Whole-cell biosensor as well as preparation method and application thereof

Abstract

The invention discloses a whole-cell biosensor as well as a preparation method and application thereof. The whole-cell biosensor contains a ThcpcB gene with a GenBanK login number of BA000039, a SpcpcB gene with a GenBanK login number of AY804216, a NocpcB gene with a GenBanK login number of NC003272 or a ThcpcB gene with a GenBanK login number of WP009454964. Protein does not need to be extracted from an organism through a complex procedure, the cells directly react with the copper ions, and the sensor cells are very simple to prepare and low in cost; the time is short, and the detection is completed within 15 minutes; the finally prepared biosensor is high in cell sensitivity (low in detection limit), high in specificity and excellent in copper ion detection performance; the technical effect is obvious, and huge social effect can be brought due to low cost.

Description

Technical Field

[0001] The invention relates to a whole-cell biosensor and a preparation method and application thereof, and belongs to the field of molecular biology. Background Art

[0002] Copper is an important micronutrient that is toxic at high concentrations, and copper deficiency can lead to neurological and blood diseases. On the other hand, copper is also widely used as a fungicide, algaecide, insecticide and wood preservative. Therefore, copper monitoring is of vital importance. Current methods for copper monitoring, such as inductively coupled plasma mass spectrometry (ICP-MS), optical emission spectroscopy (ICP-OES) and flame atomic absorption (FAA), are sensitive but time-consuming, expensive and require specialized personnel. Although various chemical and biological sensors have solved some of the problems, these methods still cannot distinguish between total copper and bioavailable copper. When assessing pollution, the priority is to quantify bioavailable copper, which can be very different from total copper. Bioavailable copper can only be measured by organisms, which has prompted the development of whole-cell copper biosensors based on transcription factors. This living cell sensor can absorb and specifically bind copper in the environment and initiate the transcription of a reporter element to produce a detectable signal. Although this type of whole-cell biosensor has satisfactory analytical parameters, it is also severely limited. Because they require optimal nutrients and growth conditions (pH, temperature) to produce appropriate signals and reproducibility, and signal generation depends on the growth and reproduction of bacteria and the maturation of reporter elements, the detection time is often up to several hours.

[0003] Phycocyanin and copper can interact and quench the fluorescence of phycocyanin. This quenching effect is dose-dependent. Therefore, phycocyanin α subunit extracted from organisms has been developed for the detection of copper. However, due to the recognition of the limitations of the above-mentioned biosensors based on proteins rather than cells, the development of biosensors based on cell systems has greater advantages. In addition, there are great differences in the gene and protein structure between phycocyanin and different subunits of phycocyanin from different cyanobacteria. This structural difference will inevitably lead to differences in the response performance to copper.

[0004] In the prior art, the copper whole-cell biosensor based on transcription factors is realized by using the fluorescence quenching effect of copper on the β-subunit of thermophilic algae phycocyanin, which can be called the light-extinguishing type; while the previous technology is realized by the fluorescence enhancement effect of transcriptional regulation, which can be called the light-on type. There is also a technology that uses the fluorescence quenching effect of certain fluorescent proteins caused by copper to achieve the detection of copper. This technology is essentially based on proteins rather than cell systems, and the extraction of proteins requires complex procedures and costs. Moreover, the phycocyanin β-subunit and the phycocyanin α-subunit, and even the phycocyanin β-subunits of different cyanobacteria, have large differences in the coding gene sequence and protein structure, resulting in significant differences in their responses to copper.

[0005] Therefore, it is also of great significance to obtain special molecular sequences with better sensing performance for copper. 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 and its preparation method and application.

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

[0008] Among them, the nucleotide sequence includes a sequence as shown in any one of SEQ ID NOs. 1 to 4.

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

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

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

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

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

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

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

[0016] The OD of the whole-cell biosensor is 600 The value is 0.2~1.

[0017] Further preferably, the OD of the whole cell biosensor is 600 The value is 0.2~0.6.

[0018] Further preferably, the OD of the whole cell biosensor is 600 The value is 0.2~0.4.

[0019] Wherein, the response time of the whole-cell biosensor to copper ions is 15 to 150 minutes.

[0020] The fluorescence quenching rate of the whole-cell biosensor can reach about 30% within 15 minutes, so the time can be shortened as the detection time point.

[0021] Among them, the detection temperature is 10~55℃.

[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 when the temperature is 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, wherein y is the fluorescence quenching rate of the whole-cell biosensor, and x is the concentration of copper ions.

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

[0028] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Compared with the copper whole-cell biosensor based on transcription factors, the copper whole-cell biosensor in the present invention does not rely on the growth and reproduction of bacteria. Copper ions enter the cells and directly and quickly combine with phycocyanin to quench the fluorescence of phycocyanin. Therefore, the detection is completed within only 15 minutes; 3. Compared with the protein-based biosensor, the present invention does not need to extract proteins from the organism through complicated procedures. The cells directly react with copper ions. The preparation of sensor cells is very simple, low-cost and reproducible; 4. The biosensor cells finally prepared by the present invention have high sensitivity (low detection limit) and strong specificity, and have excellent copper ion detection performance; 5. The technical effect of the present invention is obvious, and due to its low cost, it will bring huge social effects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0034] Figure 6 is the temperature-effect relationship of the response of recombinant CpcB cells to copper (30 μM);

[0035] Figure 7 The linear relationship between the fluorescence of recombinant CpcB cells and copper response. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with 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 PCR cloned from the Thermosynechococcus vestitus BP-1 genome and named ThcpcB (GenBanK accession number: BA000039), the gene encoding the phycocyanin β subunit CpcB was PCR cloned from the Spirulina subsalsa genome and named SpcpcB (GenBanK accession number: AY804216), the gene encoding the phycocyanin β subunit CpcB was PCR cloned from the Nostoc PCC 7120 genome and named NocpcB (GenBanK accession number: NC_003272), and the gene encoding the phycocyanin β subunit MacpcB was PCR cloned from the Mastigocladus laminosus genome and named ThcpcB (GenBanK accession number: WP_009454964).

[0039] The four genes were respectively 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 lyase, hol and pcyA encode phycocyanin PCB synthase, the construction process of the fusion enzyme encoding gene cpcS::ho1::pcyA can be found in the article: XJWu, H.Yang, YTChen, PPLi, Biosynthesis of fluorescent βsubunits 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.) were subcloned into the second multiple cloning site of the expression vector pETDuet-1, and finally the series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA were constructed. The structure of the series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA is shown in Figure 1 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 SpcpcB (the sequence of SpcpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.2) and the expression vector pETDuet-1 are double-digested with restriction endonucleases SacΙ and HindⅢ, the digestion products are connected with T4 ligase, and are transferred 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), the PCR product of MacpcB (the sequence of MacpcB-cpcS::ho1::pcyA is shown in SEQ ID NO.4) and the expression vector pETDuet-1 are double-digested with restriction endonucleases SacΙ and HindⅢ, the digestion products are connected with T4 ligase, and are transferred into Escherichia coli DH5α strain to obtain cloning transformants; IDNO.4) and the expression vector pETDuet-1 were double-digested with restriction endonucleases SacΙ and SalΙ, the digested products were ligated with T4 ligase, and transformed into Escherichia coli DH5α strain to obtain clone transformants. Among them, the PCR primers and amplification systems of ThcpcB, SpcpcB, NocpcB and MacpcB are shown in Tables 1-2, and the PCR reaction procedures are: 4℃ pre-denaturation for 3min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 72℃ extension for 1min, a total of 30 cycles; final extension at 72℃ for 5min.

[0041] Table 1 Gene CpcB amplification information

[0042]

[0043] Table 2 PCR amplification system

[0044]

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

[0046] Table 3 Enzyme digestion system 1

[0047]

[0048] Table 4 Enzyme Digestion System 2

[0049]

[0050] Table 5 Connection system

[0051]

[0052] After the above process, four new plasmids pETDuet-NocpcB, pETDuet-MacpcB, pETDuet-ThcpcB, and pETDuet-SpcpcB were generated. These four plasmids were double-digested with NdeΙ and XhoΙ, and then connected with the gene sequence cpcS::ho1::pcyA double-digested with the same enzymes, and finally four expression plasmids pETDuet-NocpcB-cpcS::ho1::pcyA, pETDuet-MacpcB-cpcS::ho1::pcyA, pETDuet-ThcpcB-cpcS::ho1::pcyA, and pETDuet-SpcpcB-cpcS::ho1::pcyA were constructed. Among them, the enzyme digestion and connection 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 Escherichia coli (E.coli BL21 (DE3)) and induced to express with IPTG at 16 degrees Celsius for 12 hours. The shaking speed was 200 rpm. The recombinant cells were collected to prepare 4 whole-cell biosensors EThCpcB, ESpCpcB, EMaCpcB and ENoCpcB. The cells were suspended in deionized water. Under the excitation of light with a wavelength of 580nm, the cell suspension produced a fluorescence emission spectrum with a maximum fluorescence emission wavelength of about 644nm. Equal amounts of EThCpcB, ESpCpcB, EMaCpcB and EnoCpcB were reacted with a final concentration of 30μM copper at room temperature for 30 minutes, and the fluorescence emission spectrum of the suspension was detected. Among them, the protein concentration of EThCpcB, ESpCpcB, EMaCpcB and EnoCpcB was 0.4μM, 198μL of protein and 2μL of Cu 2+ Mix, the final concentration of copper is 30 μM. Figure 2 As shown in the figure, the quenching rate of ESpCpcB is about 30%, which is much higher than other cell sensors and shows the greatest degree of fluorescence quenching. Therefore, ESpCpcB was selected for the next experiment.

[0058] Step 3: Add various metal ion solutions with a final concentration of 30 μM to the equal parts of recombinant cells EspCpcB (concentration of 0.4 μM) produced in step 2, incubate at room temperature (25 degrees Celsius) for 30 minutes, detect the fluorescence intensity of the suspension at 644 nm under 580 nm light excitation, and use the suspension without heavy metals as a control. Figure 3 As shown, only Cu 2+ The fluorescence of ESpCpcB was quenched, and there was no significant difference between other ions and the blank control without metal. 2+ Highly selective.

[0059] Step 4: The aliquots of recombinant EspCpcB cells produced in step 2 were suspended in different volumes of deionized water to prepare various cell densities (OD 600 The suspensions with a final concentration of 30 μM copper ion solution were added to the suspensions at room temperature (25 degrees Celsius) for 30 minutes. The fluorescence intensity of the suspensions at 644 nm was detected under 580 nm light excitation. The suspensions without copper were used as controls, and the fluorescence intensity of the control group was normalized. Figure 4 As shown, the suspension OD 600 The cells with an OD of 0.2 had the highest fluorescence quenching rate, which was about 50%. 600 The optimal test condition is 0.2, and the next experiment is carried out.

[0060] Step 5: According to the test results of step 4, 600 The cell suspension with a value of 0.2 was added with a copper ion solution with a final concentration of 30 μM to react at room temperature, and the fluorescence intensity at 644 nm was detected at different time points. The results are as follows Figure 5 As shown in the figure, the fluorescence quenching rate has reached about 30% at 15 minutes, so the time can be shortened as the detection time point.

[0061] Step 6: According to the test results of step 5, the OD 600 The cell suspension with a value of 0.2 was added with a copper ion solution with a final concentration of 30 μM and reacted at different temperatures for 15 minutes. The fluorescence intensity at 644 nm was detected, and the suspension without copper was used as a control. The fluorescence intensity of the control group was normalized. Figure 6 As shown, the fluorescence quenching rate is the highest when the temperature is 42 degrees Celsius, reaching about 60%. The temperature of 42 degrees Celsius is selected as the optimal test condition for the next experiment.

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

[0063] Through the above experimental scheme, the prepared whole-cell biosensor ESpCpcB has a specific fluorescence response to copper ions. The preferred reaction conditions are cell OD 600 The value is 0.2, the temperature is 42 degrees Celsius, and the detection time is 15 minutes. The copper ion concentration and the cell fluorescence quenching rate show a good linear relationship. The prepared whole-cell biosensor has the ability to detect copper ions, and the detection time is short (15 minutes). The cell preparation is simple, the operation is convenient, it does not rely on bacterial growth, and the detection process does not require nutrition. It can detect biologically effective copper.

[0064] Example 2

[0065] Under optimal conditions, ESpCpcB was used to detect copper ions in spiked tap water. The tap water sample was filtered through a microporous filter membrane with a pore size of 0.22μM. First, standard copper ions were added to 3 portions of the same tap water, so that the concentrations of copper ions were 0, 1, and 5mmol / L respectively to prepare spiked tap water samples. 2uL of the spiked tap water sample was added to the 198uL suspension of the whole cell biosensor to react under optimal conditions, and the fluorescence quenching rate of the cells was detected. The Cu of the spiked tap water was calculated by a linear equation. 2+ concentration, and according to (calculated concentration - original concentration) / added concentration, the spike recovery rate was 96.3%. Among them, the original concentration is the concentration without Cu 2+ The calculated concentration of the tap water sample was not detected here, i.e., below the detection limit of the biosensor and therefore equal to 0.

[0066] Example 3

[0067] Under optimal conditions, ESpCpcB was used to detect copper ions in spiked Yangtze River water. The river water samples were filtered through a microporous filter membrane with a pore size of 0.22μM. First, standard copper ions were added to 3 portions of filtered river water to make the copper ion concentrations 0, 1, and 5mmol / L respectively to prepare spiked river water samples. Then, 2uL of the spiked river water sample was added to 198uL of the suspension of the whole cell biosensor and reacted under optimal conditions. The fluorescence quenching rate of the cells was detected, and the Cu of the spiked lake water was calculated by a linear equation. 2+concentration, and according to (calculated concentration - original concentration) / added concentration, the spike recovery rate was 101.3%. Among them, the original concentration is the concentration without Cu 2+ The calculated concentration of the river water sample was not detected here, i.e., it was below the detection limit of the biosensor and was therefore 0.

[0068] Example 4

[0069] Under optimal conditions, ESpCpcB was used to detect copper ions in the spiked water of Nanjing Xuanwu Lake. The lake water samples were filtered through a microporous filter membrane with a pore size of 0.22μM. First, standard copper ions were added to 3 portions of filtered lake water to make the concentrations of copper ions 0, 1, and 5mmol / L, respectively. Then 2uL of the spiked lake water sample was added to the 198uL suspension of the whole cell biosensor to react under optimal conditions, and the fluorescence quenching rate of the cells was detected. The Cu of the spiked lake water was calculated by a linear equation. 2+ concentration, and according to (calculated concentration - original concentration) / added concentration, the spike recovery rate was 104.5%. Among them, the original concentration is the concentration without Cu 2+ The calculated concentration of the lake water sample was not detected here, i.e., it was below the detection limit of the biosensor and was therefore 0.

[0070] Table 8

[0071]

[0072] As shown in Table 8, the accuracy and practicality of the detection method proposed in the present invention are verified based on the spike recovery rate. 2 + concentration and actual Cu added 2+ The concentrations are basically the same, that is, the recovery rate is about 100%. This shows that the method is effective in the detection of Cu in environmental water. 2 + Accuracy in detection.

Claims

1. A whole-cell biosensor, characterized in that: It contains the ThcpcB gene of GenBanK Accession No.: BA000039, the SpcpcB gene of GenBanK Accession No.: AY804216, the NocpcB gene of GenBanK Accession No.: NC_003272 or the ThcpcB gene of GenBanK Accession No.: WP_009454964.

2. The whole-cell biosensor according to claim 1, characterized in that: It contains cpcB-cpcS::ho1::pcyA, and the nucleotide sequence of cpcB-cpcS::ho1::pcyA is shown in any one of SEQ ID NOs. 1-4.

3. A method for preparing the whole-cell biosensor according to claim 1 or 2, characterized in that: The following steps are involved: (1) inserting the ThcpcB, SpcpcB, NocpcB or ThcpcB gene described in claim 1 into the first multiple cloning site of the expression vector pETDuet-1 respectively; and subcloning the fusion enzyme encoding gene cpcS::ho1::pcyA into the second multiple cloning site of the expression vector pETDuet-1, and finally constructing a series of plasmids pETDuet-cpcB-cpcS::ho1::pcy A; (2) The series of plasmids pETDuet-cpcB-cpcS::ho1::pcyA described in step (1) are transformed into Escherichia coli respectively, and the expression is induced by IPTG to obtain the whole-cell biosensor.

4. A kit for detecting the concentration of copper ions in water, characterized in that: It contains the whole-cell biosensor according to claim 1 or 2.

5. Use of the whole-cell biosensor according to claim 1 or 2 in preparing a kit for detecting the copper ion concentration in water.

6. Use of the whole-cell biosensor according to claim 1 or 2 in detecting the copper ion concentration in water.

7. The use according to claim 5 or 6, characterized in that: The fluorescence emission spectrum of the whole-cell biosensor was detected at 644 nm.

8. The use according to claim 5 or 6, characterized in that: The OD of the whole cell biosensor 600 The value is 0.2~1.

9. The use according to claim 5 or 6, characterized in that: The detection temperature is 10~55℃.

10. The use according to claim 5 or 6, 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, wherein y is the fluorescence quenching rate of the whole-cell biosensor, and x is the concentration of copper ions.

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