Mercury response reporting carrier and visual monitoring and early warning microbial sensor cells and their applications

Abstract

The present invention discloses a mercury response reporting vector and a visual monitoring and early warning microbial sensor cell and its application. p2_244 The expression level is finely regulated and high-intensity ribosome binding site RBS is used GroEL A double-copy fluorescent protein gene is used as a reporter element, with a wide host vector as the backbone; the constitutive promoter sequence is shown in any one of SEQ ID NO. 4-9; the RBS GroEL The sequence is shown in SEQ ID NO.10; the green fluorescent protein gene sequence after codon optimization is shown in SEQ ID NO.11. Microbial sensor cells have lower Hg 2+ ELISA detection limit and Hg 2+ The visual detection limit provides a simple, feasible, portable and efficient solution for the rapid detection and visual early warning of environmental heavy metal mercury pollution.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a mercury response reporting carrier and a visual monitoring and early warning microbial sensor cell and applications thereof. Background Art

[0002] Mercury, cadmium, lead, chromium, copper, zinc, nickel, and arsenic, a heavy metal with similar properties, are common heavy metal contaminants in aquaculture water. These contaminants primarily originate from agricultural and industrial pollution of source water, the release of heavy metals from pond sediments, and the use of heavy metal pesticides during the aquaculture process. Excessive levels of heavy metals in aquaculture water can accumulate in aquatic organisms and affect the growth and reproduction of aquatic products. Enrichment through the food chain can pose serious risks to human food safety and health. Furthermore, a growing body of research indicates that heavy metal contamination in aquatic environments can increase bacterial antibiotic resistance, posing a broader global health threat. Therefore, rapid and efficient monitoring and early warning of heavy metal contamination in aquaculture are crucial for ensuring food safety for consumers and protecting the health of the ecological environment.

[0003] Currently, a variety of chemical analysis methods are used for heavy metal detection, such as inductively coupled plasma mass spectrometry and atomic fluorescence spectrometry. These methods offer high sensitivity and strong specificity. However, they often require expensive instrumentation and specialized personnel, tedious sample preparation, and complex testing procedures. This makes them unaffordable in some regions and even impossible for ordinary users. To overcome these limitations, research has focused on biosensors that use biomaterials such as enzymes, antibodies, and cells as recognition modules. Biosensors are biomonitoring technologies that integrate biological recognition modules with physical sensing modules to produce a measurable signal that is linearly proportional to the analyte concentration. They offer advantages such as speed, simplicity, low cost, high efficiency, and high sensitivity, providing a highly effective solution for rapid monitoring and early warning of environmental heavy metal pollution. However, currently, there are few highly sensitive and specific heavy metal-responsive elements available. Heavy metal biosensors based on known natural elements still face limitations in practical applications, such as insufficient sensitivity and low specificity. Furthermore, the application of biosensor arrays for simultaneous detection of multiple metals in environmental heavy metal pollution monitoring remains to be expanded. Summary of the Invention

[0004] In response to these major issues faced by heavy metal pollution monitoring and early warning in aquaculture waters, the present invention aims to provide a user-friendly, sensitive, efficient, fast and simple monitoring and early warning technology. Based on the MerR family transcriptional regulatory factor P2_244 on the plasmid, the mercury-responsive biosensor gene circuit is designed and optimized with a focus on promoter induction mode and reporter gene assembly. By replacing the mercury-inducible promoter with a constitutive promoter of different strengths, the mercury-responsive biosensor gene circuit is optimized. p2_244The expression level is finely regulated, and a high-intensity ribosome binding site is used in combination with a double-copy fluorescent protein element in series. A mercury-responsive biosensor gene circuit is assembled using a broad-host vector as a skeleton, thereby constructing a mercury-responsive reporter vector and a visual monitoring and early warning microbial sensor cell, which is applied to the visual monitoring and early warning of environmental heavy metal mercury pollution.

[0005] The first object of the present invention is to provide a mercury response reporter vector comprising a constitutive promoter pair. p2_244 The expression level is finely regulated and high-intensity ribosome binding site RBS is used GroEL The tandem double copy fluorescent protein gene is used as a reporter element, with a wide host vector as the backbone; the constitutive promoter is P groEL(P) 、P dnaK-1 、P rpmB 、P rpsB 、P dnaK-2 or P groES The PgroEL (P) sequence is shown in SEQ ID NO. 4, and the P dnaK-1 The sequence is shown in SEQ ID NO.5, the P rpmB The sequence is shown in SEQ ID NO.6, the P rpsB The sequence is shown in SEQ ID NO.7, the P dnaK-2 The sequence is shown in SEQ ID NO.8, the P groES The sequence is shown in SEQ ID NO.9; the RBS GroEL The sequence is shown as SEQ ID NO.10.

[0006] Preferably, the constitutive promoter is P dnaK-1 or P groES , the P dnaK-1 The sequence is shown in SEQ ID NO.5, the P groES The sequence is shown as SEQ ID NO.9.

[0007] Preferably, the fluorescent protein gene is a codon-optimized green fluorescent protein gene, the sequence of which is shown in SEQ ID NO.11.

[0008] Preferably, the inducible promoter P merT Drives the expression of the reporter element, the P merT The sequence is shown as SEQ ID NO.3.

[0009] Preferably, the p2_244 The sequence is shown as SEQ ID NO.1.

[0010] Preferably, the broad-host vector is pBBR1MCS-5.

[0011] Preferably, the constitutive promoter P dnaK-1 or P groES Replace the mercury-inducible promoter P merR drive p2_244 Gene expression, with high intensity ribosome binding site RBS GroEL Two tandem copies of the codon-optimized green fluorescent protein gene served as reporter elements, driven by the inducible promoter P merT Drives expression of the reporter element; the P dnaK-1 The sequence is shown in SEQ ID NO.5, the P groES The sequence is shown in SEQ ID NO.9, the RBS GroEL The sequence is shown in SEQ ID NO.10, the green fluorescent protein gene sequence after codon optimization is shown in SEQ ID NO.11, and the P merT The sequence is shown as SEQ ID NO.3.

[0012] The second object of the present invention is to provide a mercury visual monitoring and early warning microbial sensor cell containing the mercury response reporting carrier.

[0013] Preferably, the cells are mercury resistant but have no endogenous p2_244-p2_241 Sphingobacterium spp. Sphingobium abikonense NBRC 16140.

[0014] The third object of the present invention is to provide the use of the above-mentioned mercury response reporting carrier or the above-mentioned mercury visual monitoring and early warning microbial sensor cell in the rapid detection and visual early warning of environmental heavy metal mercury pollution.

[0015] The fourth object of the present invention is to provide a sequence as shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID NO.11 for constructing a mercury response reporting vector, constructing a visual monitoring and early warning microbial sensor cell or detecting Hg 2+ Application in content.

[0016] The present invention is based on the xenobiotic sphingomyelinase Sphingobium xenophagum The MerR family transcriptional regulatory factor P2_244 on the plasmid of the C1 strain was used as the mercury recognition element, and the constitutive promoter P with different expression strengths was used. C (P groEL(P) 、P dnaK-1 、P rpmB 、P rpsB 、P dnaK-2 、P groES )as well as p2_244 The inducible promoter of the gene itselfmerR Induce p2_244 Gene expression; with high intensity ribosome binding site RBS GroEL Two copies of the green fluorescent protein gene were used as reporter elements and located in S. xenophagum MerT family protein encoding genes on the C1 plasmid p2_243 The inducible promoter P merT Induce the expression of reporter elements; Using the broad-host vector pBBR1MCS-5 as the backbone, seven mercury-responsive reporter vectors (pTFmerR, pTFgroEL(P), pTFdnaK-1, pTFrpmB, pTFrpsB, pTFdnaK-2, and pTFgroES) were constructed. Then, mercury-resistant but endogenous p2_244-p2_241 Sphingobacterium spp. S. abikonense NBRC 16140 was used as a base cell, and seven mercury-responsive reporter vectors were introduced to obtain seven types of mercury-monitoring microbial sensor cells. By analyzing the mercury ion monitoring performance of the seven microbial sensor cells with different promoter induction modes, two microbial sensor cells with superior performance were discovered and obtained: S. abikonense NBRC 16140 (pTFdnaK-1) and S. abikonense NBRC 16140 (pTFgroES), which S. abikonense Hg in NBRC 16140 (pTFdnaK-1) 2+ The induced fluorescence signal intensity was correlated with Hg concentration in the range of 0.005-6.0 μmol / L 2+ There is a linear correlation, and the correlation coefficient R in the low concentration range 2 =0.976, correlation coefficient R in the high concentration range 2 =0.995,Hg 2+ The detection limit of the enzyme marker is 0.005 μmol / L (1.0 μg / L). S. abikonense Hg in NBRC 16140 (pTFgroES) 2+ The induced fluorescence signal intensity was correlated with Hg concentration in the range of 0.001-6.0 μmol / L 2+ Shows excellent dose response, with correlation coefficient R in the low concentration range 2 =0.995, correlation coefficient R in the high concentration range 2 =0.994,Hg 2+ The detection limit of the enzyme marker is as low as 0.001 μmol / L (0.2 μg / L), which is much lower than the detection limit of most chemical instruments and can meet the requirements of Hg in the Fishery Water Quality Standard (GB 11607-89). 2+Trace detection requirements (≤0.5 μg / L). In addition, S. abikonense NBRC 16140 (pTFdnaK-1) sensor cells for Hg 2+ The visual detection limit of is 2.0 μmol / L (400.0 μg / L). S. abikonense NBRC 16140 (pTFgroES) sensor cells for Hg 2+ The visual detection limit is 0.4 μmol / L (80.0 μg / L), which means that Hg concentrations above 0.4 μM (80.0 μg / L) can be observed with the naked eye. 2+ , providing a simple, feasible, portable and efficient solution for the rapid detection and visual early warning of environmental heavy metal mercury pollution.

[0017] The Sphingobium abikonense NBRC 16140 strain involved in the present invention is disclosed in the reference: Sphingobium cupriresistens sp. nov., a copper-resistant bacterium isolated from copper mine soil, and emended description of the genus Sphingobium. DOI: 10.1099 / ijs.0.040865-0. The applicant also holds and guarantees that the strain will be made available to the public within 20 years from the filing date of this application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : Schematic design of mercury-responsive biosensor gene circuits.

[0019] Figure 2 : Time-dependent response performance of different mercury-responsive biosensor cells.

[0020] Figure 3 : Detection performance of different mercury-responsive biosensor cells.

[0021] Figure 4 : Detection performance of mercury-responsive microbial sensing cells with superior performance.

[0022] Figure 5 :Detection standard curve of mercury-responsive microbial sensor cells with superior performance. A and B are S. abikonense NBRC 16140 (pTFdnaK-1) in Hg 2+ Standard curves of concentrations 0.005-1.0 μmol / L and 1.0-6.0 μmol / L; C and D are S. abikonenseNBRC 16140 (pTFgroES) in Hg 2+ Standard curves for concentrations of 0.005-1.0 μmol / L and 1.0-6.0 μmol / L.

[0023] Figure 6 : Visual detection of different mercury-responsive biosensor cells. DETAILED DESCRIPTION

[0024] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention.

[0025] Example 1: Sphingobium xenophagum Obtaining core elements and sequences in the C1 genome

[0026] Xenophagous Sphingobacteria S. xenophagum C1 (=CCTCC AB 2015198 =KCTC 42740; previously named S. hydrophobicum Comparison and analysis of the whole genome information of C1 (NCBI accession numbers CP022745, CP022746, CP022747, CP022748, CP022749, CP022750, and CP022751) revealed that there were six MerR family protein elements in its genome, namely, chr1_387 、 chr1_530 and on the plasmid p2_244 、 p2_ 246 、 p4_31 and p4_86 .in p2_246 The gene encodes an incomplete MerR family protein. chr1_387 Genes and chr1_ 388 、 chr1_389 The genes are located in the same operon, which encodes the C-terminal domain protein of acyl-CoA dehydrogenase and amino acid permease respectively. chr1_530 The genes are located within multiple operons encoding transposases, exonucleases, and DNA polymerases. p4_31 The genes are located within multiple operons encoding cation transporters. p4_86 The gene is located in the same operon as multiple hypothetical protein genes. p2_244 The gene is located in the same operon as the genes encoding MerT, MerA and heavy metal related domain proteins, and is likely to have mercury resistance function, so the p2_244 (The sequence is shown in SEQ ID NO.1) as a candidate recognition element for sensor circuit design.

[0027] Use the BPROM module from the Softberry website (www.softberry.com) to S. xenophagum C1 bacteria p2_244-p2_241 The potential promoter sequence of the gene cluster was predicted and the p2_244 Potential promoter sequence of gene (FPKM=25) TAGCCACTACAGACTCAAGAGGTATTTT , and a sequence containing a ribosome binding site is selected to initiate gene expression as an inducible P merR Promoter (sequence shown in SEQ ID NO.2). p2_243 Potential promoter sequence of gene (FPKM=25) TAGCGCCCTGACGTCCGGTTAGGCTATACT , combined with p2_244 Gene location analysis and selection p2_244 Genes containing ribosome binding sites initiate gene expression as inducible P merT Promoter (sequence shown in SEQ ID NO.3).

[0028] Xenophagous Sphingobacteria S. xenophagum C1 mRNA transcriptome data under different conditions (https: / / www.biosino.org / node / , accession numbers: OEP002610) were analyzed, and constitutive genes with different intensities were selected. p1_155 (FPKM=14), chr1_874 (FPKM=23), chr1_1881 (FPKM=152), chr1_308 (FPKM=1439), chr1_391 (FPKM=8887), chr1_236 (FPKM=21430), the potential promoter sequences of different constitutive genes were predicted using the above-mentioned Softberry website, and the p1_155 The potential promoter sequence of the gene is ATGAAGGAGAGCGACATCCTCGGCATCAT, chr1_874 The potential promoter sequence of the gene is TTGACTTGGCGGAAGTTCGCAAAAT, chr1_1881 The potential promoter sequence of the gene is TTTCGTTGCGGGTGCCAACCTCTACCTT, chr1_308 The potential promoter sequence of the gene is TTGCCATTTGCACGGCTTGTCCCACAAT, chr1_391 The potential promoter sequence of the gene TTGCCCCGGCTGATCGCGGTATCAT and chr1_236The potential promoter sequence of the gene ATGCATCCTCTGTCCTTTCCTGTTATGAC was analyzed, and the sequence containing the respective ribosome binding site was selected to start gene expression, respectively as the constitutive P groEL(P) Promoter (sequence shown in SEQ ID NO.4), P dnaK-1 Promoter (sequence shown in SEQ ID NO.5), P rpmB Promoter (sequence shown in SEQ ID NO.6), P rpsB Promoter (sequence shown in SEQ ID NO.7), P dnaK-2 Promoter (sequence shown in SEQ ID NO.8) and P groES Promoter (sequence shown in SEQ ID NO.9). chr1_236 Gene, screening chr1_236 Genes and chr1_235 The strong ribosome binding site sequence AAGAAAGAGGG between genes (FPKM=24540) does not contain a promoter, and two reporter genes are connected in series as RBS GroEL Ribosome binding site (sequence shown in SEQ ID NO.10).

[0029] Example 2: Design of mercury-responsive biosensor gene circuits and assembly of reporter vectors

[0030] MerR protein binds to Hg ions and induces mer The characteristics of gene cluster expression are used to design mercury-responsive biosensor gene circuits. First, the green fluorescent protein gene is matched S. xenophagum C1 and S. abikonense The codons of NBRC16140 were optimized. The optimized sequence is shown in SEQ ID NO.11. The unoptimized green fluorescent protein gene sequence is shown in SEQ ID NO.12 (the unoptimized codons of GFP are in S. xenophagum C1 and S. abikonense NBRC16140 had no mercury-induced fluorescence signal, with a value of 0). Then, a gene circuit with an inducible promoter was designed and modified. merR (Sequence as SEQ ID NO.2) induced p2_244 The gene (sequence shown in SEQ ID NO.1) is expressed; with a high-intensity ribosome binding site RBS GroEL (Sequence shown in SEQ ID NO.10) Two copies of the codon-optimized green fluorescent protein gene (sequence shown in SEQ ID NO.11) were used as reporter elements, with the inducible promoter P merT(Sequence as shown in SEQ ID NO.3) Induce the expression of the reporter element and assemble the biosensor gene circuit sequence TFmerR ( Figure 1 A). The large biosensor gene circuit sequence TFmerR was synthesized by Sangon Biotech (Shanghai) Co., Ltd., with Sac I (GAGCTC) and Kpn I (GGTACC) restriction sites designed at both ends of the TFmerR biosensor gene fragment. Plasmid DNA from the broad-host vector pBBR1MCS-5 was extracted using a plasmid extraction kit (Cat. No. DC201-01) from Nanjing Novozymes Biotech Co., Ltd. The pBBR1MCS-5 plasmid DNA was digested with FastDigest Sac I (Cat. No. FD1134) and FastDigest Kpn I (Cat. No. FD0524) restriction enzymes from Thermo Fisher Scientific (China) Co., Ltd. at 37°C. The digested plasmid fragment was then purified using a purification kit (Cat. No. DC301-01) from Nanjing Novozymes Biotech Co., Ltd. The biosensor gene fragment TFmerR was ligated into the linearized pBBR1MCS-5 vector using the Homologous Recombination Seamless Cloning Kit (Cat. No. CU201-02) from Beijing TransGen Biotech Co., Ltd., and then heat-shocked into competent Escherichia coli TOP10 cells. Positive clones were screened on LB solid medium containing 50 mg / L gentamicin. Positive clones were selected, and plasmid DNA was extracted using a plasmid extraction kit (Cat. No. DC201-01) from Nanjing Novozymes Biotech Co., Ltd. PCR amplification of the biosensor gene fragment TFmerR was performed using Taq enzyme premix (Cat. No. P131-01) from Nanjing Novozymes Biotech Co., Ltd. and primers bbr-U (CCAGGGTTTTCCCAGTCACG) and bbr-D (CTTTACACTTTATGCTTCCG). The PCR fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification, resulting in the successful assembly of the mercury-responsive inducible reporter vector pTFmerR.

[0031] Then, the gene circuit of constitutive promoter was designed and modified. C (P groEL(P) 、P dnaK-1 、P rpmB 、P rpsB 、P dnaK-2 、P groES ) (sequences shown in SEQ ID NO.4 - NO.9) to replace the mercury-inducible promoter P merR (Sequence as SEQ ID NO.2), respectively p2_244The expression level of the gene (sequence shown in SEQ ID NO.1) is finely regulated; the high-intensity ribosome binding site RBS is also used GroEL (Sequence shown in SEQ ID NO.10) Two copies of the codon-optimized green fluorescent protein gene (sequence shown in SEQ ID NO.11) were used as reporter elements, with the inducible promoter P merT (Sequence as shown in SEQ ID NO.3) induced the expression of reporter elements, and assembled the biosensor gene circuit sequences TFgroEL(P), TFdnaK-1, TFrpmB, TFrpsB, TFdnaK-2, TFgroES ( Figure 1B). These large biosensor gene circuit sequences were synthesized by Sangon Biotech (Shanghai) Co., Ltd., with Sac I (GAGCTC) and Kpn I (GGTACC) restriction sites designed at both ends of each biosensor gene fragment. Plasmid DNA from the broad-host vector pBBR1MCS-5 was extracted using a plasmid extraction kit (Cat. No. DC201-01) from Nanjing Novozymes Biotech Co., Ltd. The pBBR1MCS-5 plasmid DNA was digested with FastDigest Sac I (Cat. No. FD1134) and FastDigest Kpn I (Cat. No. FD0524) restriction enzymes from Thermo Fisher Scientific (China) Co., Ltd. at 37°C. The digested plasmid fragments were then purified using a purification kit (Cat. No. DC301-01) from Nanjing Novozymes Biotech Co., Ltd. The biosensor gene fragments TFgroEL(P), TFgroES, TFdnaK-1, TFrpmB, TFrpsB, and TFdnaK-2 were ligated with the linearized pBBR1MCS-5 vector using the homologous recombination seamless cloning kit (Cat. No. CU201-02) from Beijing TransGen Biotech Co., Ltd., and then heat-shocked into Escherichia coli TOP10 competent cells. Positive clones were screened using 50 mg / L gentamicin-resistant LB solid medium. Positive clones were picked and plasmid DNA was extracted using the plasmid extraction kit (Cat. No. DC201-01) of Nanjing Novozymes Biotech Co., Ltd. The Taq enzyme premix (Cat. No. P131-01) and primers bbr-U (CCAGGGTTTTCCCAGTCACG) and bbr-D (CTTTACACTTTATGCTTCCG) of Nanjing Novozymes Biotech Co., Ltd. were used to perform PCR amplification of the biosensor gene fragments TFgroEL(P), TFdnaK-1, TFrpmB, TFrpsB, TFdnaK-2, and TFgroES, respectively. The PCR fragments were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification. The six mercury-responsive constitutive reporter vectors pTFgroEL(P), pTFdnaK-1, pTFrpmB, pTFrpsB, pTFdnaK-2, and pTFgroES were successfully assembled.

[0032] Example 3: Construction and performance analysis of mercury-responsive microbial sensor cells

[0033] Selection of mercury-resistant but endogenous p2_244-p2_241 Genetic elements S. abikonenseNBRC 16140 was used as the chassis to construct competent cells, and seven recombinant reporter vectors pTFmerR, pTFgroEL(P), pTFdnaK-1, pTFrpmB, pTFrpsB, pTFdnaK-2, pTFgroES and the empty vector pBBR1MCS-5 were transformed into S. abikonense Positive clones were screened in competent cells of NBRC 16140 using 50 mg / L gentamicin-resistant LB solid medium. DNA from these clones was then used for PCR amplification and verification of the TFmerR, TFgroEL(P), TFdnaK-1, TFrpmB, TFrpsB, TFdnaK-2, and TFgroES gene fragments (using the same method as in Example 2). Seven microbial sensor cells containing different biosensor reporter vectors were ultimately obtained. S. abikonense NBRC 16140 (pTFmerR), S. abikonense NBRC 16140 (pTFgroEL(P)), S. abikonense NBRC 16140 (pTFdnaK-1), S. abikonense NBRC 16140 (pTFrpmB), S. abikonense NBRC 16140 (pTFrpsB), S. abikonense NBRC 16140 (pTFdnaK-2), S. abikonense NBRC 16140 (pTFgroES) and empty vector control cells S. abikonense NBRC 16140 (pBBR1MCS-5).

[0034] Inoculate the above seven microbial sensor cells and empty vector control cells into LB medium (10.0 g peptone, 5.0 g yeast extract, 5.0 g NaCl per liter of medium, with water as the solvent) containing 50 mg / L gentamicin and culture overnight in a shaking incubator at 30°C and 180 rpm. The next day, transfer the overnight culture to a fresh LB medium tube at a volume ratio of 2% inoculum and continue to culture until the bacterial solution OD reaches 0. 600 =0.6-0.8. Collect the bacterial solution into a 10 mL centrifuge tube and centrifuge at room temperature and 6000× gThe cells were centrifuged under 4% CO 2 for 3 min, the supernatant was discarded, and the cells were washed once with an inorganic salt medium (containing per liter Na2HPO4·12H2O 2.0 g, KH2PO4 0.7 g, NH4Cl 0.5 g, NaCl 0.3 g, MgSO4·7H2O 0.1 g, CaSO4·2H2O 0.05 g, FeCl3·6H2O 0.2 mg, NaMoO4 0.2 mg, MnCl2·4H2O 0.2 mg, CuCl2·2H2O 0.2 mg, ZnSO4 0.2 mg, H3BO3 0.3 mg, CoCl2·6H2O 0.4 mg, peptone 0.2 g / L, yeast powder 0.1 g / L, glucose 5.0 g / L, and water as solvent) at room temperature. g After centrifugation for 3 min under the same conditions, the supernatant was discarded, and the bacteria were resuspended in inorganic salt medium until the OD 600 ≈0.8. In a 96-well all-black polystyrene microplate, different bacterial cultures were added sequentially at a volume of 200 μL per well, followed by the addition of 2.0 μL of HgCl2 stock solution (stock solution concentrations were 0, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, and 0.5 mmol / L), respectively, to achieve final HgCl2 concentrations of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 μmol / L, respectively. Each concentration gradient was set up in triplicate, and a bacterial culture without HgCl2 was used as the control group. The bacterial culture was cultured at 30°C using a multifunctional microplate reader, and the fluorescence value of green fluorescent protein was measured under the conditions of excitation wavelength Ex = 489 nm and emission wavelength Em = 508 nm. The total number of kinetic cycles was set to 16, with an interval of oscillation on for 10 s and off for 59 min 50 s. The fluorescence value and OD of the bacterial culture were measured every 1 h. 600 The OD value is measured at each time 600 The values ​​were normalized and the fluorescence data were calibrated.

[0035] The fluorescence responses of mercury-responsive microbial sensor cells driven by constitutive promoters and inducible promoters of different strengths at different time points were measured. The results showed that the seven mercury-responsive microbial sensor cells constructed had the highest fluorescence response to Hg 2+ The fluorescence intensity increased with the extension of incubation time and reached the peak value at 6-8 h, indicating that MerR protein can stimulate Hg 2+ The recognition of the downstream reporter gene and the activation of the expression of the downstream reporter gene require a certain reaction time ( Figure 2By comparing the performance of mercury-responsive microbial sensor cells driven by constitutive promoters and inducible promoters of different strengths, it was found that the promoter expression strength and pattern have an impact on the sensitivity and dynamic range of the sensor cells. dnaK-1 (FPKM=23), P groEL(P) (FPKM=14), P rpmB (FPKM=152) driven by a moderately strong constitutive promoter P rpsB (FPKM=1439), P dnak-2 The mercury response fluorescence signal obtained by driving the high-intensity constitutive promoter P was comparable to that obtained by driving the low-intensity constitutive promoter (FPKM=8887), which was slightly improved compared to that obtained by driving the low-intensity constitutive promoter P. groES (FPKM=21430) the mercury response fluorescence signal obtained by driving the Hg concentration increased significantly. 2+ , P groEL(P) (FPKM=14), P rpmB (FPKM=152), P rpsB (FPKM=1439), P dnak-2 (FPKM=8887), P groES (FPKM=21430) driven by mercury response fluorescence signals of 3.75, 3.97, 6.15, 6.31, 22.98 RFU / OD, which is higher than that of p2_244 The inducible promoter of the gene itself merR (FPKM=25) driven by the mercury response fluorescence signal of 4.83 RFU / OD increased by 0.78, 0.82, 1.27, 1.31, and 4.76 times, respectively ( Figure 3 ). This shows that under the same low-intensity expression level conditions, the mercury-responsive fluorescence signal driven by the inducible promoter is better than that driven by the constitutive promoter. However, as the expression intensity of the constitutive promoter increases, the mercury-responsive performance of the biosensor gene circuit driven by it is significantly enhanced. However, it is worth noting that the low-intensity constitutive promoter P dnaK-1 The mercury response fluorescence signal obtained by driving the promoter (FPKM=23) was significantly different from that of other low-intensity promoters. The mercury response fluorescence signal obtained by driving the promoter to 5.0 μmol / L was 20.66 RFU / OD, which was significantly higher than that of the promoter using p2_244 The inducible promoter of the gene itself merR The mercury-responsive fluorescence signal of 4.83 RFU / OD was driven, which increased by 4.28 times, second only to P groES , most likely due to the prediction results from the Softberry website. dnaK-1 Gene promoter and actual dnaK-1The gene promoter is inconsistent, and its expression FPKM value may not be 23. The specific expression intensity needs further confirmation. But in general, using different strength constitutive promoters to replace mercury inducible promoters has a positive effect on p2_244 The expression level was finely regulated to obtain mercury-responsive microbial sensor cells with superior performance.

[0036] Example 4: Detection Performance Analysis of Superior Mercury-Response Microbial Sensor Cells

[0037] Choose a wider Hg 2+ Concentration range (0-10.0 μmol / L), further explore the two excellent performance of mercury-responsive microbial sensor cells S. abikonense NBRC 16140 (pTFdnaK-1) and S. abikonense The detection performance, culture and testing methods of NBRC 16140 (pTFgroES) were the same as those in Example 3, wherein the HgCl2 mother solution concentrations were 0, 0.0001, 0.0005, 0.001, 0.002, 0.004, 0.006, 0.008, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mmol / L), the final HgCl2 concentrations were 0, 0.001, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 µmol / L. The results showed that S. abikonense NBRC 16140 (pTFdnaK-1) is not only 2+ Highly linear response (R 2 =0.976, and also had excellent linear response (R 2 =0.995), Hg 2+ The detection limit of the microplate reader was 0.005 μmol / L (1.0 μg / L) ( Figure 4 , Figure 5 AB). S. abikonense The fluorescence intensity of NBRC 16140 (pTFgroES) in Hg 2+ Excellent linear responses were observed in both the low concentration range (0.001−1.0 μmol / L) and the high concentration range (1.0-6.0 μmol / L), with correlations of R 2 = 0.995 and R 2 =0.994,Hg2+ The detection limit of the microplate reader is as low as 0.001 μmol / L (0.2 μg / L) ( Figure 4 , Figure 5 CD), which is far below the detection limit of most chemical instruments. According to the Fishery Water Quality Standard (GB 11607-89), Hg 2+ The upper limit of the permissible concentration of mercury is 0.0005 mg / L (i.e. 0.5 μg / L). S. abikonense Hg from NBRC 16140 (pTFgroES) 2+ The detection limit of the enzyme marker is 0.2 μg / L, which can fully meet the requirements of trace Hg in aquaculture water. 2+ detection and early warning needs.

[0038] In addition, the development of visual biosensors can simplify operations and improve the efficiency of actual detection applications. Therefore, all the mercury-responsive microbial sensor cells constructed above were placed in a 0-10.0 μmol / L Hg concentration gradient. 2+ Under induction, compare the green fluorescence output intensity of each sensor cell under UV excitation. Transfer all sensor cells to LB tubes containing 50 mg / L gentamicin and culture overnight in a shaking incubator at 30°C and 180 rpm. The next day, transfer the overnight culture to a fresh 5 mL LB medium tube at a 2% volume ratio and continue to culture until the bacterial solution OD reaches 0. 600 =0.6-0.8. 50.0 μL of HgCl2 stock solution (stock solution concentrations were 0, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L) were added sequentially to make the final HgCl2 concentrations 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 μmol / L, respectively. The bacterial solution without HgCl2 was used as the control group and cultured for another 8 h. Collect all bacterial solutions into 1.5 mL centrifuge tubes and incubate at room temperature at 6000× g The cells were centrifuged for 3 min under the same conditions, the supernatant was discarded, the cells were washed once with inorganic salt medium, and finally resuspended with 200 μL inorganic salt medium. The cells were irradiated with ultraviolet light and visually detected. The results showed that with the increase of Hg 2+ As the concentration increases, only the cells with superior performance S. abikonense NBRC 16140 (pTFdnaK-1) and S. abikonense The change in green fluorescence intensity emitted by NBRC 16140 (pTFgroES) can be observed with the naked eye ( Figure 6 A). In order to further verify the sensitivity threshold of sensor cell visualization detection, S. abikonense NBRC 16140 (pTFdnaK-1) and S. abikonense NBRC 16140 (pTFgroES) sensor cells were transferred to a black 96-well plate and observed again under UV light. S. abikonense The visual detection limit of NBRC 16140 (pTFdnaK-1) was 2 μmol / L (400.0 μg / L), while S. abikonense NBRC 16140 (pTFgroES) in Hg 2+ It can be detected visually with the naked eye at a concentration of 0.4 μmol / L (80.0 μg / L) Figure 6 B), provides a simple, feasible, portable and efficient solution for the rapid detection and visual early warning of environmental heavy metal mercury pollution.

[0039] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

[0040] SEQ ID NO.1( p2_244 nucleotide sequence)

[0041] ATGGAGCAACAGGTCGGCATCTTGCGTGCCCAGCTTGCCCGGAAAACAGGCTGCAATCTCGAAACCATCCGCTATTACGAGAAGGTGGGATTGCTGCCGGGGCCGCCTCGCAGTTCCAACGGCTACCGCGTCTATTCGCCGGAACTGGTGCAAAGGTTGCAGTTCATCCTGCGCGCGCGCGACCTTGGCTATGCAATGGATGAGATACGGTCATTGTTGTCGCTCACCGATACCGGTGCACAAACCTGCGCGGAGGTTATGGCGAGAACCGAACTCCACCTTGAAGATGTCCGCCGCCGCATTGCAGATTTGCAGAAGATAGAGGTGACGCTGGCGACCACGTTAGCCAGATGCACTGGAGATGACGTTGCCGAATGTCCCATCCTGGAAGCACTCCAGTTTTTACCCCATCAAGGCAATTGA

[0042] SEQ ID NO.2 (P merR Promoter sequence)

[0043] GGCTTGAATCCCTTGTTCCAACTGAGTGATTCGCAGTGTAGGCTCTG TAGCCACTACAGACTCAAGAGG TATTTT C

[0044] SEQ ID NO.3 (P merT Promoter sequence)

[0045] TGCGCACTCTCTCCATCAAAATGTCAGGTTGAAATCTATGATACGCAACGACATTCGTCAACAAATTCCGTTTAGTACCCTAATCTGACAGAAACCGCGG TAGCGCCCTGACGTCCGGTTAGGCTATACT CCAAGCTGACATCACAAAATCAAATCCCTCAAAGATGGCGGAAAATACCTCTTGAGTCTGTAGTGGCTACAGAGCCTACACTGCGAATCACTCAGTTGGAACAAGGGATTCAAGCC

[0046] SEQ ID NO.4 (P groEL(P) Promoter sequence)

[0047] GCAAATGGTCGGGCAGCGAGGTTCGCATCGATGGCGAGGATCTGCTCATC ATGAAGGAGAGCGACATCC TCGGCATCAT CGAGCCCGTCGCGGAGCTCAAGCAGGCCGCCTGACGGGTCGCCCCCATCCCTCGAACATCAGGAAGGACAAAGGAAAGGAGTTAGGCAG

[0048] SEQ ID NO.5 (P dnaK-1 promoter sequence)

[0049] CCTAATCCAGTATGTCTGAAAAAAGTCTTTCGCCAGCATTCGTGGTGGAA TTGACTTGGCGGAAGTTCG CAAAAT TCGCCCATGTTGCGCATGGCAAGTTTTTCGCAAGATCGACGATCGGAAAGAGCCGGGGCAGAGCATGGCAGCATAGTCGCGAGTAGGACAGCCTTGTCCCGATGCGATGGCGATGCCATAGGGCGCGCCCCCCCTTCCCCGGAGACAAAAGC

[0050] SEQ ID NO.6 (P rpmB promoter sequence)

[0051] TCAGGGTTTCTTGACGGCGACAGTCGGCACTTCGACGG TTTCGTTGCGGGTGCCAACCTCTACCTT GGCGACGCTGGCTTCATATTTGGGAAGCGCGCCTTCCTTGACGGCGATTTCGGGAAGCCGTCCGTCCCGCGTTTTCTGCAAGTCGATGAAGCCGGTGGCGATGCCGCCAGCCAGCACGAGCAACAGAATGATCAACAGCCCGCCAATGAATCGCATTTCAACCTCCCATATGTTACGGGGCGACAACGCTTCGCCGCGCAAAAGGGTTGCGACACGCCGAGCGGCGGGTTGACGGCCAGAGGGAATCCCGTTATCGGCGCGGCTTTCCGAACGAACCCGAATTACAAGGTTGATTGACT

[0052] SEQ ID NO.7 (P rpsB promoter sequence)

[0053] GGTAAACAGGGTCACTGCAACGAATGTGAACATGGAGATCACTTTCTATC TTGCCATTTGCACGGCTTG TCCCACAAT GACGGTCCAAACGGCGGTTAAGCCTATTTGTTCCGCTTGTTCACGTTATGTTCCTATCTTTGGGGCAAGTCAACCCCTTGCGCAATGTTTCGCCTGCCGTTAAAGGCGCGCCCTATTCCACATGGGAATCGACATATCCGGTGCCGGCGGGGTCTTTGAGGCTCCTTCCCGGTTCCTGATTCCCAGAGGCACAACCGGAAGGAGAATATCTT

[0054] SEQ ID NO.8 (P dnaK-2 Promoter sequence)

[0055] CATCGACCAGCAAGCGGTCGCCATTGGCCATCAGGAAGGTCAGCTGCATC TTGCCCCGGCTGATCGCGG TATCAT TGACGCCCCACAGCATCAGATGCTTGCCGCCGGGGGCGAAGGCGACCTCACCCTTGGCCGGAATATCGACACTGTCGATTCGCTCCATCGTCATCATGCCGTCCTTGTCGACGCTTTCATGCATCTCGACCTTGAGCGCGTAATCGGTCAGCACGCCGCGTATCTGCCCGCCCGCATCGCCGCCATGCGCGACGAAATAGCCGGCCGATGGCGTTTCCTTATTGGGGCTCAACCGTACCCAGGCCTGGTCGATATAGGTCGGTGCCGGGTCGCCGCACGCGGCCAGCGCCAGCGGAGCCAAGAGGGCGAAAAGGGCAAAGGGGGCGCGCATCAACTCATTCCTGACTGTCTCGTTTCCGGTCAGCGTACATAGGTAATCGCCCCAGCTTCTTGTGCCAAGCAAAAGCGCCCCTATATCGCCCTTGCAAACCGGCCTTGCCTTGGCGCTTTCGTGAGGTATGGGGCCATCAACCGCTGAATAGAATTTGGGGTTCAAAGAGGACACA

[0056] SEQ ID NO.9 (PgroES Promoter sequence

[0057] TTTGCATTGTCCGGATCGTTGATGTCGACCGCGACGACGATTGTGCCGAA ATGCATCCTCTGTCCTTTC CTGTTATGAC TAGACAACGAGGACGGCGGAAAAGCGATGCGACCGCCATCAAAGAATGCGCTGGCGACATATCTCGCCGGAATGCGCAACAGACGGGAAATGTCGGCCGAAAAAATCACCGCGAAAATTTCGCTACTAATCCTTGACCCGCCTTTTGGTTGCACCATATGAGGGCGCGCTAGCACTCTCCTTCA

[0058] SEQ ID NO.10

[0059] CCCGATCCTTTTCCTGACTATTCCCATTTGC AAGAAAGAGGG TTTTTCCC

[0060] SEQ ID NO.11 (Nucleotide sequence of gfp - Sphingobium)

[0061] ATGCGTAAGGGCGAAGAGCTGTTCACCGGTGTCGTGCCGATCCTGGTCGAACTGGACGGCGACGTTAATGGCCATAAGTTCAGCGTCTCCGGCGAAGGCGAAGGCGATGCTACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACGACCGGCAAGCTGCCCGTGCCGTGGCCCACCCTCGTCACCACGTTCGGTTATGGCGTTCAGTGCTTCGCGCGCTATCCGGACCACATGAAGCAGCATGACTTCTTCAAGTCGGCCATGCCCGAAGGCTATGTCCAGGAACGTACCATCTTCTTTAAGGACGACGGCAACTATAAGACGCGCGCTGAAGTCAAGTTCGAAGGCGACACCCTGGTTAACCGCATCGAACTGAAGGGCATCGACTTTAAGGAAGACGGCAACATCCTGGGTCACAAGCTGGAATACAACTATAACTCGCACAACGTCTATATCATGGCCGACAAGCAGAAGAATGGCATCAAGGTCAACTTCAAGATCCGTCACAATATCGAAGACGGCTCGGTTCAGCTCGCGGACCACTATCAGCAGAACACCCCCATCGGCGACGGCCCGGTCCTGCTGCCCGACAACCATTACCTGTCGACCCAGTCGGCCCTGTCGAAGGACCCGAACGAAAAGCGCGATCACATGGTCCTGCTGGAGTTCGTCACCGCGGCCGGTATCACCCATGGCATGGACGAGCTGTACAAGTAA

[0062] SEQ ID NO.12 (Nucleotide sequence of gfp)

[0063] ATGCGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAA

[0064] SEQ ID NO.13 (Amino acid sequence of p2_244)

[0065] MEQQVGILRAQLARKTGCNLETIRYYEKVGLLPGPPRSSNGYRVYSPELVQRLQFILRARDLGYAMDEIRSLLSLTDTGAQTCAEVMARTELHLEDVRRRIADLQKIEVTLATTLARCTGDDVAECPILEALQFLPHQGN

[0066] SEQ ID NO.14 (Amino acid sequence of GFP - Sphingobium)

[0067] MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0068] SEQ ID NO.15 (Amino acid sequence of GFP)

[0069] MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

Claims

1. A mercury response reporting carrier, characterized in that: Contains constitutive promoter pairs p2_244 Finely regulate gene expression levels and use high-intensity ribosome binding sites (RBS) GroEL Tandem double copies of fluorescent protein genes serve as reporter elements, with a broad-host vector as the backbone; The constitutive promoter P dnaK-1 or P groES Replace the mercury-inducible promoter P merR drive p2_244 Gene expression, with high intensity ribosome binding site RBS GroEL Two tandem copies of the codon-optimized green fluorescent protein gene served as reporter elements, driven by the inducible promoter P merT Drives expression of the reporter element; the P dnaK-1 The nucleotide sequence is shown in SEQ ID NO.5, and the P groES The nucleotide sequence is shown in SEQ ID NO.9, and the RBS GroEL The nucleotide sequence is shown in SEQ ID NO.10, the nucleotide sequence of the green fluorescent protein gene after codon optimization is shown in SEQ ID NO.11, and the P merT The nucleotide sequence is shown in SEQ ID NO.3, p2_244 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The mercury response reporting carrier according to claim 1, characterized in that The broad-host vector is pBBR1MCS-5.

3. A mercury visual monitoring and early warning microbial sensor cell, characterized in that: Containing the mercury response reporter vector according to any one of claims 1-2, the cell is mercury-resistant but has no endogenous p2_244-p2_241 Sphingobacterium spp. Sphingobium abikonense NBRC 16140.

4. Use of the mercury-responsive reporting vector according to any one of claims 1 to 2 or the mercury visual monitoring and early warning microbial sensor cell according to claim 3 in rapid detection and visual early warning of environmental heavy metal mercury pollution.

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