A whole-cell biosensor and its preparation method and application

Abstract

The application discloses a kind of whole cell biosensor and its preparation method and application, and whole cell biosensor contains GenBanK registration number: BA000039 ThcpcB, GenBanK registration number: AY804216 SpcpcB, GenBanK registration number: NC_003272 NocpcB or GenBanK registration number: WP_009454964 ThcpcB gene.The application does not need to extract protein from organism by complex procedure, cell directly and copper ion reaction, the preparation of sensor cell is very simple, low in cost;Short time, only in 15min detection is completed;The biosensor cell prepared finally in the application is high in sensitivity (low detection limit), strong in specificity, has excellent copper ion detection performance;Technical effect is obvious, since cost is low, will bring huge social effect.

Description

Technical Field

[0001] This invention relates to a whole-cell biosensor, its preparation method, and its application, belonging to the field of molecular biology. Background Technology

[0002] Copper is an important micronutrient, but high concentrations can be toxic, and copper deficiency can lead to neurological and hematological disorders. On the other hand, copper is also widely used as a fungicide, algaecide, insecticide, and wood preservative. Therefore, copper monitoring is crucial. Current methods for monitoring copper, such as inductively coupled plasma mass spectrometry (ICP-MS), optical emission spectrometry (ICP-OES), and flame atomic absorption spectrometry (FAA), while highly sensitive, are time-consuming, expensive, and require specialized personnel. Although various chemical and biological sensors address some issues, these methods still cannot distinguish between total copper and bioavailable copper. When assessing pollution, a priority is to quantify bioavailable copper, whose quantity can differ significantly from total copper. Bioavailable copper can only be measured through living organisms, which has driven the development of whole-cell copper biosensors based on transcription factors. These live-cell sensors absorb and specifically bind copper in the environment and initiate transcription of reporter elements to generate detectable signals. Although these whole-cell biosensors offer satisfactory analytical parameters, they are also subject to significant limitations. This is because they require optimal nutritional and growth conditions (pH, temperature) to produce appropriate signals and reproducibility. Moreover, signal generation depends on bacterial growth and reproduction, as well as the maturation of the reporter element, and detection time often takes several hours.

[0003] Phycocyanin interacts with copper, quenching its fluorescence in a dose-dependent manner. Based on this, phycocyanin α-subunits extracted from organisms have been developed for copper detection. However, recognizing the limitations of these protein-based rather than cell-based biosensors, developing cell-based biosensors offers greater advantages. Furthermore, significant differences exist in the genetic and protein structures of phycocyanin from different cyanobacteria and among its various subunits; these structural differences inevitably lead to variations in copper response performance.

[0004] In existing technologies, transcription factor-based copper whole-cell biosensors utilize the fluorescence quenching effect of copper on the β subunit of phycocyanin, a thermophilic algae, which can be called photoquenching. Previous technologies achieved fluorescence enhancement through transcriptional regulation, which can be called photo-on. Other technologies utilize the fluorescence quenching effect of copper on certain fluorescent proteins to achieve copper detection. This technology is essentially protein-based rather than cellular, and protein extraction requires complex procedures and is costly. Furthermore, the β and α subunits of phycocyanin, and even the β subunits of phycocyanin from different cyanobacteria, differ significantly in their encoding gene sequences and protein structures, leading to significant differences in their responses to copper.

[0005] Therefore, obtaining specific molecular sequences that have better sensing performance for copper is also of great significance. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a whole-cell biosensor, its preparation method and application.

[0007] Technical solution: To solve the above technical problems, the present invention provides a whole-cell biosensor, which includes the ThcpcB gene with GenBanK accession number: BA000039, the SppcB gene with GenBanK accession number: AY804216, the NocpcB gene with GenBanK accession number: NC_003272, or the ThcpcB gene with GenBanK accession number: WP_009454964.

[0008] This includes nucleotide sequences such as any one of SEQ ID NO. 1 to 4.

[0009] The present invention also provides a method for preparing the whole-cell biosensor, comprising the following steps:

[0010] (1) Insert the ThcpcB, SppcB, NocpcB or ThcpcB genes into the first multiple cloning site of the expression vector pETDuet respectively; and subclone the fusion enzyme encoding gene cpcS::ho1::pcyA into the second multiple cloning site of the expression vector pETDuet, and finally construct a series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA;

[0011] (2) The series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA described in step (1) were transformed into Escherichia coli and expressed by IPTG to obtain the whole-cell biosensor.

[0012] The present invention also provides a kit for detecting the concentration of copper ions in water, which contains the whole-cell biosensor.

[0013] The present invention also provides the application of the whole-cell biosensor in the preparation of a kit for detecting the concentration of copper ions in water.

[0014] The present invention also provides the application of the whole-cell biosensor in detecting copper ion concentration in water.

[0015] Among them, the fluorescence emission spectrum of the whole-cell biosensor at 644 nm was detected.

[0016] Among them, the OD of the whole-cell biosensor 600 The value is between 0.2 and 1.

[0017] More preferably, the OD of the whole-cell biosensor 600 The value is 0.2 to 0.6.

[0018] More preferably, the OD of the whole-cell biosensor 600 The value is 0.2 to 0.4.

[0019] The response time of the whole-cell biosensor to copper ions is 15–150 min.

[0020] The whole-cell biosensor can achieve a fluorescence quenching rate of about 30% within 15 minutes, which can shorten the detection time.

[0021] The detection temperature ranges from 10 to 55°C.

[0022] More preferably, the detection temperature is 25–55°C.

[0023] More preferably, the detection temperature is 37–55°C.

[0024] More preferably, the detection temperature is 25–42°C.

[0025] The fluorescence quenching rate is highest at a temperature of 42 degrees Celsius, reaching about 60%.

[0026] The linear relationship between the fluorescence quenching rate of the whole-cell biosensor and the concentration of copper ions is: y = 0.0217x - 0.0525, r = 0.9999, where y is the fluorescence quenching rate of the whole-cell biosensor and x is the concentration of copper ions.

[0027] Mechanism of action: After incubation with a copper-containing sample (water or soil sample) and the whole-cell biosensor prepared by this technology, at optimal temperature and cell density, bioavailable copper ions enter the cell through ion channels in the cell membrane. Within the cell, phycocyanin CpcB and phycobilichrome PCB bind specifically to Cu. 2+ This alters the interaction between PCB and CpcB, triggering quenching of CpcB fluorescence. Higher concentrations of bioavailable copper in the sample result in higher concentrations of copper ions entering the cells, leading to more copper ions binding to the corresponding CpcB sites and greater quenching of cell fluorescence, until the copper ion binding sites of CpcB are completely saturated. Therefore, a linear relationship can be established between copper ion concentration and cell fluorescence quenching rate. The copper ion concentration is calculated based on the degree of fluorescence quenching in the sample and the linear equation.

[0028] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. Compared with transcription factor-based copper whole-cell biosensors, the copper whole-cell biosensor in this invention does not rely on bacterial growth and reproduction. Copper ions directly and rapidly bind to phycocyanin upon entering the cell, causing phycocyanin fluorescence quenching. Therefore, detection is completed within only 15 minutes; 3. Compared with protein-based biosensors, this invention does not require complex procedures to extract proteins from organisms. Cells react directly with copper ions, making the preparation of sensor cells very simple, low-cost, and reproducible; 4. The biosensor cells ultimately prepared by this invention have high sensitivity (low detection limit) and strong specificity, possessing excellent copper ion detection performance; 5. The technical effects of this invention are significant, and due to its low cost, it will bring about a huge social impact. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the construction of a series of plasmids, where cpcB represents four phycocyanin encoding genes;

[0030] Figure 2 The fluorescence quenching effect of copper ions (30 μM) on recombinant CpcB cells;

[0031] Figure 3 To demonstrate the selective response of recombinant CpcB cells to heavy metals (30 μM);

[0032] Figure 4 The effect of cell density (OD) on the response to copper (30 μM);

[0033] Figure 5 To reconstruct the time-effect relationship of the response of CpcB cells to copper (30 μM);

[0034] Figure 6 To reconstruct the temperature-response relationship of CpcB cells' response to copper (30 μM);

[0035] Figure 7 To determine the linear relationship of fluorescence response to copper in recombinant CpcB cells. Detailed Implementation

[0036] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0037] Example 1: Construction of cell sensor and screening of fluorescence quenching conditions

[0038] Step 1: Using molecular cloning technology, the gene encoding the phycocyanin β subunit CpcB was cloned by PCR from the genome of Thermosynechococcus vestitus BP-1 and named ThcpcB (GenBanK accession number: BA000039); the gene encoding the phycocyanin β subunit CpcB was cloned by PCR from the genome of Spirulina subsalsa and named SppcB (GenBanK accession number: AY804216); the gene encoding the phycocyanin β subunit CpcB was cloned by PCR from the genome of Nostoc PCC 7120 and named NocpcB (GenBanK accession number: NC_003272); and the gene encoding the phycocyanin β subunit MacpcB was cloned by PCR from the genome of Mastigocladus laminosus and named ThcpcB (GenBanK accession number: WP_009454964).

[0039] These four genes were inserted into the first multiple cloning site of the expression vector pETDuet-1 (purchased from Novagen); then the fusion enzyme encoding gene cpcS::ho1::pcyA (cpcS gene encodes a lyase, hol and pcyA encode phycocyanin PCB synthase) stored in our laboratory was used. The construction process of the fusion enzyme encoding gene cpcS::ho1::pcyA can be found in the following articles: XJWu, H.Yang, YTChen, PPLi, Biosynthesis of fluorescent β-subunits of C-phycocyanin from Spirulina subsalsa in Escherichia coli, and their antioxidant properties, Molecules 23(2018)1369, https: / / doi.org / 10.3390 / molecules23061369 or XJWu, K.Chang, J.Luo, M.Zhou, H.Scheer, KHZhao, Modular generation of fluorescent (phycobiliproteins, Photochem. Photobiol. Sci. 12(2013)1036-1040, https: / / doi.org / 10.1039 / c3pp25383j.) Subcloning was performed into the second multiple cloning site of the expression vector pETDuet-1, ultimately constructing the series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA. The structure of the series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA is as follows: Figure 1 As shown.

[0040] The specific steps are as follows: The PCR product of ThcpcB (the sequence of ThcpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.1), the PCR product of SppcB (the sequence of SppcB-cpcS::ho1::pcyA is shown in SEQ ID NO.2), and the expression vector pETDuet-1 were all digested with restriction endonucleases SacI and HindIII. The digested products were ligated with T4 ligase and transformed into Escherichia coli DH5α strain to obtain cloning transformants; the PCR product of NocpcB (the sequence of NocpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.3), and the PCR product of MacpcB (the sequence of MacpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.2) were all digested with restriction endonucleases SacI and HindIII. Both ID No. 4 and the expression vector pETDuet-1 were digested with restriction endonucleases SacI and SalI. The digestion products were ligated with T4 ligase and transformed into Escherichia coli DH5α strain to obtain cloning transformants. The PCR primers and amplification systems for ThcpcB, SppcB, NocpcB and MacpcB are shown in Tables 1-2. The PCR reaction program was as follows: 4℃ pre-denaturation for 3 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; and a final extension at 72℃ for 5 min.

[0041] Table 1. Information on CpcB gene amplification

[0042]

[0043] Table 2 PCR amplification system

[0044]

[0045] The enzyme digestion system and ligation system are shown in Tables 3-5:

[0046] Table 3 Enzyme digestion system 1

[0047]

[0048] Table 4 Enzyme digestion system 2

[0049]

[0050] Table 5 Connection System

[0051]

[0052] Following the above process, four new plasmids were generated: pETDuet-NocpcB, pETDuet-MacpcB, pETDuet-ThcpcB, and pETDuet-SpcpcB. These four plasmids were double-digested with NdeI and XhoI, and then ligated to the gene sequence cpcS::ho1::pcyA, which had also been double-digested with the same enzymes, to construct four expression plasmids: pETDuet-NocpcB-cpcS::ho1::pcyA, pETDuet-MacpcB-cpcS::ho1::pcyA, pETDuet-ThcpcB-cpcS::ho1::pcyA, and pETDuet-SpcpcB-cpcS::ho1::pcyA. The enzyme digestion and ligation systems are shown in Tables 6 and 7.

[0053] Table 6 Enzyme digestion system

[0054]

[0055] Table 7 Connection System

[0056]

[0057] Step 2: The series of plasmids constructed in Step 1 were transformed into *E. coli* (BL21(DE3)) and induced with IPTG at 16°C for 12 h with a shaking speed of 200 rpm. Recombinant cells were collected to prepare four whole-cell biosensors: EThCpcB, ESpCpcB, EMaCpcB, and ENOCpcB. Cells were suspended in deionized water, and under excitation at 580 nm, the cell suspensions produced fluorescence emission spectra with maximum fluorescence emission wavelengths around 644 nm. Equal amounts of EThCpcB, ESpCpcB, EMaCpcB, and ENOCpcB were reacted with a final concentration of 30 μM copper at room temperature for 30 min, and the fluorescence emission spectra of the suspensions were detected. The protein concentrations of EThCpcB, ESpCpcB, EMaCpcB, and ENOCpcB were 0.4 μM, and 198 μL of protein was reacted with 2 μL of copper. 2+ Mixed, the final copper concentration was 30 μM. (As follows) Figure 2 As shown, ESpCpcB exhibits a quenching rate of approximately 30%, significantly higher than other cell sensors, demonstrating the highest degree of fluorescence quenching. Therefore, ESpCpcB was chosen for the next stage of experiments.

[0058] Step 3: Add various metal ion solutions to a final concentration of 30 μM to equal aliquots of recombinant EspCpcB cells (concentration 0.4 μM) generated in Step 2, and incubate at room temperature (25 degrees Celsius) for 30 minutes. Under 580 nm light excitation, detect the fluorescence intensity of the suspension at 644 nm, using a suspension without added heavy metals as a control. Figure 3 As shown, only Cu 2+ This quenches the fluorescence of ESpCpcB, while other ions and the blank control without metals show no significant difference. Therefore, EspCpcB has the effect of quenching the fluorescence of Cu. 2+ High selectivity.

[0059] Step 4: The equal portions of recombinant cells EspCpcB produced in Step 2 were suspended in different volumes of deionized water to prepare cells at various densities (OD). 600 Suspensions of (value) were incubated with a final concentration of 30 μM copper ion solution at room temperature (25 degrees Celsius) for 30 minutes. The fluorescence intensity of the suspensions was then measured at 644 nm under 580 nm light excitation. A suspension without copper was used as a control, and the fluorescence intensity was normalized using the control group. Figure 4 As shown, the OD of the suspension 600 Cells with an OD value of 0.2 exhibited the highest fluorescence quenching rate, approximately 50%. Therefore, OD was selected. 600 The optimal test condition is 0.2, and the next experiment will proceed.

[0060] Step 5: Based on the test results of Step 4, adjust the OD in Step 4. 600 Cell suspension with a concentration of 0.2 was added to a final concentration of 30 μM copper ion solution and reacted at room temperature. Fluorescence intensity at 644 nm was measured at different time points, and the results are as follows: Figure 5 As shown, the fluorescence quenching rate reaches approximately 30% after 15 minutes, allowing the detection time to be shortened.

[0061] Step 6: Based on the test results of Step 5, adjust the OD from Step 4. 600 Cell suspensions with a concentration of 0.2 were added to a final concentration of 30 μM copper ion solution and reacted at different temperatures for 15 minutes. Fluorescence intensity at 644 nm was measured, with a suspension without copper added serving as a control. The fluorescence intensity was normalized using the control group. Figure 6 As shown, the fluorescence quenching rate is highest at 42 degrees Celsius, reaching approximately 60%. Therefore, 42 degrees Celsius was selected as the optimal testing condition for the next stage of the experiment.

[0062] Step 7: In step 3, OD 600Different concentrations of copper ions were added to cell suspensions with a pH of 0.2, and the cells were incubated at 42°C for 15 minutes. The fluorescence intensity at 644 nm was measured, with a suspension without copper added serving as a control. Figure 7 As shown, there was a significant linear relationship between copper ion concentration and cell fluorescence quenching rate, with a detection limit of 7.3 μM, which was significantly lower than the maximum allowable concentration of copper ions in drinking water (31.5 μM).

[0063] The whole-cell biosensor ESpCpcB prepared using the above experimental protocol exhibits a specific fluorescence response to copper ions. The preferred reaction conditions are cellular OD... 600 The optimal concentration (V) was 0.2, the temperature was 42 degrees Celsius, and the detection time was 15 minutes. A strong linear relationship was observed between copper ion concentration and cell fluorescence quenching rate. The prepared whole-cell biosensor demonstrated the ability to detect copper ions, with a short detection time (15 minutes). It was simple to prepare using cells, easy to operate, did not rely on bacterial growth, required no nutrients during the detection process, and could detect bioavailable copper.

[0064] Example 2

[0065] Under optimal conditions, copper ions in spiked tap water were detected using ESpCpcB. Tap water samples were filtered through a 0.22 μM microporous membrane. Standard copper ions were first added to three identical tap water samples to prepare copper ion concentrations of 0, 1, and 5 mmol / L, respectively, to prepare spiked tap water samples. 2 μL of the spiked tap water sample was added to 198 μL of whole-cell biosensor suspension, and the reaction was carried out under optimal conditions. The fluorescence quenching rate of the cells was detected, and the Cu ion concentration in the spiked tap water was calculated using a linear equation. 2+ The concentration was calculated, and the recovery rate was 96.3% based on (calculated concentration - original concentration) / added concentration. The original concentration was the concentration before Cu addition. 2+ The concentration of the tap water sample was not detected, meaning it was below the detection limit of the biosensor, and therefore the value was 0.

[0066] Example 3

[0067] Under optimal conditions, copper ions in spiked Yangtze River water were detected using ESpCpcB. The river water samples were filtered through a 0.22 μM microporous membrane. First, standard copper ions were added to three portions of the filtered river water to prepare spiked samples with concentrations of 0, 1, and 5 mmol / L. Then, 2 μL of the spiked river water sample was added to 198 μL of a whole-cell biosensor suspension and reacted under optimal conditions. The fluorescence quenching rate of the cells was detected, and the Cu content in the spiked river water was calculated using a linear equation. 2+The concentration was calculated, and the recovery rate was 101.3% based on (calculated concentration - original concentration) / added concentration. The original concentration was the concentration before Cu addition. 2+ The concentration calculated from the river water sample was not detected, meaning it was below the detection limit of the biosensor, and therefore was 0.

[0068] Example 4

[0069] Under optimal conditions, copper ions in spiked lake water from Xuanwu Lake in Nanjing were detected using ESpCpcB. The lake water samples were filtered through a 0.22 μM microporous membrane. First, standard copper ions were added to three portions of the filtered lake water to achieve concentrations of 0, 1, and 5 mmol / L, respectively. Then, 2 μL of the spiked lake water sample was added to 198 μL of whole-cell biosensor suspension and reacted under optimal conditions. The fluorescence quenching rate of the cells was detected, and the Cu concentration in the spiked lake water was calculated using a linear equation. 2+ The concentration was calculated, and the recovery rate was 104.5% based on (calculated concentration - original concentration) / added concentration. The original concentration was the concentration before Cu addition. 2+ The concentration calculated from the lake water sample was not detected here, meaning it was below the detection limit of the biosensor, and therefore was 0.

[0070] Table 8

[0071]

[0072] As shown in Table 8, the accuracy and practicality of the detection method proposed in this invention were verified based on the spiked recovery rate. Through spiked experiments, Cu was detected in various water bodies using the biosensor method. 2 +Concentration and Actual Cu Added 2+ The concentrations were basically consistent, meaning the recovery rate was around 100%. This indicates that the method is effective in environmental water bodies containing Cu. 2 +Accuracy in testing.

Claims

1. The application of a whole-cell biosensor in the preparation of a reagent kit for detecting copper ion concentration in water, characterized in that, The preparation method of the whole-cell biosensor includes the following steps: (1) Insert the SppcB gene into the first multiple cloning site of the expression vector pETDuet-1; and subclone the fusion enzyme encoding gene cpcS::ho1::pcyA into the second multiple cloning site of the expression vector pETDuet-1, and finally construct the plasmid pETDuet-SpcpcB-cpcS::ho1::pcyA. The nucleotide sequence of SppcpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.2; (2) The plasmid pETDuet-SpcpcB-cpcS::ho1::pcyA described in step (1) was transformed into Escherichia coli and expressed by IPTG to obtain the whole-cell biosensor.

2. The application of a whole-cell biosensor in detecting copper ion concentration in water, characterized in that, The preparation method of the whole-cell biosensor includes the following steps: (1) Insert the SppcB gene into the first multiple cloning site of the expression vector pETDuet-1; and subclone the fusion enzyme encoding gene cpcS::ho1::pcyA into the second multiple cloning site of the expression vector pETDuet-1, and finally construct the plasmid pETDuet-SpcpcB-cpcS::ho1::pcyA. The nucleotide sequence of SppcpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.2; (2) The plasmid pETDuet-SpcpcB-cpcS::ho1::pcyA described in step (1) was transformed into Escherichia coli and expressed by IPTG to obtain the whole-cell biosensor.

3. The application according to claim 2, characterized in that, The fluorescence emission spectrum of the whole-cell biosensor at 644 nm was detected.

4. The application according to claim 3, characterized in that, The OD of the whole-cell biosensor 600 The value is 0.2 to 1.

5. The application according to claim 3, characterized in that, The detection temperature is 10~55℃.

6. The application according to claim 3, characterized in that, The linear relationship between the fluorescence quenching rate of the whole-cell biosensor and the concentration of copper ions is: y = 0.0217x - 0.0525, r = 0.9999, where y is the fluorescence quenching rate of the whole-cell biosensor and x is the concentration of copper ions.

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