter P narG365 The nucleotide sequence of promoter J23109 is shown in SEQ ID NO.4, the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.5, and the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.6.

[0017] In one implementation, promoter P narG365 In the promoter P narG515 It is obtained by truncating it by 150 bp.

[0018] In one embodiment, one or more of the following amino acids are knocked out in the genome of the genetically engineered Escherichia coli: NarX (amino acid sequence as shown in SEQ ID NO.1), NarL (amino acid sequence as shown in SEQ ID NO.2), NarU (amino acid sequence as shown in SEQ ID NO.8), NarK (amino acid sequence as shown in SEQ ID NO.9), NapA (amino acid sequence as shown in SEQ ID NO.10), and NapB (amino acid sequence as shown in SEQ ID NO.11) to enhance the low-concentration nitrate detection response.

[0019] In one embodiment, NarX and NarL are knocked out of the genome of the genetically engineered Escherichia coli, and one or both of NarU and NarK are further knocked out.

[0020] The fifth objective of this invention is to provide a two-component biosensor device for detecting nitrate content, comprising a bioreaction module, a fluorescence detection module, a data processing module, a wireless communication module, and a smart terminal; The bioreaction module is used to culture the above-mentioned Escherichia coli genetically engineered bacteria. After being cultured to the logarithmic growth phase, it is mixed with the sample to be tested and incubated. After incubation, it is centrifuged, diluted, and resuspended to obtain the bacterial solution to be tested. The fluorescence detection module includes an excitation module, a light signal acquisition module, and a signal output module. The excitation module uses a light source with a wavelength of 470 nm to excite sfGFP fluorescence. The light signal acquisition module converts the acquired fluorescence signal into a photocurrent through a silicon photodiode, and then converts the photocurrent into a voltage signal through a transimpedance amplifier circuit. The signal output module converts the voltage signal into a digital signal. The data processing module collects digital signals and transmits them to the smart terminal via the wireless communication module; The intelligent terminal processes the received data, including: blank control subtraction, background correction, outlier removal, duplication averaging, cell density normalization, standard curve fitting and concentration conversion, and outputs the qualitative or quantitative detection results of the target substance in the sample to be tested.

[0021] In one implementation, the samples to be tested in the bioreactor module include surface water, groundwater, agricultural irrigation water, and domestic sewage. The incubation conditions for the test sample and the genetically engineered Escherichia coli were 37℃ for 1-6 h. After incubation, the OD of the genetically engineered E. coli 600 Dilute to 0.1~0.5.

[0022] A sixth object of the present invention is to provide the application of any of the above-mentioned nitrate-responsive histidine kinase NarX mutants, or the above-mentioned polynucleotides, or the above-mentioned cells, or any of the above-mentioned genetically engineered Escherichia coli bacteria, or the above-mentioned two-component biosensor devices in the detection of nitrates.

[0023] Beneficial effects of the present invention (1) The present invention uses the two-component signal transduction system NarX-NarL to identify nitrate, which has natural signal recognition specificity and cascade amplification effect. Compared with single-component biosensors, such as single protein or nucleic acid sensors, it can identify low concentration targets outside the cell and reduce cross-interference in complex sample matrices.

[0024] (2) This invention uses a dual-component biosensor in conjunction with a smart terminal, eliminating the need for large desktop equipment such as enzyme-linked immunosorbent assay (ELISA) readers and fluorescence spectrophotometers. Signal acquisition and data analysis can be completed with just a portable fluorescence reading unit and a smartphone, significantly reducing the dependence on laboratory environment and professional operators. Attached Figure Description

[0025] Figure 1 Optimization of the sensor output promoter; where A: Schematic diagram of the gene circuit of the natural NarX-NarL TCSB biosensor; B: NarX-NarL TCSB at different concentrations of NO3 - Curve showing the change in induced sfGFP expression level over time; C:P narG515 The output and dynamic range of the promoter-truncated NarX-NarL TCSB, where the dynamic range is the ratio of the maximum output to the minimum output, is indicated above each bar chart.

[0026] Figure 2 Fluorescence output of NarX-NarL TCSB constructed from 36 promoter combinations; where A: 6 promoter sequences replace the promoters in NarX-NarL TCSB respectively; B: 36 NarX-NarL TCSB in NO3 - The maximum and minimum outputs are at 0 mM and 20 mM.

[0027] Figure 3 The effects of NarX and NarL expression levels on sensor performance are shown in section A: the effect of relative NarX and NarL mRNA expression levels on dynamic range, where Pn and Pm represent the promoters controlling NarX and NarL, respectively; and section B: analysis of the detection limit and detection range of 5-1XL. The red dashed line indicates the detection limit is three times the minimum output of NO3 corresponding to the TCSB signal. - concentration.

[0028] Figure 4 For NarX S562R / L490M / M418I NarX L507S / R446H / N439K NarX P492A / V541A NarX L507F / S562R Maximum output, minimum output, and dynamic range.

[0029] Figure 5 Analysis of the response characteristics of mutants; where A; NarX S562R / L490M / M418I - Analysis of the detection limit and linear detection range of NarL TCSB; B: NarX L507S / R446H / N439K -Analysis of the detection limit and linear detection range of NarL TCSB; C: NarX P492A / V541A Analysis of the detection limit and linear detection range of NarL TCSB; D: NarX L507F / S562R -Analysis of the detection limit and linear detection range of the NarL TCSB. The black curve represents the location of the detection limit, defined as the NO3 required for the sensor output to reach three times the minimum output. - concentration.

[0030] Figure 6 Characterization of the biosensor performance of NarX combined mutants; A: Spatial distribution of the 9 amino acid mutation sites on the NarX kinase domain, the NarX-ATP complex structure was predicted by AlphaFold 3; B: Box plot of the dynamic range of libraries with different numbers of mutation sites; C: Analysis of significant differences in the dynamic range of the combined mutants, significant differences were assessed using a two-tailed, unpaired t-test; D: Schematic diagram of the 2-30XL plasmid; E: Detection characteristic analysis of 2-30XL, the black curve represents the detection limit, which is defined as the NO3 required for the sensor output to reach 3 times the minimum output. - concentration.

[0031] Figure 7 For 2-30XL sensors, NO3 knockout - NO3 in the chassis of the transport and reduction pathway - The response characteristics; where A; dose-response curves for N1~N7. The control refers to the sensor chassis cells as... ΔnarX / ΔnarL A green background indicates 0–1 mM NO3. - The low concentration range; B: N1~N7 in 1 mM NO3 - Comparison of dynamic range under the given conditions, with a dynamic range of 1 mM NO3 - The sensor output under induced conditions is related to 0 mM NO3. - The ratio of sensor outputs under induced conditions.

[0032] Figure 8This is a schematic diagram of the working process of the fluorescence detection device of the present invention, wherein A: schematic diagram of the device appearance; B: schematic diagram of the structure and optical path of the photoelectric conversion module; C: schematic diagram of the working process of the fluorescence detection device; D: influence of different sample volumes on voltage output; E: cell density (OD). 600 The voltage values ​​of the control strain MG1655 (without the sensor plasmid) are shown on the right-hand vertical axis. ΔnarX / narL The voltage of the sensor; F: The comparison results of the fluorescence output detection of the sensor by the fluorescence detection device of the present invention and the laboratory enzyme-linked immunosorbent assay (ELISA) reader; Figure 9 This diagram illustrates the spiked response curve and quantitative analysis results of the nitrate concentration detection method of the present invention. A: Schematic diagram of sampling point distribution; B-F: Comparison results of nitrate content determination in different water bodies using the biosensor method and ultraviolet spectrophotometry, with the light blue shaded area representing a spiked recovery rate of 80%-120%; G: Results of nitrate content detection in wastewater using a biosensor, with samples marked in green indicating spiked samples. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] In this invention, the dose-response curve is a characteristic curve for evaluating the performance of TCSB. Taking an activation-type two-component sensor as an example, it reflects the correspondence between inducer concentration (input signal) and reporter gene expression level (output signal). This curve can typically be described by the following quantitative parameters: (1) Baseline output (also known as minimum output) refers to the baseline signal output of TCSB in the uninduced state; (2) Dynamic range, which is the ratio of maximum output to minimum output, is often used as a measure of signal-to-noise ratio; (3) Detection threshold (EC50) refers to the concentration of inducer required for the sensor output to reach half of its maximum value; (4) Limit of detection (LOD) indicates the lowest concentration at which the target signal can be reliably monitored. These parameters are key indicators for TCSB performance adjustment.

[0035] Raw materials used in the examples: pJKR-H-CdaR and pACYCDuet-1 are both commercially available. The primers used for constructing plasmids, knocking out genes, and mutations in the examples are shown in Table 1.

[0036] Table 1 Primers

[0037] Test method: Nitrate-induced fluorescence detection method Escherichia coli MG1655 and its derived knockout strains ΔnarX / narL , ΔnarU , ΔnarK , ΔnarU / narK , ΔnapA , ΔnapB , ΔnapA / napB and ΔnarU / narK / napA / napB As a chassis cell, it was used for performance characterization of a nitrate two-component sensor.

[0038] The biosensor plasmid was transformed into competent cells of these strains and plated on LB agar plates containing chloramphenicol resistance. The plates were incubated overnight at 37°C. Single colonies were then picked and inoculated into LB liquid medium and cultured at 37°C with shaking at 250 rpm for 10–12 h. The seed culture was then transferred at 5% to 96-well plates containing fresh LB liquid medium and cultured with shaking at 250 rpm for 2–3 h to allow the strains to reach the logarithmic growth phase. A gradient concentration of sodium nitrate solution (final concentration 0–20 mM) was then added, and the plates were covered with an airtight sealing film. After incubation at 37°C for 3 h, the cells were collected by centrifugation at 3900 rpm, washed three times with pre-cooled sterile PBS, and resuspended in PBS. The OD600 was appropriately diluted to approximately 0.5. 200 μL of the bacterial culture was placed in a 96-well plate and readjusted using a Biotek HT microplate reader at an excitation wavelength of 485 nm. The fluorescence intensity was detected at an emission wavelength of 528 nm, and the absorbance OD600 of the bacterial culture was detected at 600 nm. The normalized fluorescence intensity was expressed as the ratio of the detected fluorescence intensity to OD600, in au. The normalized fluorescence intensity can be used to evaluate the output of TCSB (natural two-component system-based biosensor).

[0039] Example 1: Construction of biosensor plasmids 1. Gene knockout To remove interference from NarX and NarL expression in the *E. coli* genome, the narX / narL operons in *E. coli* MG1655 were knocked out using CRISPR-Cas9 technology to construct MG1655 (… ΔnarX / narL ) strain.

[0040] In MG1655 ( ΔnarX / narL Based on this, individual and combined NO3 knockout - Transporter protein-related genes narU / narK, NO3 -The reductase encoding genes napA / napB were used to obtain strain MG1655. ΔnarU ), MG1655 ΔnarK ), MG1655 ΔnapA ), MG1655 ΔnapB ), MG1655 ΔnarU / narK ), MG1655 ΔnapA / napB ) and MG1655 ( ΔnarU / narK / napA / napB ).

[0041] 2. Constructing plasmids Use narX-F / narX-R, narL-F / narL-R, P narG515 Three primer pairs were used to amplify two-component protein-coding genes from Escherichia coli MG1655. narX , narL and the controlled promoter P of NarL narG515 ; Use primer pairs sfgfp -F / sfgfp -R was amplified from the pJKR-H-CdaR plasmid containing a fluorescent reporter gene. sfgfp Gene fragments including terminators; Simultaneously, using pACYCDuet-1 as a template, PCR amplification was performed using primer pairs VXF / VXR, VLF / VLR, and VGF / VGR to obtain vector fragments linked to different genes; The purified gene fragment and vector fragment were mixed sequentially at a molar ratio of 3:1, and Gibson assembly was performed with the addition of homologous recombinase. Ligation was carried out at 50°C for 1 h. The ligation product was transformed into E. coli JM109 competent cells, and after overnight culture, positive transformants were screened by colony PCR and Sanger sequencing to construct the plasmid pACYC-P. narG515 - sfgfp and pACYC- narX - narL - sfgfp Sensor plasmid.

[0042] Example 2: Truncation of non-coding regions In plasmid pACYC-P narG515 - sfgfp Based on this, the promoter P narG515 The segments are truncated at 50 bp intervals and named sequentially according to their length, P. narG465 P narG415 P narG365 and P narG315 .

[0043] pACYC-P narG515Using -sfgfp as a template, the gene fragments were amplified using primer pairs Tru465-F / Tru465-R, Tru415-F / Tru415-R, Tru365-F / Tru365-R, and Tru315-F / Tru315-R, respectively. The amplified gene fragments were then assembled and cloned into pACYC-P using Gibson assembly. narG515 - sfgfp In the carrier sfgfp Upstream replacement P narG515 The non-coding region truncated plasmid pACYC-P was obtained. narG465 - sfgfp, pACYC-P narG415 - sfgfp, pACYC-P narG365 - sfgfp, pACYC-P narG315 - sfgfp .

[0044] The plasmid with the non-coding region truncated was transferred into E. coli MG1655. ΔnarX / narL The sensor, which detects plasmids carrying different truncated segments, operates in the range of 0–20 mM NO3. - The output and dynamic range after 10 hours of conditioned induction are shown in the following figures. Figure 1 As shown.

[0045] The results show that after deleting 151 b upstream of the promoter, P narG365 Its dynamic range is the highest, which is P of the original length. narG515 The dynamic range was increased from 13 times to 21 times; while based on P narG365 Further removal of adjacent RstA binding sites significantly reduced the dynamic range to 6-fold. Therefore, subsequent selection of P... narG365 It serves as the output promoter of the sensor.

[0046] Example 3: Different promoter combinations To adjust the expression levels of NarX and NarL, six promoters were selected: BBa_J23109 (J23109), BBa_J23114 (J23114), BBa_J23105 (J23105), BBa_J23107 (J23107), BBa_J23108 (J23108), and BBa_J23101 (J23101), obtained from the Anderson promoter library (http: / / parts.igem.org / Promoters / Catalog / Anderson). Primers were designed based on promoter sequences, using plasmid pACYC- narX - narL - sfgfpUsing this as a template, whole-plasmid PCR was performed. After purifying the PCR product with a purification kit, it was digested with Dpn I enzyme at 37°C for 15 min, and then transformed into E. coli JM109 competent cells. Positive clones were screened by Sanger sequencing, and 36 NarX-NarL expression intensities (6 NarX and 6 NarL expression intensities) were successfully constructed. narG365 TCSB plasmid expressing fluorescent protein.

[0047] 36 plasmids were transformed into E. coli MG1655. ΔnarX / narL The chassis contains 0~20 mM NO3 - The sensor performance was tested under certain conditions, and the results are as follows: Figure 2 As shown.

[0048] The results show that when NarX is expressed under the control of J23114 or J23107, simultaneously increasing NarL expression significantly amplifies the sensor output, with the maximum output increasing from 252 au to 24776 au and the minimum output increasing from 306 au to 1847 a.u. However, the maximum output of other promoter combinations did not show an increase with increasing NarL expression. This indicates that NarL expression is not "the stronger the better," and a suitable expression ratio between NarX and NarL is required to induce a high signal output from the TCSB.

[0049] Furthermore, the dynamic range of the sensor was calculated based on the maximum and minimum outputs of these 36 promoter combinations, and the transcriptional levels of narX and narL were measured to calculate their relative transcriptional ratios. The results are as follows: Figure 3 As shown.

[0050] The results showed that when the relative transcriptional levels of NarX and NarL were below 0.5, the dynamic range generally exceeded 4-fold; however, higher expression ratios often led to a decrease in the sensor's dynamic range, and even reduced its sensitivity to NO3. - There was no response. This phenomenon may be due to NarX overexpression, which leads to a large amount of NarX being in phosphatase activity, thereby inhibiting NarL phosphorylation and its activation of downstream genes.

[0051] Among all combinations, the combination with J23109 controlling NarX and J23101 controlling NarL had the highest dynamic range (14-fold) and the lowest relative expression ratio of NarX to NarL (approximately 0.003). The combination plasmid was named 5-1XL (i.e., plasmid pACYC-P). J23109 - narX -P J23101 - narL - sfgfp -P narG365The dose-response curve results showed that the expression plasmid 5-1XL in Escherichia coli MG1655 ( ΔnarX / narL Biosensors for NO3 - The linear detection range is 1~10 mM, and the detection limit is 0.9 mM.

[0052] Example 4: Construction of NarX mutants In plasmid pACYC-P J23109 -narX-P J23101 -narL-sfgfp-P narG365 Based on this, NarX was subjected to multiple point mutations to obtain the mutant NarX. S562R / L490M / M418I NarX L507S / R446H / N439K NarX P492A / V541A NarX L507F / S562R NarX mutant plasmids were constructed separately.

[0053] The NarX mutant plasmid was transformed into E. coli MG1655. ΔnarX / narL A biosensor strain was constructed; TCSB was detected at 0 and 20 mM NO3. - The maximum output, minimum output, and dynamic range under the given conditions are as follows: Figure 4 As shown.

[0054] The results show that NarX S562R / L490M / M418I NarX L507S / R446H / N439K NarX L507F / S562R The dynamic ranges reached 17, 16, and 19 times respectively, representing improvements of 21%, 14%, and 36% compared to the control; while NarX P492A / V541A While the maximum output increased by 135%, the minimum output increased by 200% simultaneously, resulting in a decrease in dynamic range of approximately 30%.

[0055] The results of dose response curve analysis are as follows: Figure 5 As shown, NarX S562R / L490M / M418I With NarX P492A / V541A The detection limit was reduced by approximately 80% (0.5 mM), while NarX... P492A / V541A With NarX L507F / S562R The detection limits were increased to 16 mM and 1.6 mM, respectively.

[0056] Based on the nine amino acid mutation sites involved in the above combined mutations, nine single-point mutants were constructed and further combined with mutations to obtain the mutant NarX. L507F / S562R / P492A / V541A / L490M / M418I / R446H / N439K pACYC-P containing this mutant J23109 - narX -P J23101 - narL - sfgfp -P narG365 The plasmid was named 2-30XL, and the results are as follows: Figure 6 As shown.

[0057] The results showed that none of the single-point mutations significantly improved the dynamic range, meaning that individual changes were insufficient to improve sensor performance, and there was a synergistic effect among the various sites; Escherichia coli MG1655 expressing 2-30XL ( ΔnarX / narL The biosensor's detection limit is reduced to 0.4 mM, and it has a linear detection range of 0.4 to 8 mM.

[0058] Example 5: Fabrication of a biosensor Plasmids 2-30XL were transformed into E. coli MG1655, respectively. ΔnarU Biosensors N1 and MG1655 were constructed and obtained. ΔnarK Biosensors N2 and MG1655 were constructed and obtained. ΔnarU / narK Biosensors N3 and MG1655 were constructed and obtained. ΔnapA Biosensors N4 and MG1655 were constructed and obtained. ΔnapB Biosensors N6 and MG1655 were constructed and obtained. Δ napA / napB Biosensors N6 and MG1655 were constructed and obtained. ΔnarU / narK / napA / napB The biosensor N7 was constructed, and different biosensors were used to detect NO3. - Response characteristics, results as follows Figure 7 As shown.

[0059] The results showed that knocking out the transporter protein encoding gene narU or narK ,as well as narU / narK Both knockouts resulted in the sensor output being at a low concentration of NO3. - The response range becomes steeper and saturates earlier, leading to an increase in minimum output. The dynamic range of N1, N2, and N3 decreases from 21x to 10x, 5x, and 8x, respectively. However, N3 exhibits a higher dynamic range in 1 mM NO3. - The output signal under induction changed by 7 times, which was significantly improved by 4 times compared with the control, and its detection limit was reduced to 0.1 mM (linear range 0.1~1 mM).

[0060] In contrast, knocking out the periplasmic reductase gene napA / napB (N4~N6) failed to improve the sensor's response to low concentrations. Further simultaneous blocking of transport and reduction (N7) revealed that the sensor's response to NO3... - There was no obvious response, indicating a constitutive activated state. These results suggest that simultaneous knockout... narU and narK It can significantly reduce the detection limit. The mechanism may originate from perimass NO3. - The increase in net accumulation boosted NarX and NO3. - The binding rate drives NarX conformational changes and autophosphorylation, thereby enhancing sensing and amplifying downstream output signals.

[0061] Therefore, the biosensor N3 was used for all subsequent performance tests.

[0062] Example 6: A method for detecting nitrate concentration using a biosensor 1. A device, detection method, and signal acquisition process based on a two-component biosensor, such as... Figure 8 As shown.

[0063] (1) Biosensing response N3 was inoculated into LB liquid medium and cultured at 37°C with shaking at 250 rpm for 10–12 h. The seed culture was then transferred at a 5% inoculation rate to 96-well plates containing fresh LB liquid medium and cultured at 250 rpm for 2–3 h to allow the strain to reach the logarithmic growth phase. Gradual concentrations of sodium nitrate solution (final concentration 0–20 mM) were added, and the plates were covered with an airtight sealing film. After incubation at 37°C for 3 h, the cells were collected by centrifugation at 3900 rpm, washed three times with pre-cooled sterile PBS, and finally resuspended in PBS, with an OD600 of approximately 0.5.

[0064] (2) Fluorescence output of the ELISA reader sensor 200 μL of bacterial culture was placed in a 96-well plate, and the fluorescence intensity was detected using a Biotek HT microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Simultaneously, the absorbance (OD) of the bacterial culture was measured at 600 nm. 600 Normalized fluorescence intensity is calculated by comparing the detected fluorescence intensity with OD. 600 The ratio is expressed in au, and the normalized fluorescence intensity can be used to evaluate the sensor output.

[0065] (3) Fluorescence signal acquisition The fluorescence output of the sensor is measured using a fluorescence detection device; the device converts the fluorescence signal into a voltage signal output, and within a certain range, the voltage and fluorescence intensity exhibit a linear relationship. The linear range and sensitivity are analyzed before measurement.

[0066] Experimental results show that within the concentration range of 0.1–1 mM, the detection signal exhibits a good linear relationship with nitrate concentration, with a correlation coefficient R0. 2 The detection limit is greater than 0.98. The detection limit can reach 0.1 mM, indicating that the method of the present invention has good sensitivity.

[0067] The detection volume was optimized. By comparing the signal output under different detection volume conditions, it was found that the detection signal gradually increased with the increase of sample volume. When the volume reached about 800 μL, the signal tended to stabilize. Therefore, the preferred detection volume is 800 μL.

[0068] The effect on cell density was analyzed. (OD) 600 Within the range of 0.1 to 0.5, the detection signal showed an approximately linear relationship with cell density (R0). 2 >0.95). To reduce the impact of cell density on the detection results, the detection signal was normalized (voltage / OD). 600 ).

[0069] (4) Combined measurement process of biosensor and fluorescence detection device The induced sensor cell suspension was adjusted to OD600=0.5, and 800 μL was taken into a 5 mL centrifuge tube; all measurements were performed in the light-shielding chamber of the device, and the average value of the sample was obtained from at least three independent biological replicates; a quality control sample was inserted for every 8-10 samples to monitor signal drift; the fluorescence detection device includes an excitation module, a light signal acquisition module and a signal output module; The working process is as follows: the excitation module uses a light source with a wavelength of 470 nm to excite sfGFP fluorescence, and the fluorescence signal is filtered by optical filtering components (including excitation light filter, dichroic mirror and emission filter); the optical signal acquisition module converts the fluorescence signal into photocurrent through silicon photodiode; the photocurrent is converted into a voltage signal by transimpedance amplifier circuit and noise is reduced by low-pass filter circuit; the signal output module converts the voltage signal into a digital signal through analog-to-digital converter.

[0070] (5) Data transmission and processing The digital signal is packaged by the processor and then sent to the smart terminal via the wireless communication module; preferably, Wi-Fi communication is used for data transmission; the smart terminal processes the received data, including: blank control subtraction, background correction, outlier removal, duplication averaging, cell density normalization, standard curve fitting and concentration conversion.

[0071] (6) Output of results The smart terminal outputs the concentration value of nitrate in the sample to be tested and generates a test report, which includes the test time, sample number, test items and test results.

[0072] (7) Method comparison and verification The method was compared with the conventional microplate detection method, and the results showed that the two methods had good consistency under the same sample conditions, indicating that the method of the present invention can achieve accurate detection of nitrate.

[0073] 2. Detect the nitrate content in actual water samples. Spiking experiments were conducted using nitrate standard solutions of different concentrations, such as... Figure 9 As shown.

[0074] (1) Sample collection and pretreatment Environmental water samples were collected from Changguangxi River in Wuxi City, Jiangsu Province. Five sampling points were selected along the river, covering wetlands, residential areas and agricultural irrigation areas, as well as a sewage treatment plant (Yixing Urban Sewage Resource Concept Plant). The samples were centrifuged at 12,000 rpm for 5 min at room temperature, and the supernatant was filtered through a 0.22 μm filter membrane to remove suspended particles.

[0075] (2) Establishment of standard curve Prepare nitrate standard solutions with different concentration gradients, react them with a two-component biosensor module, and perform detection under the same conditions to record the corresponding electrical or fluorescence signals; A standard curve was established by fitting the detection signal to the nitrate concentration. In the spiked recovery experiment, different concentrations of NaNO3 standard solution were added to the environmental water sample, and a standard curve was prepared in parallel in ultrapure water-LB. The fluorescence value of the spiked environmental sample was substituted into the ultrapure water-LB standard curve for interpolation to obtain the NO3 concentration. - The concentration was compared with the spiked amount to calculate the recovery rate; the background NO3 of unspiked environmental samples was also calculated based on the same standard curve. - content.

[0076] (3) Actual sample testing Actual water samples were tested using the above method, and the results showed that the spiked recovery rate was between 80% and 120%. Figure 9 As shown, the method of the present invention has good accuracy and applicability.

[0077] Sequences used in the examples Nitrate-responsive histidine kinase NarX starting amino acid sequence SEQ ID NO.1: MLKRCLSPLTLVNQVALIVLLSTAIGLAGMAVSGWLVQGVQGSAHAINKAGSLRMQSYRLLAAVPLSEKDKPLIKEMEQTAFSAELTRAAERDGQLAQLQGLQDYWRNELIPALMRAQNRETVSADVSQFVAGLDQLVSGFDRTTEMRIETVVLVHRVMAVFMALLLVFTIIWLRARLLQPWRQLLAMASAVSHRDFTQRANISGRNEMAMLGTALNNMSAELAESYAVLEQRVQEKTAGLEHKNQILSFLWQANRRLHSRAPLCERLSPVLNGLQNLTLLRDIELRVYDTDDEENHQEFTCQPDMTCDDKGCQLCPRGVLPVGDRGTTLKWRLADSHTQYGILLATLPQGRHLSHDQQQLVDTLVEQLTATLALDRHQERQQQLIVMEERATIARELHDSIAQSLSCMKMQVSCLQMQGDALPESSRELLSQIRNELNASWAQLRELLTTFRLQLTEPGLRPALEASCEEYSAKFGFPVKLDYQLPPRLVPSHQAIHLLQIAREALSNALKHSQASEVVVTVAQNDNQVKLTVQDNGCGVPENAIRSNHYGMIIMRDRAQSLRGDCRVRRRESGGTEVVVTFIPEKTFTDVQGDTHE* Response regulator protein NarL amino acid sequence SEQ ID NO.2: MSNQEPATILLIDDHPMLRTGVKQLISMAPDITVVGEASNGEQGIELAESLDPDLILLDLNMPGMNGLETLDKLREKSLSGRIVVFSVSNHEEDVVTALKRGADGYLLKDMEPEDLLKALHQAAAGEMVLSEALTPVLAASLRANRATTERDVNQLTPRERDILKLIAQGLPNKMIARRLDITESTVKVHVKHMLKKMKLKSRVEAAVWVHQERIF* Fluorescent protein amino acid sequence is as set forth in SEQ ID NO.3: MRIGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKDACQF* Promoter P narG365 Nucleotide SEQ ID NO.4: CGGTTTTCTCCTGTGGGAGCCTGTCGGCCCTCTGATAAATCGACATTGCTAATGTTTAAGGGCAAGGTAACGCTCTGAAACAGCGTGGGAATTGATAACGATCAAGAGTGATGGGGATGAAAAATAAAGTAAATTCCTAAGATACCCCTTTAGGATAATTAACAGCCACCACATAACAGACTGTAAATTAAGACGTTTTATACCTGTAGTTATTTAAACTATTAAGATTTTTATGCAGATGGGTATTAAGGAGTATTCCCCATGGGTAACATATTAATGGCTCTTAATGAAATCCGGCGAAATAAACCATATAACATTAGTATGTTGTAACTAAATGACGCATAAAAAAAGCGCGACATCATGCC Promoter J23109 nucleotide SEQ ID NO.5: TTTACAGCTAGCTCAGTCCTAGGGACTGTGCTAGC Promoter J23101 nucleotide SEQ ID NO.6: TTTACAGCTAGCTCAGTCCTAGGTATTATGCTAGC Promoter P narG515 Nucleotide SEQ ID NO.7: ACCGTTACTCGTCATACTTCGGGTTACATGTGCTGCGGCTGCGTTCATTCACCCCAGTCACTTACTTTAGTAAGCTCCTGGGATTCATTCACTTGCCGCCTTCCTGTAAACCGAATTATATAGAGTAAAATATTTGATTATCCTTTGCGCGGCATGATGTCGCGCTTTTTTTATGCGTCATTTAGTTACAACATACTAATGTTATATGGTTTATTTCGCCGGATTTCATTAAGAGCCATTAATATGTTACCCATGGGGAATACTCCTTAATACCCATCTGCATAAAAATCTTAATAGTTTAAATAACTACAGGTATAAAACGTCTTAATTTACAGTCTGTTATGTGGTGGCTGTTAATTATCCTAAAGGGGTATCTTAGGAATTTACTTTATTTTTCATCCCCATCACTCTTGATCGTTATCAATTCCCACGCTGTTTCAGAGCGTTACCTTGCCCTTAAACATTAGCAATGTCGATTTATCAGAGGGCCGACAGGCTCCCACAGGAGAAAACCG The amino acid sequence of NarU is shown in SEQ ID NO. 8 MALQNEKNSRYLLRDWKPENPAFWENKGKHIARRNLWISVSCLLLAFCVWMLFSAVTVNLNKIGFNFTTDQLFLLTALPSVSGALLRVPYSFMVPIFGGRRWTVFSTAILIIPCVWLGIAVQNPNTPFGIFIVIALLCGFAGANFASSMGNISFFFPKAKQGSALGINGGLGNLGVSVMQLVAPLVIFVPVFAFLGVNGVPQADGSVMSLANAAWIWVPLLAIATIAAWSGMNDIASSRASIADQLPVLQRLHLWLLSLLYLATFGSFIGFSAGFAMLAKTQFPDVNILRLAFFGPFIGAIARSVGGAISDKFGGVRVTLINFIFMAIFSALLFLTLPGTGSGNFIAFYAVFMGLFLTAGLGSGSTFQMIAVIFRQITIYRVKMKGGSDEQAHKEAVTETAAALGFISAIGAVGGFFIPQAFGMSLNMTGSPVGAMKVFLIFYIVCVLLTWLVYGRRKFSQK* The amino acid sequence of NarK is shown in SEQ ID NO.9 MSHSSAPERATGAVITDWRPEDPAFWQQRGQRIASRNLWISVPCLLLAFCVWMLFSAVAVNLPKVGFNFTTDQLFMLTALPSVSGALLRVPYSFMVPIFGGRRWTAFSTGILIIPCVWLGFAVQDTSTPYSVFIIISLLCGFAGANFASSMANISFFFPKQKQGGALGLNGGLGNMGVSVMQLVAPLVVSLSIFAVFGSQGVKQPDGTELYLANASWIWVPFLAIFTIAAWFGMNDLATSKASIKEQLPVLKRGHLWIMSLLYLATFGSFIGFSAGFAMLSKTQFPDVQILQYAFFGPFIGALARSAGGALSDRLGGTRVTLVNFILMAIFSGLLFLTLPTDGQGGSFMAFFAVFLALFLTAGLGSGSTFQMISVIFRKLTMDRVKAEGGSDERAMREAATDTAAALGFISAIGAIGGFFIPKAFGSSLALTGSPVGAMKVFLIFYIACVVITWAVYGRHSKK* The amino acid sequence of NapA is set forth in SEQ ID NO.10 MKLSRRSFMKANAVAAAAAAAGLSVPGVARAVVGQQEAIKWDKAPCRFCGTGCGVLVGTQQGRVVACQGDPDAPVNRGLNCIKGYFLPKIMYGKDRLTQPLLRMKNGKYDKEGEFTPITWDQAFDVMEEKFKTALKEKGPESIGMFGSGQWTIWEGYAASKLFKAGFRSNNIDPNARHCMASAVVGFMRTFGMDEPMGCYDDIEQADAFVLWGANMAEMHPILWSRITNRRLSNQNVTVAVLSTYQHRSFELADNGIIFTPQSDLVILNYIANYIIQNNAINQDFFSKHVNLRKGATDIGYGLRPTHPLEKAAKNPGSDASEPMSFEDYKAFVAEYTLEKTAEMTGVPKDQLEQLAQLYADPNKKVISYWTMGFNQHTRGVWANNLVYNLHLLTGKISQPGCGPFSLTGQPSACGTAREVGTFAHRLPADMVVTNEKHRDICEKKWNIPSGTIPAKIGLHAVAQDRALKDGKLNVYWTMCTNNMQAGPNINEERMPGWRDPRNFIIVSDPYPTVSALAADLILPTAMWVEKEGAYGNAERRTQFWRQQVQAPGEAKSDLWQLVQFSRRFKTEEVWPEDLLAKKPELRGKTLYEVLYATPEVSKFPVSELAEDQLNDESRELGFYLQKGLFEEYAWFGRGHGHDLAPFDDYHKARGLRWPVVNGKETQWRYSEGNDPYVKAGEGYKFYGKPDGKAVIFALPFEPAAEAPDEEYDLWLSTGRVLEHWHTGSMTRRVPELHRAFPEAVLFIHPLDAKARDLRRGDKVKVVSRRGEVISIVETRGRNRPPQGLVYMPFFDAAQLVNKLTLDATDPLSKETDFKKCAVKLEKV* The amino acid sequence of NapB is set forth in SEQ ID NO.11 MKSHDLKKALCQWTAMLALVVSGAVWAANGVDFSQSPEVSGTQEGAIRMPKEQDRMPLNYVNQPPMIPHSVEGYQVTTNTNRCLQCHGVESYRTTGAPRISPTHFMDSDGKVGAEVAPRRYFCLQCHVPQADTAPIVGNTFTPSKGYGK* Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A nitrate-responsive histidine kinase NarX mutant, characterized in that, The nitrate-responsive histidine kinase NarX has one or more mutations at position 507 or 562, based on the amino acid sequence as shown in SEQ ID NO.

1.

2. The nitrate-responsive histidine kinase NarX mutant according to claim 1, characterized in that, The nitrate-responsive histidine kinase mutant has the following amino acid sequence as shown in SEQ ID NO.1: leucine at position 507 is mutated to phenylalanine and serine at position 562 is mutated to arginine.

3. A polynucleotide encoding the nitrate-responsive histidine kinase NarX mutant of claim 1 or a cell expressing the nitrate-responsive histidine kinase NarX mutant of claim 1.

4. A genetically engineered Escherichia coli bacterium, characterized in that, Express the nitrate-responsive histidine kinase NarX mutant as described in any one of claims 1 to 2.

5. The genetically engineered Escherichia coli according to claim 4, characterized in that, It also expresses the response regulatory protein NarL and the fluorescent protein sfGFP; The amino acid sequence of the response regulatory protein NarL is shown in SEQ ID NO.2, and the amino acid sequence of the fluorescent protein sfGFP is shown in SEQ ID NO.

3.

6. The Enterobacter genetically engineered bacterium according to claim 5, characterized in that, Use the promoter P narG365 It expresses fluorescent proteins; promoter J23109 expresses the nitrate-responsive histidine kinase NarX mutant; promoter J23101 expresses the responsive regulatory protein NarL. Among them, promoter P narG365 The nucleotide sequence of promoter J23109 is shown in SEQ ID NO.4, the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.5, and the nucleotide sequence of promoter J23101 is shown in SEQ ID NO.

6.

7. The genetically engineered *Escherichia coli* strain according to any one of claims 4 to 6, characterized in that, The genome of the genetically engineered Escherichia coli has had narX and narL knocked out, and one or both of narU and narK have been further knocked out.

8. A two-component biosensor device for detecting nitrate content, characterized in that, It includes a bioreaction module, a fluorescence detection module, a data processing module, a wireless communication module, and a smart terminal; The bioreaction module is used to culture the Escherichia coli genetically engineered bacteria as described in claims 4-7. After being cultured to the logarithmic growth phase, the bacteria are mixed with the sample to be tested and incubated. After incubation, the bacteria are centrifuged, diluted, and resuspended to obtain the bacterial solution to be tested. The fluorescence detection module includes an excitation module, a light signal acquisition module, and a signal output module. The excitation module is used to excite fluorescence. The light signal acquisition module converts the acquired fluorescence signal into a photocurrent through a silicon photodiode, and then converts the photocurrent into a voltage signal through a transimpedance amplifier circuit. The signal output module converts the voltage signal into a digital signal. The data processing module collects digital signals and transmits them to the smart terminal via the wireless communication module; The intelligent terminal processes the received data, including: blank control subtraction, background correction, outlier removal, duplication averaging, cell density normalization, standard curve fitting and concentration conversion, and outputs the qualitative or quantitative detection results of the target substance in the sample to be tested.

9. The apparatus according to claim 8, characterized in that, In the bioreactor module, the samples to be tested include surface water, groundwater, agricultural irrigation water, and domestic sewage; The incubation conditions for the test sample and the genetically engineered Escherichia coli were 37℃ for 1-6 h. After incubation, the OD of the genetically engineered E. coli 600 Dilute to 0.1~0.

5.

10. The application of the nitrate-responsive histidine kinase NarX mutant according to any one of claims 1-2, or the polynucleotide or cell according to claim 3, or the Escherichia coli genetically engineered bacteria according to any one of claims 4-7, or the two-component biosensor device according to any one of claims 8-9 in the detection of nitrate.

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