Whole-cell biosensor for detecting tetracycline in environmental water body and application of whole-cell biosensor

Abstract

The invention discloses a whole-cell biosensor for detecting tetracycline in an environmental water body and application of the whole-cell biosensor. The invention firstly provides a functional plasmid, wherein the functional plasmid is constructed based on a repressor protein TetR and a super-folded green fluorescent protein as main bodies; the functional plasmid is further utilized to construct the tetracycline specific whole-cell biosensor, the fluorescence intensity signal of the whole-cell biosensor is positively correlated with the tetracycline concentration in a low concentration range (the tetracycline concentration range is 0-0.4 mg / L), the correlation coefficient R2 is 0.998, the lowest detection limit can reach 0.01 mg / L, and the tetracycline specific whole-cell biosensor can be used for detecting the tetracycline concentration of the whole-cell biosensor. Good sensitivity is shown in the aspect of detection of low-concentration tetracycline. When being used for detecting tetracycline, the whole-cell biosensor has the advantages of simplicity, convenience and rapidness, is high in specificity and sensitivity, and has the potential of being applied to environment monitoring.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular, relates to a whole-cell biosensor for detecting tetracycline in environmental water and an application thereof. Background Art

[0002] As a new type of pollutant, antibiotics continue to receive widespread attention at home and abroad. With the development of society and the continuous improvement of living standards, antibiotics are widely used in human medicine, livestock, poultry and aquaculture. However, waste and wastewater generated in the production process and antibiotics that are not fully absorbed after use may be discharged into the environment in their original state and metabolite state, causing environmental pollution. Among them, tetracycline antibiotics (TCs), as one of the most widely used antibiotics in the world, are a class of broad-spectrum antibacterial agents that have been widely used in medicine, agriculture and aquaculture. However, the long-term improper use of tetracyclines has also brought about environmental problems such as water pollution. Studies have shown that about 30% to 90% of TCs cannot be absorbed and flow into the natural environment through different pathways, leading to chemical pollution of groundwater, surface water and drinking water.

[0003] At present, conventional detection methods for tetracycline in the environment mainly include spectrophotometry, atomic absorption spectroscopy, various types of chromatography and mass spectrometry. However, the above methods generally rely on expensive detection instruments, which are costly, cumbersome to operate, complex sample pretreatment, and long detection cycles, and are not suitable for rapid screening needs. Compared with traditional detection technologies, biosensors have the advantages of high sensitivity, good selectivity, simple operation, rapid response, reusability, and convenience for in situ detection. They are a very promising detection method. In addition, whole-cell biosensors use bacteria as recognition elements. While measuring the concentration of pollutants, they can also evaluate the biological effectiveness of pollutants. Since living biological cells are easy to culture and simple to operate, these biosensors can be reused if the nutritional conditions for cell survival can be provided, which greatly reduces the preparation and use costs of biosensors. At present, there are few research and development of whole-cell biosensors for detecting tetracycline in the environment. Patent CN118773226A provides a tetracycline engineered microbial cell sensor adsorber, which can adsorb low-concentration tetracycline in the environment, but cannot achieve quantitative detection of low-concentration tetracycline. In order to achieve quantitative detection of low-concentration tetracycline in contaminated water, it is of great significance to provide a biosensor that can quantitatively detect tetracycline. Summary of the invention

[0004] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide a functional plasmid.

[0005] The second object of the present invention is to provide a whole-cell biosensor.

[0006] The third object of the present invention is to provide an application of the whole-cell biosensor for detecting the content of tetracycline in environmental water.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention first provides a functional plasmid, comprising a plasmid vector and a reporter element and a regulatory element connected in the plasmid vector; the reporter element comprises a reporter gene and a promoter driving the reporter gene expression, and the regulatory element comprises a tetR gene, a tet operator and a promoter driving the tetR gene expression; the reporter gene is a superfolded green fluorescent protein gene; there is a spacer region with a sequence length of 2 to 6 bp between the promoter driving the reporter gene expression and the tet operator; the following elements are operably connected on the plasmid vector from 5' to 3' direction: a promoter driving the reporter gene expression, a spacer region, a tet operator, a ribosome binding site (RBS), a reporter gene, a terminator, a promoter driving the tetR gene expression and the tetR gene.

[0009] Related studies have pointed out that under environmental stresses such as heavy metals, antibiotics, and organic pollutants, transcriptional regulators, as proteins that can specifically bind to cis-acting elements in the promoter region of a gene, can specifically respond to stress signals and convert them into cellular responses, thereby enhancing cell resistance. The repressor protein TetR belongs to the first family of TetR transcriptional regulators found to be associated with bacterial resistance. It can specifically respond to tetracycline in the environment and regulate gene expression by binding to the tetO sequence in the promoter region of the resistance gene.

[0010] The invention uses tetR gene as a regulatory element, fuses a promoter with various gene elements and a plasmid skeleton to construct a new functional plasmid, screens the length of a spacer sequence (0-8 bp) and a reporter protein gene, and the results show that when the length of the spacer sequence is 2-6 bp, when TetR is present, the fluorescence activation of the reporter protein is reduced to a baseline level; when TetR is absent, strong fluorescence activation is observed, while no obvious fluorescence activation is detected in spacers of other lengths, or the repressor protein cannot inhibit the expression of the reporter gene; when the reporter gene is a gene encoding a purple pigment protein amilCP, the expression intensity of amilCP does not respond to the regulation of the TetR repressor protein under different spacers, therefore, 2-6 bp can be used as a suitable functional spacer length between the promoter and the repressor protein binding region tetO, and a superfolded green fluorescent protein is used as a reporter gene to construct a tetracycline biosensor to achieve the response to tetracycline in a low concentration range in environmental water.

[0011] Furthermore, the nucleotide sequence of the tetR gene is shown in SEQ ID No.1, the nucleotide sequence of the tet operon is shown in SEQ ID No.2, and the nucleotide sequence of the superfolded green fluorescent protein gene is shown in SEQ ID No.3.

[0012] Furthermore, the plasmid vector is placP-sfGFP.

[0013] Furthermore, the promoter driving the expression of the tetR gene is the natural promoter of the rpsL gene.

[0014] Furthermore, the promoter driving the expression of the reporter gene is a T7 promoter, and the nucleotide sequence of the T7 promoter is shown in SEQ ID No.4.

[0015] The T7 promoter has a fast expression speed and strong specificity and is often used for gene expression of recombinant proteins. Therefore, the present invention uses the T7 promoter to initiate the expression of the reporter gene.

[0016] Furthermore, the spacer nucleotide sequence is selected from the natural downstream sequence of the T7 promoter in the T7 phage nucleotide sequence.

[0017] Preferably, the spacer nucleotide sequence is selected from any one of the sequences: "GG", "GGGA" or "GGGAGA".

[0018] Furthermore, the terminator is a T7 terminator, which blocks the reporter gene from the promoter driving the expression of the tetR gene to prevent mutual interference between their expression and regulation.

[0019] The present invention provides the use of any of the above-mentioned functional plasmids in preparing a biosensor for detecting the content of tetracycline antibiotics.

[0020] Furthermore, the tetracycline antibiotics include tetracycline (TC) and doxycycline (DC).

[0021] The present invention provides a whole-cell biosensor, comprising any one of the above-mentioned functional plasmids and chassis cells for expressing the functional plasmid.

[0022] The tetracycline-specific whole-cell biosensor based on the repressor protein TetR and superfolded green fluorescent protein has a high response to tetracycline. In the low concentration range (tetracycline concentration range is 0-0.4 mg / L), its fluorescence intensity signal is positively correlated with the tetracycline concentration, and the correlation coefficient R 2 The value of the tetracycline whole cell biosensor provided by the present invention is 0.998, and the minimum detection limit can reach 0.01 mg / L. This indicates that the tetracycline whole cell biosensor provided by the present invention has good sensitivity in detecting low concentration tetracycline.

[0023] Furthermore, the chassis cell is E. coli Bl21. E. coli Bl21 (DE3) contains T7 RNA polymerase required by T7 promoter, and does not contain tetR gene in its genome, so E. coli Bl21 (DE3) is used as the chassis cell for functional plasmid expression.

[0024] The present invention provides the use of any of the above-mentioned whole-cell biosensors in detecting the content of tetracycline antibiotics in environmental water.

[0025] Furthermore, the tetracycline antibiotics include tetracycline (TC) and doxycycline (DC).

[0026] Furthermore, the detection is a quantitative detection.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention first provides a functional plasmid, wherein the functional plasmid is constructed based on the repressor protein TetR and the superfolded green fluorescent protein as the main body; further, the functional plasmid is used to construct a tetracycline-specific whole-cell biosensor, wherein the fluorescence intensity signal of the whole-cell biosensor is positively correlated with the tetracycline concentration in a low concentration range (the tetracycline concentration range is 0 to 0.4 mg / L), and the correlation coefficient R 2 The detection limit is 0.998, and the minimum detection limit can reach 0.01 mg / L, showing good sensitivity in the detection of low-concentration tetracycline. The whole-cell biosensor is simple and rapid in detecting tetracycline, and has high specificity and sensitivity, and has the potential to be applied to environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the plasmid map of the tetracycline whole-cell biosensor.

[0030] Figure 2 The diagram is a model mechanism diagram for constructing a tetracycline whole-cell biosensor; wherein, (a) is a diagram of the mechanism of action when there is no repressor protein, (b) is a diagram of the mechanism of action when there is a repressor protein, and (c) is a diagram of the mechanism of action when both tetracycline and repressor protein are present.

[0031] Figure 3 The regulatory function of the spacer region between the T7 promoter and the tetO sequence; (a) is a schematic diagram of the spacer region, (b) is the effect of the spacer length on the performance of the biosensor using sfGFP as a reporter gene; (c) is the effect of the spacer length on the performance of the biosensor using amilCP as a reporter gene.

[0032] Figure 4Fitting curves of the biosensor under different concentrations of lincomycin (a~d), tetracycline (e~h), CuSO4 (i~l) and ethanol C2H5OH (m~p).

[0033] Figure 5 This is the growth curve of chassis cells.

[0034] Figure 6 This is the sensitivity measurement result of the specific tetracycline whole cell biosensor.

[0035] Figure 7 This is the specificity measurement result of the specific tetracycline whole cell biosensor. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] Plasmids and strains: The green fluorescent protein gene plasmid placP-sfGFP was purchased from Shanghai Baosai Biotechnology Co., Ltd. The purple pigment protein gene amilCP was synthesized by Nanjing Novozyme Biotechnology Co., Ltd. The promoter sequence was obtained by PCR amplification technology; Escherichia coli competent cells E. coli BL21 (DE3) and DH5α were purchased from Shanghai Shenggong Biotechnology Co., Ltd.

[0039] Experimental reagents and equipment: Tetracycline (60-54-8), lincomycin (154-21-2), tylosin (1401-69-0), doxycycline (564-25-0), and kanamycin (8063-07-8) were purchased from Shanghai Aladdin Reagent Co., Ltd., China. Tetracycline stock solution (10 mg / mL) was prepared with Milli-Q water, filtered and sterilized with a sterile 0.22 μm filter, and diluted with sterile water before use. High temperature (121°C) high pressure steam sterilization for 20 min. Single fragment seamless cloning kit (ClonECpressⅡOneStep Cloning Kit) was purchased from Nanjing Novozyme Biotechnology Co., Ltd., DNA gel recovery kit, high-fidelity DNA polymerase, and other reagents were purchased from Shanghai Shenggong Biotechnology Co., Ltd. Microplate reader SpectraMaC M5 (Molecular Devices).

[0040] Water samples: Water samples were collected from groundwater in Chongming, Shanghai, and stored at 4°C immediately after collection.

[0041] Example 1

[0042] 1. Construction of Tetracycline Whole-cell Biosensor

[0043] The present invention uses TetR as a response element to construct a whole-cell biosensor that can specifically respond to tetracycline. Through seamless cloning technology, the promoter and various gene elements are fused with the plasmid skeleton to construct a new functional plasmid ( Figure 1 ). The plasmid mainly includes a reporter element and a regulatory element, and its gene circuit is designed by multiple assembly and combination of basic elements. The reporter element is composed of a promoter and a reporter gene, and the reporter gene is selected from the purple pigment protein amilCP gene or the superfolded green fluorescent protein (sfGFP) gene (SEQ ID No.3). The T7 promoter (SEQ ID No.4) is selected to initiate the expression of the reporter gene. The regulatory element is derived from the tetR gene encoding the TetR repressor protein in the genome of Escherichia coli E.coli DH5α, and is driven by the promoter of the highly expressed housekeeping gene rpsL to ensure the efficient expression of the tetR gene (SEQ ID No.1). The T7 terminator separates the reporter element from the regulatory element to prevent mutual interference between their expression and regulation. Escherichia coli E.coliBl21 (DE3) contains the T7 RNA polymerase required by the T7 promoter, and does not contain the tetR gene in the genome. Therefore, E.coliBl21 (DE3) is used as the chassis cell for functional plasmid expression, and its mechanism of action is as follows Figure 2 In the absence of tetR gene, the reporter gene is expressed normally ( Figure 2 In the presence of tetR gene, when tetracycline is absent, the repressor protein TetR produced by tetR gene expression will bind to the promoter binding region tetO (SEQ ID No. 2), thereby inhibiting the expression of downstream reporter genes ( Figure 2 (b)); When tetracycline enters E. coli Bl21 (DE3) containing a functional plasmid, tetracycline can bind to TetR, causing it to detach from the tetO promoter binding region, resulting in changes in the expression level of the downstream reporter gene, and then producing corresponding changes in color or fluorescence signals ( Figure 2 (c)).

[0044] The tetR gene was amplified by PCR using primers TetR-F, TetR-R, and high-fidelity enzymes on a PCR instrument (Thermo Fisher Scientific, USA). The amplification program was as follows: 98°C pre-denaturation for 30s, 98°C denaturation for 10s, 60°C annealing for 5s, 72°C extension for 10s, repeated 35 cycles, 72°C extension for 1min, and stored at 4°C. The PCR amplification products were gel-recovered and purified, and then sequenced. Primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd. Finally, the concentration of the obtained products was determined by Nanodrop one ultra-micro spectrophotometer. The promoter region, tetR gene, and sfGFP or amilCP gene were connected by reverse PCR and seamless cloning technology, introduced into E. coli BL21 (DE3) and transformed by chemical method, and positive clones were picked for verification. The relevant primers are shown in Table 1.

[0045] Table 1 Related primers

[0046]

[0047] Induction of biosensor: Based on the ampicillin resistance of the plasmid, the biosensor chassis cells (E. coli BL21 (DE3)) stored in the glycerol tube were inoculated into 6 mL of Luria-Bertani (LB) liquid medium containing 100 μg / mL ampicillin resistance, and cultured at 37°C 200 rpm for 12 hours to activate the chassis cells. Take the overnight bacterial solution and inoculate it into fresh LB medium at a ratio of 1%, take 6 mL of the diluted bacterial solution and add a certain concentration of tetracycline, and culture at 37°C 200 rpm until the OD 600 =0.6. Take a sample, centrifuge at 15000×g for 10 min, remove the supernatant, resuspend the cells with an equal volume of PBS buffer, repeat once, and use a microplate reader to measure the fluorescence intensity and optical density OD 600 . Experiments were performed three times and included at least three replicates.

[0048] Data processing: When using an ELISA reader for detection, the fluorescence intensity is measured at an excitation / emission wavelength of 488 / 511 nm, and the purple pigment protein absorbance and optical density OD are measured at wavelengths of 588 nm and 600 nm. 600 The unit fluorescence intensity and unit absorbance are expressed as fluorescence intensity and purple pigment protein absorbance and OD, respectively. 600 The experimental results were analyzed and plotted using Origin 2024 and GraphPad Prism 10 software.

[0049] 2. Study on the regulatory effect of TetR repressor protein under different reporter genes and different spacers

[0050] The distance between the promoter and the reporter gene is an important factor affecting the promoter effect and the intensity of gene expression. If the distance between the two is too far or too close, it may affect the inhibitory effect of the repressor protein and the strength of gene expression. Exploring this distance is crucial to optimizing the sensor's response to antibiotics. At the same time, the response sensitivity of the biosensor will be affected by the response strength of different reporter genes.

[0051] A series of plasmids (Plac-T7-gap-sfGFP, Plac-T7-gap-amilCP) were constructed using sfGFP and amilCP as reporter genes, respectively, and using different lengths of spacer regions between the T7 promoter and the tetO sequence. The source of the spacer region was the natural sequence downstream of the T7 promoter in the T7 phage nucleotide sequence, ranging from 0 to 8 bp, with an increment of 2 bp ( Figure 3 (a)). The results showed that in the Plac-gap-sfGFP sensor, no obvious fluorescence activation was detected when the gap was missing (0gap), regardless of the presence or absence of TetR (inhibited or not). When the spacer was 2bp to 6bp, the fluorescence activation decreased to the baseline level in the presence of TetR; strong fluorescence activation was observed in the absence of TetR, and the fluorescence intensity was highest at 4bp. When the spacer was 8bp, strong fluorescence values ​​were still detected in the presence of TetR, indicating that the repressor protein could not inhibit the expression of the reporter gene under the 8bp spacer ( Figure 3 (b)). However, the expression intensity of amilCP in Plac-gap-amilCP did not respond to the regulation of TetR repressor protein under different spacer regions ( Figure 3 Therefore, 4 bp was selected as the appropriate functional spacer length between the promoter and the repressor protein binding region tetO, and superfolded green fluorescent protein was used as the reporter gene to construct Plac-T7-4-sfGFP tetracycline biosensor for research.

[0052] Comparative Example 1

[0053] Studies have pointed out that stress proteins such as heat shock proteins and cold shock proteins are related to biological stress resistance. Under various environmental stress conditions such as heat, ethanol, sodium arsenite, and heavy metals, the promoter activity of their gene promoters is enhanced, the intensity of gene expression is increased, and protein synthesis is promoted, thereby enhancing the cell's stress resistance and maintaining the stability of physiological and biochemical functions. This study intends to construct a universal whole-cell biosensor based on the response of stress protein promoters to environmental stress. Through PCR amplification technology, the promoters of stress protein genes (dnaJ, ibpB, hspQ, grpe, cspA, clpB, rpoS) in Escherichia coli E. coli DH5α were obtained, and the promoter was used to activate green fluorescent protein to construct a universal biosensor. Using E. coli DH5α as the chassis cell, its response to antibiotics (lincomycin (LCM), tetracycline (TC)), heavy metals (Cu 2+ ), the response under ethanol environmental stress. The relevant primers are shown in Table 2.

[0054] Table 2 Primer sequence list

[0055]

[0056]

[0057] The results are as follows Figure 4 As shown in the figure, the sensors constructed by the promoters of the cold shock protein gene cspA and the heat shock protein gene hspQ showed high fluorescence intensity, indicating good activation activity. However, with the increase of pollutant concentration, the fitting curve R between pollutant concentration and fluorescence intensity 2 The value is less than 0.95, indicating that the universal promoter element construction sensor has a poor response effect to environmental pollutants. Although the stress caused by the addition of pollutants can enhance the promoter activity and enhance the expression of fluorescent protein genes, the toxicity of pollutants will inhibit cell growth and may weaken the expression intensity of fluorescent proteins. The interaction of the two reactions may result in the inability to present a linear response relationship. Moreover, under complex environmental conditions, universal promoters respond synergistically to multiple environmental pollutants, and specific pollution problems are difficult to determine. Based on the requirements of sensor accuracy, the promoter of stress proteins is not suitable as a response element for constructing sensors.

[0058] The present invention determines that the specific cell sensor has more advantages in responding to environmental pollution by comparing the general cell sensor and the specific cell sensor.

[0059] Example 2

[0060] Since antibiotics have an inhibitory effect on cell growth, the growth of chassis cells under antibiotic stress conditions will affect the performance of the biosensor. In order to determine the growth of Plac-T7-4-sfGFP tetracycline biosensor chassis cells E.coli Bl21 (DE3) in different concentrations of tetracycline, the tetracycline concentration was set at 0-20 mg / L. Within 14 hours, samples were taken in units of 1 hour, and the OD was measured using an ELISA reader. 600 , evaluate the growth of chassis cells and analyze the inhibitory effect of tetracycline on chassis cells. In different tetracycline concentration environments, the cell growth curves are as follows Figure 5 shown.

[0061] When the chassis cells were cultured at a tetracycline concentration of <0.1 mg / L, the cell growth was similar to that without tetracycline addition, entering the exponential phase after 2 hours and the stable phase at about 9 hours. There was no significant effect of tetracycline on the growth of chassis cells. When the tetracycline concentration was 0.15-0.4 mg / L, the time for cells to enter the logarithmic growth phase was prolonged, and there was a small inhibitory effect on cell growth. When the tetracycline concentration was >0.4 mg / L, the growth of chassis cells was significantly inhibited, and the toxic effect of tetracycline on cells was extremely obvious. In order to avoid tetracycline from significantly inhibiting cell growth and to test the sensor performance under the widest possible tetracycline concentration, tetracycline with a final concentration of 0-0.4 mg / L was selected for subsequent biosensor fluorescence detection research.

[0062] Example 3

[0063] Sensitivity is an important evaluation index for the performance of cell sensor construction. According to the tetracycline concentration range with weak growth inhibition effect obtained in the experiment, Plac-T7-4-sfGFP tetracycline biosensor was selected for sensitivity test. The diluted overnight bacteria were cultured to OD 600 =0.6, and the fluorescence intensity and OD were measured by microplate reader 600 Absorbance, calculate the unit fluorescence intensity, the result is as follows Figure 6 As shown in (a). When the tetracycline concentration range is 0-0.4 mg / L, the fluorescence intensity increases significantly with the increase of tetracycline concentration, showing a good linear relationship. The linear equation is y=37620.7[C(Tc)]+4048.5, and the correlation coefficient is R 2 is 0.998( Figure 6 (b)). The results showed that the modified tetracycline whole-cell biosensor showed good sensitivity in detecting low concentrations of tetracycline.

[0064] Example 4

[0065] Specificity is one of the important indicators for evaluating sensor performance. Taking the common antibiotics lincomycin (LCM, lincosamides), tylosin (TYL, macrolides), doxycycline (DC, tetracyclines), and kanamycin (KANA, aminoglycosides) as representatives, the specificity of tetracycline whole-cell biosensor was tested at antibiotic concentrations of 0.01, 0.05, and 0.1 mg / L. The method is as follows:

[0066] Take 1% of the overnight culture solution and inoculate it into fresh LB medium. Take 6 mL and add TC, LCM, TYL, DC, and KANA with final concentrations of 0.01, 0.05, and 0.1 mg / L, respectively. Place the medium in a 37°C shaker at 200 rpm for induction culture until OD 600 =0.6, sample and centrifuge, resuspend the cells with an equal volume of PBS buffer, and measure the fluorescence intensity and optical density OD with an enzyme-labeled instrument 600 .

[0067] The results are as follows Figure 7 As shown, when tetracycline is added, the fluorescence intensity is much higher than other types of antibiotics. When doxycycline is added to the system, a strong fluorescence response is generated. When lincomycin (LCM), tylosin (TYL), and kanamycin (KANA) are added to the system, no strong fluorescence response is generated, and the fluorescence intensity is equivalent to that of the control group, proving that the sensor is specific to tetracycline antibiotics.

[0068] Example 5

[0069] In order to test the application potential of the constructed tetracycline biosensor in actual water sample analysis, the sensor was used to detect actual groundwater samples (CM1, CM2, and CM3) collected from three sampling points in Chongming, Shanghai. After the collected water samples were filtered with a 0.22μm filter membrane, no tetracycline was detected by high performance liquid chromatography, indicating that the tetracycline concentration in the water sample was lower than the detection limit and suitable for spike recovery experiments. The spike recovery experiment method is as follows:

[0070] The collected water samples were centrifuged at 15000×g for 10 min, and the supernatant was collected and filtered through a 0.22μm filter membrane and stored at 4°C in the dark. The overnight cultured bottom plate cell culture was mixed with natural water samples with designated concentrations of tetracycline in a 1:1 volume ratio, so that the final concentrations of tetracycline were 0.01, 0.03, 0.05, 0.1, 0.2, and 0.4 mg / L. The culture was induced at 37°C and 200 rpm until the OD 600 =0.6, centrifuge the sample, and resuspend it in PBS to measure the fluorescence intensity and optical density OD 600 .

[0071] Under the induction of 6 kinds of tetracycline spiked concentrations, the response results of the sensor are shown in Table 3. In the concentration range of 0.01-0.4 mg / L, the tetracycline recovery rate detected by the tetracycline biosensor was 91%-108.8%, and the relative standard deviation was 0.85%-2.89%, indicating that the sensor has strong anti-interference and good stability in the determination of tetracycline in groundwater, and can be used for actual water sample detection.

[0072] Table 3 Results of tetracycline spike recovery test in groundwater samples

[0073]

[0074]

[0075] In summary, the present invention demonstrates that biosensors using stress protein gene promoters do not respond well to pollutants. By screening transcription factors and reporter genes and optimizing the length of the spacer region, we developed a biosensor that is highly sensitive to tetracycline. The sensor is able to detect low concentrations of tetracycline with a detection limit of 0.01 mg / L. In addition, it exhibits good stability and anti-interference properties in natural water samples. The development of this sensor provides a valuable paradigm for the design and construction of biosensors for other environmental pollutants and provides a promising approach to advance environmental monitoring technology.

Claims

1. A functional plasmid, characterized in that: The invention comprises a plasmid vector and a reporter element and a regulatory element connected to the plasmid vector; the reporter element comprises a reporter gene and a promoter driving the reporter gene expression, and the regulatory element comprises a tetR gene, a tet operator and a promoter driving the tetR gene expression; the reporter gene is a superfolded green fluorescent protein gene; there is a spacer region with a sequence length of 2 to 6 bp between the promoter driving the reporter gene expression and the tet operator; the following elements are operably connected to the plasmid vector from the 5' to 3' direction: a promoter driving the reporter gene expression, a spacer region, a tet operator, an RBS, a reporter gene, a terminator, a promoter driving the tetR gene expression and a tetR gene.

2. The functional plasmid according to claim 1, characterized in that: The promoter driving the expression of the tetR gene is the rpsL promoter.

3. The functional plasmid according to claim 1, characterized in that: The promoter driving the expression of the reporter gene is a T7 promoter.

4. The functional plasmid according to claim 3, characterized in that: The spacer nucleotide sequence is selected from the natural sequence downstream of the T7 promoter in the T7 phage nucleotide sequence.

5. The functional plasmid according to claim 4, characterized in that: The spacer nucleotide sequence is selected from any one of the sequences: "GG", "GGGA" or "GGGAGA".

6. The functional plasmid according to claim 1, characterized in that: The plasmid vector is placP-sfGFP.

7. Use of the functional plasmid according to any one of claims 1 to 6 in the preparation of a biosensor for detecting the content of tetracycline antibiotics.

8. A whole-cell biosensor, characterized in that The invention comprises the functional plasmid according to any one of claims 1 to 6 and chassis cells for expressing the functional plasmid.

9. The whole-cell biosensor according to claim 8, characterized in that: The chassis cells are E.coli Bl21.

10. Use of the whole-cell biosensor according to claim 8 or 9 in detecting the content of tetracycline antibiotics in environmental water.

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