Arsenic-responsive whole-cell biosensor and construction method and application thereof

Abstract

The present application belongs to the field of synthetic biology and biosensor technology, and relates to an arsenic-responsive whole-cell biosensor and a construction method and application thereof. The biosensor takes Escherichia coli as a host, comprises an arsenic-responsive regulation module of arsR1 gene and ars operon from Corynebacterium glutamicum, and a cascade signal amplification device composed of hrpR gene, hrpS gene and PhrpL promoter in series; wherein the cascade signal amplification device is located downstream of the regulation of the arsenic-responsive regulation module. The biosensor further comprises a report output module, which can be an EmGFP fluorescent report module or a lacZ / X-gal visual color development module. The former is used for fluorescent quantitative detection of trivalent arsenic, and the latter is used for naked eye judgment or semi-quantitative analysis combined with RGB channel values of a smart phone. The detection limit of the biosensor for trivalent arsenic is ≤0.1 ug / L, and the biosensor is used for detection of trivalent arsenic in water samples or food and beverages.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and biosensor technology, and relates to an arsenic-responsive whole-cell biosensor, its construction method, and its application. Specifically, it relates to a highly sensitive fluorescent and visualized whole-cell biosensor based on a heterologous arsenic-responsive sensing module derived from Corynebacterium glutamicum and a cascade transcription amplification device, as well as its construction method and application. Background Technology

[0002] Arsenic pollution is a persistent global public health challenge. Arsenic can be released into the environment through natural and anthropogenic processes such as mining, smelting, and agricultural activities. Its toxicity depends primarily on its chemical form; As(III) is approximately 40-60 times more toxic than As(V) due to its strong interaction with protein sulfhydryl groups. Long-term intake of As(III) can cause skin lesions, nerve damage, cardiovascular dysfunction, and multiple organ cancers. Inorganic arsenic has been classified as a Group 1 carcinogen. Currently, As(III) detection mainly relies on atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS). While these methods are accurate and reliable, they generally rely on expensive instruments, cumbersome pretreatment, and specialized personnel, making them unsuitable for rapid on-site detection. Therefore, developing a portable sensing platform that is easy to operate, low-cost, highly sensitive, and selective for As(III) is of great significance.

[0003] Whole-cell microbial biosensors specifically bind to target analytes through molecular recognition elements and convert recognition events into quantifiable optical or electrical signals. With advantages such as low cost, ease of operation, and direct reflection of As(III) bioavailability, these sensors have become a promising research direction in the field of As(III) detection. The rapid development of synthetic biology has provided abundant tools for gene circuit design and optimization, driving the development of high-performance arsenic-responsive whole-cell biosensors. Currently, most arsenic-responsive biosensors rely on the ArsR regulatory system derived from *Escherichia coli*, such as the ArsRBC operon on chromosomes or the ars operon in plasmid R773. Although studies have explored sensing modules such as *Geobacter sulfurreducens*, the available sensing modules remain severely lacking in terms of source and functional diversity. This limitation in element source restricts the expansion of the biological element library and further improvement of sensor performance. Regarding sensitivity enhancement strategies, modular transcription cascade amplification technology can significantly enhance signal output at the gene circuit level. However, almost all existing amplification devices are used in conjunction with E. coli homologous ArsR sensing modules, and their compatibility with heterologous sensing elements remains unknown.

[0004] Corynebacterium glutamicum is one of the most arsenically resistant microorganisms known, with its genome containing two complete arsenic resistance operons, ars1 and ars2, possessing a unique arsenic sensing and regulatory mechanism. This genetic characteristic makes it a highly promising but underdeveloped source of heterologous arsenic-responsive elements.

[0005] Therefore, this invention provides a highly sensitive, visualized whole-cell biosensor constructed based on an arsenic-responsive regulation system derived from Corynebacterium glutamicum. This invention expands the range of arsenic-responsive sensing elements and successfully couples a heterogeneous sensing module with a cascaded signal amplification module, significantly improving sensing performance. Specifically, the detection limit of the constructed fluorescent detection system is 0.011 ug / L, which is better than the lowest reported value of 0.018 ug / L of the existing E. coli homology system (Li, J., et al. (2023). A self-amplifying plasmid based ultrasensitive biosensor for the detection of As(Ⅲ) in water. Biosensors and Bioelectronics, 221, 114937.); the detection limit of the visualization detection system is 0.043 ug / L, which is nearly an order of magnitude lower than the lowest detection limit of 0.39 ug / L of the existing visualization arsenic sensor (Sheng-Yan Chen, et al.; De Novo Design of the ArsR Regulated ParsPromoter Enables a Highly Sensitive Whole-Cell Biosensor for ArsenicContamination. Anal. Chem. 24 May 2022;94(20):7210-7218.), truly achieving an organic combination of high sensitivity and portable detection. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing arsenic whole-cell biosensors, such as the single source and lack of diversity of sensing elements, and to provide a highly sensitive fluorescent and visualized whole-cell biosensor based on a heterologous arsenic sensing module from Corynebacterium glutamicum, as well as its construction method and application.

[0007] The technical solution of the present invention:

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a recombinant nucleic acid construct, the recombinant nucleic acid construct comprising:

[0010] (a) An arsenic response regulatory module comprising at least the gene encoding the As(III) response regulatory protein ArsR1 and the promoter ParsB1 that responds to said As(III);

[0011] (b) A reporter gene expression module, which is connected downstream of the arsenic response regulation module, contains a reporter gene that is expressed via the ParsB1 promoter in the presence of As(III);

[0012] The protein encoded by the ArsR1 encoding gene contains the amino acid sequence shown in SEQ ID NO:1, or is a functional variant that has at least 80% sequence identity with SEQ ID NO:1 and retains the As(III) binding activity, or is a functional mutant that has one or more amino acid substitutions, deletions or insertions in SEQ ID NO:1 and retains the As(III) binding activity.

[0013] The recombinant nucleic acid construct as described above further includes a constitutive promoter and a ribosome binding site, wherein the constitutive promoter and the ribosome binding site are sequentially linked to the ArsR1 coding gene; preferably, the constitutive promoter is J23119 and the ribosome binding site is B0034; more preferably, the constitutive promoter has a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; preferably, the ribosome binding site has a nucleotide sequence as shown in SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

[0014] The recombinant nucleic acid construct as described above further includes one or more signal amplification elements, which constitute a cascaded amplification device capable of amplifying the transcription signal triggered by As(III) binding;

[0015] Preferably, the cascade amplification device is Amp30Syn, which contains functional variants or equivalents of the hrpR gene, hrpS gene and their response promoter PhrpL from Pseudomonas syringae.

[0016] In the recombinant nucleic acid construct described above, the reporter gene is either the fluorescent protein encoding gene EmGFP or the β-galactosidase encoding gene lacZ;

[0017] Preferably, the lacZ encoding gene is derived from Escherichia coli K-12MG1655.

[0018] The recombinant nucleic acid construct vector, host cell, or arsenic whole-cell biosensor as described above, preferably, is Escherichia coli, and more preferably, TOP10 strain, DH5α strain, or BL21(DE3) strain.

[0019] Secondly, the present invention provides a method for constructing a host cell or arsenic whole-cell biosensor as described above, comprising the following steps:

[0020] (1) Provide a recombinant nucleic acid construct as described above;

[0021] (2) The recombinant nucleic acid construct is operatively inserted into the expression vector to obtain a recombinant expression plasmid;

[0022] (3) The recombinant expression plasmid is introduced into the host cell to obtain the recombinant host cell, which is the biosensor.

[0023] As described above, the recombinant nucleic acid construct in step (1) is obtained by chemical synthesis or by PCR amplification and assembly of genomic DNA from Corynebacterium glutamicum and Pseudomonas syringae. Preferably, the host cell in step (3) is Escherichia coli, preferably a TOP10 strain.

[0024] The application of host cell or whole-cell arsenic biosensors as described above in the detection of trivalent arsenic.

[0025] Thirdly, the present invention provides a method for detecting trivalent arsenic in a liquid sample, comprising the following steps:

[0026] (1) The biosensor described above is brought into contact with the liquid sample to be tested and co-incubated under suitable conditions to express the reporter gene. Preferably, the liquid sample is filtered through a filter membrane before detection, more preferably through a 0.22 μm filter membrane.

[0027] (2) Detect the signal of the reporter gene in the co-incubation product obtained in step (1), and determine the content of trivalent arsenic in the sample to be tested according to the standard curve;

[0028] Preferably, the biosensor is recombinant Escherichia coli; more preferably, in step (1), the sample to be tested is added to the recombinant Escherichia coli culture and co-incubated; more preferably, the recombinant Escherichia coli culture is Escherichia coli cultured to the logarithmic phase; more preferably, the Escherichia coli is co-incubated with shaking at 30℃-37℃.

[0029] As described above, when the reporter gene is lacZ, the co-incubation temperature is 37°C, and the time is 0.2-4 hours, preferably 0.5-2 hours, more preferably 1 hour; and in step (1), 0.005%-0.02%, preferably 0.01% SDBS and 50-200 μg / mL, preferably 70 μg / mL X-gal substrate are added to the co-incubation system, with the X-gal substrate concentration preferably being 70 μg / mL; the signal detected in step (2) is the 255-R value of the RGB channel, preferably, the RGB value of the reaction system is read using a smart application, and the red channel value (255-R) minus 255 is used as the characterization of blue intensity to establish its linear relationship with arsenic concentration, further preferably, the smart application is a smartphone application; when the reporter gene is EmGFP, the co-incubation temperature is 37°C, and the time is 2-10 hours, preferably 4-6 hours, more preferably 5 hours, and the signal detected in step (2) is the fluorescence value and OD 600 The ratio of .

[0030] In the above method, the liquid sample is drinking water, environmental water, or food and beverage; preferably, the liquid sample is filtered through a 0.22 μm filter membrane before detection. More preferably, the method further includes using inductively coupled plasma mass spectrometry (ICP-MS) as a reference method to perform parallel measurements on the same sample to verify the accuracy of the sensor's detection results. The biosensor's detection results for trivalent arsenic in actual samples show good consistency with the ICP-MS measurements.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) For the first time, the arsR1-ParsB1 sensing module derived from Corynebacterium glutamicum was functionally applied in Escherichia coli, overcoming the defect of the single source of traditional homologous ArsR system components, and providing a new high-performance sensing element for arsenic biosensors.

[0033] (2) The cascaded amplification device Amp30Syn was successfully coupled with the heterogeneous arsenic sensing module, verifying the compatibility between the two and reducing the sensor detection limit to the sub-ppb level (the detection limit in fluorescence mode is 0.011ug / L and the detection limit in visualization mode is 0.04ug / L), which is significantly better than the WHO standard of 10 ppb for drinking water.

[0034] (3) A dual-mode signal output system of fluorescence and visualization was constructed. The visualization mode can be interpreted by smartphone within 1 hour without the need for large instruments and equipment, thus realizing rapid on-site screening of As(III). Attached Figure Description

[0035] Figure 1A schematic diagram of the gene circuit construction of the signal cascade amplification circuit in the sensor of this invention.

[0036] Figure 2 Fitting plot of As(III) dose-response curves of the sensor before and after the introduction of the cascade amplification device.

[0037] Figure 3 Selective response diagram of the sensor to various metal ions.

[0038] Figure 4 Visualize the dose-response relationship of the sensor at different X-gal substrate concentrations.

[0039] Figure 5 Visualize the As(III) dose-effect standard curve of the sensor. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto. These embodiments provide a reference for those skilled in the art to implement the present invention and should not be construed as limiting the present invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc. used in the following embodiments are commercially available.

[0041] Example 1: Construction of a fluorescent arsenic whole-cell biosensor

[0042] I. Construction of recombinant plasmid pET-30a-CgarsR1-Amp30Syn-EmGFP

[0043] Using the commercial plasmid pET01, namely pET-30a(+)-EmGFP, as a backbone, an arsenic-responsive biosensor was constructed through multi-step assembly.

[0044] (1) Construction of whole-cell sensor

[0045] Using E. coli MG1655 genomic DNA as a template, the gene encoding the ArsR regulatory protein was amplified by PCR (Sheng-Yan Chen, Yan Zhang, Renjie Li, Baojun Wang, Bang-Ce Ye; De Novo Design of the ArsR Regulated Pars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination. Anal. Chem. 24 May 2022;94(20):7210-7218.). The constitutive promoter BBa_J23113 (SEQ ID NO:2) and the ribosome binding site BBa_B0031 (SEQ ID NO:3) were sequentially linked to the 5' end of the ArsR gene to form an ArsR constitutive expression cassette. At the same time, the homologous response promoter Pars sequence of the E. coli ArsR protein was amplified (Sheng-Yan Chen, Yan Zhang, Renjie Li, Baojun Wang, Bang-Ce Ye; De Novo Design of the ArsR Regulated Pars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination. Anal. Chem. 24 May 2022;94(20):7210-7218.). Ye;De Novo Design of the ArsR Regulated Pars Promoter Enables a Highly Sensitive Whole-Cell Biosensor for Arsenic Contamination. Anal. Chem. 24 May 2022;94(20):7210-7218.). Using pET-30a(+)-GFP as the backbone vector, the above-mentioned ArsR constitutive expression cassette was inserted into the corresponding site of the vector, and the original promoter upstream of the GFP reporter gene on the backbone was replaced with the Pars promoter, finally constructing the uncoupled arsenic response loop reference plasmid pET02.Using the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, the ArsR1-ParsB1 regulatory module (containing the encoding gene of the As(III) response regulatory protein ArsR1 and its promoter ParsB1, see reference Ordóñez E, Letek M, Valbuena N, Gil JA, Mateos LM. Analysis of genes involved in arsenic resistance in Corynebacterium glutamicum ATCC 13032. Appl Environ Microbiol. 2005 Oct;71(10):6206-15. doi:10.1128 / AEM.71.10.6206-6215.2005.PMID:16204540;PMCID:PMC1266000.) was obtained by PCR amplification. The *E. coli* ArsR-Pars module in plasmid pET02 (an uncoupled arsenic response loop derived from *E. coli*, in which the ArsR regulatory protein derived from *E. coli* MG1655 is expressed under the drive of the constitutive promoter BBa_J23113 and the ribosome binding site BBa_B0031, and the homologous Pars promoter is located upstream of the gfp reporter gene) was replaced with this module to obtain plasmid pET03. Further, the promoter-ribosome binding site combination (BBa_J23113-BBa_B0031) in pET03 was replaced with BBa_J23106 (SEQ ID NO:4)-BBa_B0032 (SEQ ID NO:5) and BBa_J23119 (SEQ ID NO:6)-BBa_B0034 (SEQ ID NO:7), respectively, to obtain plasmids pET04 and pET05.

[0046] The sequences are as follows:

[0047] SEQ ID NO:1 (Protein encoded by the ArsR1 gene): MRFGLAWPASELSLSCDTFRYIDTCRYSKYFQYRHSSIYSGFMTTLHTIQLANPTECCTLATGPLSSDESEHYADLFKVLGDPVRLRILSQLAAGGCGPVSVNELTDLMGLSQPTISHHLKKMTEAGFLDRVPEGRVVLHRVRPELFAELRTVLQIGSMEL.

[0048] SEQ ID NO:2 (constitutive promoter BBa_J23113): ctgatggctagctcagtcctagggattatgctagc.

[0049] SEQ ID NO:3 (ribosome binding site BBa_B0031): ggtttcctgtgtga.

[0050] SEQ ID NO: 4 (BBa_J23106): tttacggctagctcagtcctaggtatagtgctagc.

[0051] SEQ ID NO: 5 (BBa_B0032): ctttcctgtgtga.

[0052] SEQ ID NO: 6 (BBa_J23119): GCTAGCATTATACCTAGGACTGAGCTAGCTGTCAA.

[0053] SEQ ID NO: 7 (BBa_B0034): tttctcctcttt.

[0054] (2) Introduction of cascaded amplification devices

[0055] The coding sequence of the Amp30Syn whole-genome synthesis cascade signal amplification device, synthesized by Suzhou Azenta Life Sciences Co., Ltd., contains the hrpR gene, hrpS gene, and their response promoter PhrpL from *Pseudomonas syringae* (Baojun Wang, Mauricio Barahona, Martin Buck, Engineering modular and tunable genetic amplifiers for scaling transcriptional signals in cascaded gene networks, *Nucleic Acids Research*, Volume 42, Issue 14, 18 August 2014, 9484-9492). Using the Visclone Universal One Step Cloning Kit (Coolaber, China), this module was seamlessly cloned and ligated with an arsenic-sensing circuit derived from pET05 to obtain plasmid pET06 (i.e., pET-30a-CgarsR1-Amp30Syn-EmGFP).

[0056] All PCR-amplified or synthesized DNA fragments were assembled according to the kit instructions. Positive clones of the resulting plasmids were screened by colony PCR and verified for correctness by Sanger sequencing before being used in subsequent experiments.

[0057] like Figure 1 As shown, the plasmid is designed with a non-coupled loop and includes a constitutive promoter BBa_J23119 and a ribosome binding site BBa_B0034. The BBa_J23119 promoter and the BBa_B0034 ribosome binding site are operatively linked to the ArsR1 coding gene in sequence. The plasmid also includes a cascade transcription amplification module located between the ParsB1 promoter and the reporter gene EmGFP, consisting of the hrpR gene, the hrpS gene, and their response promoter PhrpL connected in series. This cascade transcription amplification module is integrated into the plasmid using seamless DNA assembly technology. Its working mechanism is as follows: when there is no As(III) in the detection system, the ArsR1 protein binds to the ParsB1 promoter region in the form of a dimer, preventing the transcription of downstream genes; when As(III) is present, As(III) specifically binds to the ArsR1 protein, causing it to dissociate from the promoter, activating the transcription of downstream cascade amplification devices and reporter genes, and outputting a fluorescence signal after being amplified step by step by Amp30Syn.

[0058] II. Construction of recombinant strain TOP10 / pET-30a-CgarsR1-Amp30Syn-EmGFP

[0059] The recombinant plasmid pET-30a-CgarsR1-Amp30Syn-EmGFP constructed in step one was transformed into *E. coli* TOP10 competent cells using the calcium chloride method. The transformed cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C. Single colonies were picked, and after verification by PCR and sequencing, the recombinant strain TOP10 / pET-30a-CgarsR1-Amp30Syn-EmGFP was obtained.

[0060] III. Dose-response detection of different concentrations of As(III)

[0061] The recombinant bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm with shaking for 14–16 hours to obtain overnight culture. The overnight culture was then transferred to fresh LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm for 2 hours to the logarithmic growth phase. Sodium arsenite (NaAsO2) was added to achieve final As(III) concentrations of 0, 0.0046, 0.0092, 0.018, 0.036, 0.073, 0.14, 0.29, 0.58, 1.16, 2.33, and 4.68 μg / L, respectively, and cultured at 37°C and 220 rpm with shaking for 5 hours. The bacterial cultures were collected, and 1 mL of each culture was centrifuged at 12000 rpm for 1 min. The supernatant was discarded, and the bacteria were resuspended in 1 mL of 1×PBS. Take 200 μL of resuspended bacterial solution into a 96-well microplate and measure the OD. 600 Calculate Fluorescence value and fluorescence value (excitation wavelength 487nm, emission wavelength 509nm), and calculate Fluor / OD. 600 ratio.

[0062] The results are as follows Figure 2 As shown, the As(III) response of the recombinant bacteria exhibited a clear dose-response relationship, with Fluo. / OD showing a significant increase within the concentration range of 0.018-2.33 ug / L. 600 The fluorescence value increased with increasing As(III) concentration; it tended to saturate after 2.33 ug / L. Compared with the blank control group, the fluorescence signal of the 0.018 ug / L and above concentration groups showed statistically significant differences (P < 0.01), and the detection limit was 0.011 ug / L.

[0063] IV. Selectivity of the sensor for metal ions

[0064] The recombinant bacteria constructed in step two were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm with shaking for 14-16 hours to obtain an overnight culture. The overnight culture was then transferred to fresh LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm for 2 hours. Then, 1 μg / L of As(III) and 100 μg / L of Fe(II), Zn(II), Mg(II), Mn(II), Cu(II), Ni(II), Pb(II), Hg(II), Cd(II), and Sb(III) were added, respectively, with a control group containing no added metal ions. The culture was then cultured at 37°C and 220 rpm with shaking for 5 hours. Fluo. / OD was determined according to the method in step three. 600 value.

[0065] The results are as follows Figure 3As shown, the sensor exhibits a significant fluorescence response only to As(III) and almost no response to the other 10 metal ions, demonstrating a high degree of specificity for As(III).

[0066] Example 2: Construction and Application of a Visualized Whole-Cell Arsenic Biosensor

[0067] I. Construction of recombinant strain TOP10 / pET-30a-CgarsR1-Amp30Syn-lacZ

[0068] The reporter gene in plasmid pET-30a-CgarsR1-Amp30Syn-EmGFP was replaced with the lacZ gene from *E. coli* K-12MG1655, yielding pET-30a-CgarsR1-Amp30Syn-lacZ. This plasmid was transformed into *E. coli* TOP10 competent cells using the calcium chloride method, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were picked, and after verification by PCR and sequencing, the recombinant strain TOP10 / pET-30a-CgarsR1-Amp30Syn-lacZ was obtained.

[0069] II. The Influence of X-gal Substrate Concentration on Visual Sensors

[0070] The recombinant bacteria constructed in step one of Example 2 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm with shaking for 14-16 hours to obtain an overnight culture. The overnight culture was transferred to fresh LB medium (containing 50 μg / mL kanamycin) at an inoculation rate of 1% (v / v) and cultured at 37°C and 220 rpm for 2 hours. Sodium arsenite was added to the final As(III) concentrations of 0, 0.07, 0.14, 0.29, 0.58, and 1.16 μg / L, respectively. At the same time, 0.01% (v / v) sodium dodecylbenzenesulfonate (SDBS) was added, and X-gal was added to the final concentrations of 50, 70, 100, and 200 μg / mL, respectively. The culture was then incubated at 37°C and 220 rpm with shaking for 1 hour. Take 200 μL of bacterial culture and place it in a 96-well transparent microplate. Take a picture with a smartphone on a shadowless base (20×20cm) and analyze the RGB channel values ​​(255-R) of each well using self-programmed software.

[0071] The results are as follows Figure 4 As shown, the signal-to-noise ratio was optimal and the color development effect was best when the X-gal concentration was 70 μg / mL; the blue color development was insufficient when the concentration was too low (50 μg / mL); and the background color was also obvious in the control group when the concentration was too high (100 and 200 μg / mL). Therefore, 70 μg / mL was selected as the working concentration of X-gal.

[0072] III. Visualizing the sensor's response to different concentrations of As(III)

[0073] The recombinant bacteria constructed in step one were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C and 220 rpm with shaking for 14-16 hours to obtain an overnight culture. The overnight culture was then transferred to fresh LB medium (containing 50 μg / mL kanamycin) at a 1% (v / v) inoculation rate and cultured at 37°C and 220 rpm for 2 hours. Sodium arsenite was then added to achieve final As(III) concentrations of 0, 0.03, 0.07, 0.14, 0.29, 0.58, 1.16, 2.33, 4.66, and 9.32 μg / L, respectively. Simultaneously, 0.01% (v / v) SDBS and 70 μg / mL X-gal were added, and the culture was incubated at 37°C and 220 rpm with shaking for 1 hour. Subsequently, images of each well were captured according to the method described in step two, and the value of the red channel (255-R) was calculated as a quantitative indicator of blue intensity. The results are as follows: Figure 5 As shown, within the concentration range of 0.07-2.33 ug / L, the 255-R value exhibits a good dose-response relationship with the As(III) concentration, with a detection limit of 0.04 ug / L. Semi-quantitative on-site detection of As(III) can be achieved by taking photos with a smartphone and analyzing the RGB channel values.

[0074] Example 3: Detection and Accuracy Verification of Trivalent Arsenic in Actual Samples

[0075] To verify the detection performance of the arsenic whole-cell biosensor described in this invention in a real complex matrix, this embodiment selected five environmental water samples (river water, drinking water, lake water, tap water, and laboratory distilled water) and five food and beverage samples (beverages A to E). Arsenic content was detected using the fluorescent whole-cell biosensor and the visual colorimetric whole-cell biosensor constructed in this invention, respectively. The detection results of inductively coupled plasma mass spectrometry (ICP-MS) were used as a reference standard. The detection results of all samples are shown in Table 1.

[0076] Table 1 Comparison of arsenic concentration detection results in actual samples (ug / L)

[0077]

[0078] Note: ND indicates not detected (below the method detection limit); NA indicates no valid interpretable signal; the test result is expressed as "mean ± standard deviation".

[0079] As shown in Table 1, the fluorescent whole-cell biosensor constructed in this invention can achieve stable quantitative detection of As(III) in different arsenic-containing real samples. Compared with the ICP-MS detection results, the relative deviation is 4.4%~23.7%, demonstrating good detection accuracy and resistance to matrix interference. The constructed visual colorimetric whole-cell biosensor can also effectively detect As(III) in most real samples, with a relative deviation of 3.6%~26.7% compared with the ICP-MS results, showing good detection consistency. For low-concentration arsenic samples, the fluorescent sensor exhibits higher detection sensitivity, while the visual sensor is suitable for rapid on-site screening.

[0080] The above results demonstrate that the whole-cell arsenic biosensor constructed in this invention is applicable to the detection of trace amounts of trivalent arsenic in various real-world samples, such as environmental water bodies and food and beverages, and the detection results are accurate and reliable. Among them, the fluorescent sensor has higher detection sensitivity and quantitative accuracy, while the visual sensor can meet the semi-quantitative detection needs of rapid on-site screening. Both can provide effective technical support for the rapid detection of arsenic pollution in different application scenarios.

[0081] Although the technical solutions of the present invention have been described in detail above with reference to specific embodiments, the scope of protection of the present invention is not limited thereto. For those skilled in the art, any modifications, equivalent substitutions, or improvements made to the technical solutions of the present invention without departing from the spirit and scope of the present invention should be considered to fall within the scope of protection of the present invention.

Claims

1. A recombinant nucleic acid construct, characterized in that, The recombinant nucleic acid construct comprises: (a) An arsenic response regulatory module comprising at least the gene encoding the As(III) response regulatory protein ArsR1 and the promoter ParsB1 that responds to said As(III); (b) A reporter gene expression module, which is connected downstream of the arsenic response regulation module, contains a reporter gene that is expressed via the ParsB1 promoter in the presence of As(III); The protein encoded by the ArsR1 encoding gene contains the amino acid sequence shown in SEQ ID NO:1, or is a functional variant that has at least 80% sequence identity with SEQ ID NO:1 and retains the As(III) binding activity, or is a functional mutant that has one or more amino acid substitutions, deletions or insertions in SEQ ID NO:1 and retains the As(III) binding activity.

2. The recombinant nucleic acid construct according to claim 1, characterized in that, The recombinant nucleic acid construct further includes a constitutive promoter and a ribosome binding site, wherein the constitutive promoter and the ribosome binding site are sequentially linked to the ArsR1 encoding gene; preferably, the constitutive promoter is J23119 and the ribosome binding site is B0034; more preferably, the constitutive promoter has a nucleotide sequence as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6; preferably, the ribosome binding site has a nucleotide sequence as shown in SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:

7.

3. The recombinant nucleic acid construct according to claim 1 or 2, characterized in that, The recombinant nucleic acid construct also includes one or more signal amplification elements, which constitute a cascade amplification device capable of amplifying the transcription signal triggered by the binding of As(III); Preferably, the cascade amplification device is Amp30Syn, which contains functional variants or equivalents of the hrpR gene, hrpS gene and their response promoter PhrpL derived from Pseudomonas syringae.

4. The recombinant nucleic acid construct according to any one of claims 1 to 3, characterized in that, The reporter gene is either the fluorescent protein encoding gene EmGFP or the β-galactosidase encoding gene lacZ. Preferably, the lacZ encoding gene is derived from Escherichia coli K-12MG1655.

5. A vector, host cell, or arsenic whole-cell biosensor comprising the recombinant nucleic acid construct according to any one of claims 1-4, preferably, the host cell or arsenic whole-cell biosensor is Escherichia coli, more preferably TOP10 strain, DH5α strain, or BL21(DE3) strain.

6. A method for constructing the host cell or arsenic whole-cell biosensor as described in claim 5, characterized in that, Includes the following steps: (1) Provide a recombinant nucleic acid construct as described in any one of claims 1-4; (2) The recombinant nucleic acid construct is operatively inserted into the expression vector to obtain a recombinant expression plasmid; (3) The recombinant expression plasmid is introduced into the host cell to obtain the recombinant host cell, which is the biosensor.

7. The method according to claim 6, characterized in that, The recombinant nucleic acid construct described in step (1) is obtained by chemical synthesis or by PCR amplification and assembly of genomic DNA from Corynebacterium glutamicum and Pseudomonas syringae. Preferably, the host cell described in step (3) is Escherichia coli, and more preferably, the TOP10 strain.

8. The application of the host cell or arsenic whole-cell biosensor according to claim 5 in the detection of trivalent arsenic.

9. A method for detecting trivalent arsenic in a liquid sample, characterized in that, Includes the following steps: (1) The biosensor of claim 5 is brought into contact with the liquid sample to be tested and co-incubated under suitable conditions to express the reporter gene. Preferably, the liquid sample is filtered through a filter membrane before detection, more preferably through a 0.22 μm filter membrane. (2) Detect the signal of the reporter gene in the co-incubation product obtained in step (1), and determine the content of trivalent arsenic in the sample to be tested according to the standard curve; Preferably, the biosensor is recombinant Escherichia coli; more preferably, in step (1), the sample to be tested is added to the recombinant Escherichia coli culture and co-incubated; more preferably, the recombinant Escherichia coli culture is Escherichia coli cultured to the logarithmic phase; more preferably, the Escherichia coli is co-incubated with shaking at 30℃-37℃.

10. The method according to claim 9, characterized in that, When the reporter gene is lacZ, the co-incubation temperature is 37℃, and the time is 0.2-4 hours, preferably 0.5-2 hours, more preferably 1 hour; and in step (1), 0.005%-0.02%, preferably 0.01% SDBS and 50-200 μg / mL, preferably 70 μg / mL X-gal substrate are added to the co-incubation system; the signal detected in step (2) is the 255-R value of the RGB channel, preferably, the RGB value of the reaction system is read using a smart application, and the red channel value (255-R) minus 255 is used as the characterization of blue intensity to establish its linear relationship with arsenic concentration, and more preferably, the smart application is a smartphone application; when the reporter gene is EmGFP, the co-incubation temperature is 37℃, and the time is 2-10 hours, preferably 4-6 hours, more preferably 5 hours, and the signal detected in step (2) is the fluorescence value and OD 600 The ratio of .

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