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Expression of Cadmium-Binding Proteins and Research on Water Environmental Heavy Metal Pollution Detection Sensor Overview
The detection of heavy metal pollution and the corresponding environmental remediation remain significant challenges today. In this study, we propose a novel, portable, paper-based biosensor for the direct detection of heavy metals such as cadmium in polluted water bodies. This sensor consists of a stem-loop structure containing a cadmium aptamer DNA sequence and the expression and regulation genes for a fluorescent protein. The DNA sequence within the stem-loop structure serves as a switch for fluorescent protein expression. When cadmium is present, the fluorescent protein is expressed, and the level of expression can partially reflect the amount of pollutants. The entire detection and protein expression system is on a paper strip, enabling rapid detection and adsorption purification of contaminated water, laying the foundation for further pollutant treatment.



Figure 1. Schematic Diagram of Cadmium Detection and Adsorption Principle

In the presence of heavy metal cadmium in the environment, it can bind to the aptamer, activating the cell-free expression system in paper (filter paper) to express cadmium-binding proteins and green fluorescent proteins. This dual-purpose system not only provides a visual effect but also adsorbs heavy metal cadmium in the environment, achieving both detection and purification.

Experimental Materials

  • Bacterial Strains and Plasmids

Escherichia coli BL21(DE3)
pET22b(+)

  • Restriction endonucleases: HindlII, EcoR I, Nde I, Mlu I, Xba I, BamH I, Sph I, etc.
  • T4 DNA ligase, PCR reagent kit, various markers
  • Water environmental DNA extraction reagent kit, purification reagent kit
  • Common culture media
    • LB medium
      • Yeast extract: 5g
      • Tryptone: 10g
      • Sodium chloride: 10g
      • Adjust pH to 7.0 with NaOH, autoclave at 121°C for 20 minutes
  • Common solution preparation
    • Alkaline plasmid extraction solution:
      • Solution 1 (lysis buffer): Glucose 50 mmol/L, EDTA 10 mmol/L, Tris·HCl 25 mmol/L (pH 8.0), autoclaved, stored at 4°C.
      • Solution 2: NaOH 0.2 mol/L, SDS 1%, prepared immediately before use.
      • Solution 3: NaAc 0.5 mol/L, pH 4.8.
    • TE buffer: Tris·HCl 10 mmol/L, EDTA 1 mmol/L, pH 8.0.
  • Antibiotic concentrations in culture media: Ampicillin (Amp) 100mg/L, Gentamicin (Gm) 30mg/L, Kanamycin (Kan) 50mg/L, Chloramphenicol (Cm) 20mg/L.

Lysozyme was purchased from Sigma, 0.22um filter membranes from Thermo Fisher, E. coli BL-21 competent cells from GenScript, and the total DNA extraction reagent kit for contaminated water from Omiga (USA). The DNA gel recovery kit was purchased from QIAGEN, and protease K, filter membranes, antibiotics, and plasmid extraction reagent kit were purchased from Shanghai Sangon Biotech Co., Ltd. Reverse transcriptase, LA-Taq DNA polymerase, dNTP, T4 DNA ligase, restriction endonucleases, and various vectors were all purchased from TaKaRa. Primer synthesis and gene sequencing were completed by Nanjing GenScript Biotechnology Co., Ltd. Cd was purchased from Aladdin Reagent Co., Ltd., and other chemical reagents were common analytical pure reagents.

Experimental Methods
1、Extraction of DNA from Polluted Water Environments
  • Take 500 mL of sewage and filter it through a 0.45μm aqueous filter membrane. Collect the filter membrane and extract total DNA using the Water DNA kit. First, add the filter membrane to the Powerbead Tubes and vortex to mix.
  • Add 60ul of solution C1 solution, invert and mix several times, then vigorously shake on a vortex shaker for about 5-10 minutes.
  • Centrifuge at room temperature, 10000g for 30 seconds, transfer the supernatant to another clean collection tube, add 250ul solution C2, vortex to mix, centrifuge at 4°C for 5 minutes, and then centrifuge at 10000g for 1 minute at room temperature.
  • Transfer the supernatant to another collection tube, add 200ul of solution C3, incubate at 4°C for 5 minutes, centrifuge at 10000g for 1 minute, transfer the supernatant to a new collection tube, add 1200ul of solution C4, vortex for 5 seconds.
  • Then, gradually transfer the solution (approximately 675ul each time) to the Spin Filter, centrifuge at room temperature, 10000g for 1 minute, discard the filtrate, and continue to transfer the supernatant to the filter membrane until all the transfer is completed.
  • Add 500ul of washing solution to the Spin Filter, centrifuge at 4°C for 5 minutes, and then centrifuge at 10000g for 1 minute at room temperature.

Carefully transfer the Spin Filter to a 2-milliliter collection tube, add 100ul of Solution C6 for DNA dissolution. After incubating at room temperature for 2 minutes, centrifuge at 10,000g for 30 seconds. The collected liquid is the DNA solution, which is reserved for UV spectrophotometer quality control.
Collect water environment samples with potential contamination, use the appropriate DNA extraction kit to extract DNA, and perform gel electrophoresis for testing. The results are as follows.




Figure.2 Gel Electrophoresis Profile of Water Environment DNA
2、PCR amplification of the target DNA fragment

Amplification and identification of the Cadmium-Binding Protein (CBP) gene segment
The amino acid sequence corresponding to this binding protein gene is as follows:
MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHCRSLDMPLSDVRTLLSYRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACSGARPAQSCGILQGLSDCVCDTRGTTAHPSDGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLE
Design primers based on its gene sequence for amplification from total DNA.



Figure.3 amplification results of the Cadmium-Binding Protein (CBP) gene

As shown in the above figure, primers were designed based on the Cadmium-Binding Protein (CBP) DNA sequence for PCR amplification. Two samples obtained the desired gene sequence with a length of 1100 bp.

4、PCR Product Ligation and Transformation

Enzymatic digestion and ligation of the vector and PCR products were performed based on predefined restriction enzyme sites. First, a double digestion was performed using HindIII and NdeI, followed by ligation with CBP. The ligated product was then transformed into E. coli BL21(DE3) competent cells and selected using ampicillin to obtain a certain quantity of transformants.

Another double digestion was carried out using HindIII and EcoRI, followed by ligation with CBP. The ligated product was similarly transformed into E. coli BL21(DE3) competent cells and selected using ampicillin to obtain a certain quantity of transformants. Plasmid verification was conducted through enzyme digestion. As shown in the following figure, the sizes of the digested target gene fragments met the expected requirements.




Figure.4 Recombinant Plasmid Enzyme Digestion Figure:
1: Double-digested target gene;
2: Plasmid.

5、Expression of the Target Protein
Transformation: The recombinant plasmid was introduced into BL21(DE3) E. coli competent cells, heat-shocked at 42°C, and then plated on agar plates containing 50 µg/mL ampicillin, followed by incubation at 37°C.
Activation: Single colonies were picked and grown in liquid medium containing 50 µg/mL ampicillin at 37°C.
Induction: When the OD value reached 0.6, 0.5 mM IPTG inducer was added, and the cells were further cultured. Cultures were incubated overnight at 20°C and for 6 hours at 37°C. Cells cultured without inducer served as a negative control.
Cell Harvest: The cells were harvested by centrifugation at 4000 rpm for 10 minutes, and the supernatant was discarded, leaving the bacterial pellet.
Expression Analysis: The bacterial pellet was suspended in Buffer A and thoroughly dissolved using an ultrasonic disruptor. The supernatant and pellet were separated by centrifugation and then dissolved in Buffer B, followed by protein preparation. Samples were prepared for gel electrophoresis analysis.

6、Detection of Expression Products
SDS-PAGE Analysis: Protein samples were processed, prepared, and separated on a 12% separating gel and 5% stacking gel. Gel electrophoresis was performed to determine molecular weights.
Western Blot Verification: Protein samples were processed and prepared. A 5% stacking gel and a 12% separating gel were used. The primary antibody was anti-His tag mouse monoclonal, and the secondary antibody was anti-mouse IgG from sheep. Visualization was done using TMB (3,3',5,5'-Tetramethylbenzidine) staining, and validation was performed using the tagged antibody.
To facilitate the successful implementation of the cell-free expression system, we initially conducted traditional cell-based expression verification.



Figure.5 Expression and Verification of Cadmium-Binding Protein
A: Growth of transformed colonies; B: SDS-PAGE analysis of fusion protein; C: Western Blot analysis of fusion protein expression.

The results above show that Cadmium-Binding Protein expressed well in Escherichia coli after transformation and can be used for subsequent experimental research.
To enable the visual detection of heavy metal cadmium, we used GFP (Green Fluorescent Protein) as a reporter gene, which emits yellow-green fluorescence. We constructed pET-22b-GFP as a positive control.
The constructed pET-22b-GFP was digested with EcoRI and HindIII restriction endonucleases, and PCR was performed using GFP's flanking sequences. The GFP gene fragment was 780 bp in length, as confirmed by agarose gel electrophoresis, which matched the expected length (see the figure below).





Figure.6  Construction and Verification of Linear Plasmids Using PCR and Restriction Enzyme Digestion
a: Double digestion of neckloop switch plasmid; b: pET-22b-neckloop switch-CBP-GFP plasmid; M: Molecular weight marker.

Transfected BL21 bacterial strains were cultured on agar plates, and the results showed normal expression of the fluorescent protein. These bacteria were used as positive controls for neckloop switch plasmid transfection. The expression results are shown in the figure below.



Figure.7  Expression of Fluorescent Protein in BL21 Bacteria Transfected with pET-22b-CBP-GFP Plasmid

Based on the GFP sequence, Cadmium-Binding Protein sequence, and cadmium adapter sequence, a neckloop switch plasmid was designed, capable of producing a yellow-green reporter protein. It was cloned into the pET-22b expression vector at EcoRI-HindIII cloning sites for cadmium detection.

This neckloop switch plasmid was validated through restriction enzyme digestion experiments. The inserted sequence was 780 bp in length, and this result was confirmed by agarose gel electrophoresis.
The pET-22b-neckloop switch-CBP-GFP plasmid was transfected into BL21 strains (see figure below). In the absence of cadmium ion induction, there was no GFP gene expression in BL21 strains, indicating no or very low GFP protein expression. This demonstrates the effectiveness of the neckloop switch. Subsequently, cadmium ions were transfected into BL21 strains containing the pET-22b-neckloop switch-CBP-GFP plasmid, and it was observed that some clones successfully transfected with cadmium ions exhibited yellow-green fluorescence.




Figure.8  Expression of GFP in BL21 Strains.
A: Strain without transfection of cadmium ions; B: BL21 strain transfected with both cadmium ions and the switch plasmid.

7、Preparation of E. coli BL21 Cell Lysates
Select a single colony of engineered bacteria constructed earlier and inoculate it into 5 mL of LB medium. Incubate overnight at 37°C with shaking at 220 rpm.
The next day, transfer the overnight culture to 300 mL of 2× YTPG medium and incubate at 37°C with shaking at 220 rpm.
When the OD600 of the bacterial culture reaches the late logarithmic growth phase, collect the bacteria by centrifugation at 10,000 g for 1 minute and remove the supernatant.
Suspend the bacteria in pre-cooled S30 buffer and mix thoroughly on a shaker. Centrifuge at 4°C and 8000 g for 7 minutes, then remove the supernatant. Repeat this step three times, discarding all traces of the supernatant.
Add 30 mL of pre-cooled S1 buffer to every 1 g of bacteria and mix thoroughly.
Use an ultrasonic cell disruptor to lyse the cells with the following settings: 2 s on, 2 s off, total time 15 min, and temperature alarm set to 40°C.
Centrifuge the tubes at 4°C and 12,000 g for 20 minutes, then transfer the supernatant to a new tube, freeze in liquid nitrogen, and store at -80°C. This can be used as a cell lysate extract.

  • Note:
  • 2× YTPG medium: 22 mM potassium dihydrogen phosphate, 40 mM dipotassium hydrogen phosphate, 100 mM glucose, 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride.
  • S30 buffer: 10 mM acetyl phosphate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, 2 mM DTT

.8、Construction of the Cell-Free System
Prepare 11 mixtures in advance: 9 mM magnesium acetate, 90 mM potassium glutamate, 80 mM ammonium acetate, 57 mM HEPES-KOH, 0.171 mg/mL tRNA, 0.034 mg/mL folate, 2 mM dithiothreitol (DTT), 1 mM putrescine, 1.5 mM spermidine, 4 mM oxalic acid, 33 mM sodium pyruvate.
Prepare a cell-free reaction system (15 μL) consisting of: 6 μL of the 11 mixtures, 1.2 mM ATP, 0.86 mM GTP, CTP, and UTP, 5% (V/V) PEG-8000, 0.1 mM phosphoenolpyruvate (PEP), 0.27 U/μL RNase inhibitor, 2 mM of all 20 amino acids, 25% (V/V) Escherichia coli BL21 Δ Lac Z cell lysate, 5% (V/V) 20 mg/mL X-gal color substrate.
Add 5 nM pET-22b-CBP-GFP circular switch plasmid.
Incubate at 37°C for 1 hour and record any changes in the solution's color.
Note: Use a colored protein as an indicator and add the corresponding circular switch plasmid; do not add X-gal substrate.
This process outlines the setup of a cell-free system for conducting experiments, and the use of colored proteins as indicators to monitor the reactions. X-gal substrate is not to be added in this process.

9、Preparation of Paper-Based Sensors Based on the Cell-Free Expression System

  • Place Whatman filter paper strips (0.6×4cm) in a culture dish, add a 5% bovine serum albumin solution to completely cover the paper, and seal it overnight at 4°C.
  • The next day, remove the 5% bovine serum albumin solution from the culture dish, add ddH2O to wet the paper, place the culture dish in a desiccator, and incubate for 5 minutes. Discard the ddH2O.
  • Repeat step 2 and wash the paper 5 times.
  • Open the lid of the culture dish, place it in an electric constant-temperature drying oven. Dry the paper for later use.
  • On spots pre-marked with a pencil, spot 7 μL volumes of Escherichia coli BL21 Δ LacZ cell lysate on the Whatman filter paper strips.
  • Place them in a -80°C ultra-low-temperature freezer for 6 hours.
  • After pre-cooling in a freeze dryer, seal the paper strips with plastic wrap and perforate the wrap for ventilation. Freeze-dry overnight.
  • Store the paper sensor strips at 4°C until use.

After transfection of the circular switch plasmid into BL21 strains, we explored the culture conditions for the strains and optimized the concentration of cadmium ions and the culture time. Since the reporter gene has color, we optimized the reaction conditions based on color.
Considering the sensitivity of detection and the subsequent paper-based detection system, it is crucial to find the lowest detectable concentration of chromium ions. We primarily conducted experiments to explore the detection concentration. The corresponding experimental results are shown in the following figure.



Figure.9  A：The impact of chromium ion concentration on the expression of fluorescent protein in BL21 strains transfected with the circular switch plasmid is depicted in the figure. From left to right, the transfection concentrations are as follows: 0.01 mg/L, 0.02 mg/L, 0.05 mg/L, 0.1 mg/L, 0.5 mg/L, and 1 mg/L. B. A linear correlation graph.
As observed in the above figure, with the increase in Cd2+ concentration, the expression of fluorescent protein becomes more pronounced. There is a good linear correlation between the concentrations ranging from 0.02 mg/L to 1 mg/L.

 

10、Exploration of Reaction Time in the Cell-Free Expression System
After determining the optimal detection concentration and related culture conditions, cell extract was treated with ultrasound to obtain the extraction solution. This extraction solution was mixed with ATP, phosphoenolpyruvate (PEP), amino acids, and other components. Cadmium ions were added to induce the expression of the target protein, and the expression was studied at different reaction times. The results showed that after 1 hour, there was no significant change in the fluorescence signal. Therefore, 1 hour was established as the most suitable reaction time.





Figure.10 Exploration of Fluorescent Protein Expression Time in the Cell-Free Expression System
A: Expression of paper-based fluorescent protein; B: Analysis of fluorescence intensity at different times.

11、Research on Paper-Based Detection Sensors Based on the Cell-Free Expression System
Filter paper is a readily available, cost-effective, and convenient-to-transport and store biological analysis material. Filter paper, after soaking in bovine serum albumin and drying, is used to apply the cell-free reaction system. After freeze-drying, it forms paper-based sensors that can be stored for an extended period. During detection, a solution containing cadmium ions is dropped onto the paper-based sensor containing the circular switch plasmid, and the reaction is carried out at 37°C for 1 hour, allowing the protein to be expressed.





Figure.11 Expression of the Reporter Gene GFP Induced by Different Concentrations of Cadmium Ions
The results show that the expression of the reporter gene GFP can be activated with a cadmium solution concentration of 0.02 mg/L, making it suitable for rapid detection of cadmium pollution in conventional water environments.

12、Detection of Actual Water Pollution Samples in Real Water Environments
To further validate the practical usability of our prepared sensor, we collected some contaminated water samples from the environment and performed tests using the paper-based sensor. The results show that there is a faint expression of fluorescent proteins (as shown in the figure below), indicating the potential for widespread application of this sensor.



Fig.12 Paper-Based Sensor Detection in Actual Water Environment Pollution Samples
The above image shows the results of testing two different contaminated water samples using our paper-based biosensor.

Summary

In this study, we have established a paper-based biosensor for the detection of cadmium pollution in water environments using a cadmium-specific aptamer. The results demonstrate that we can detect cadmium solutions with concentrations as low as 0.02 mg/L using this paper-based sensor, which is comparable in sensitivity to traditional methods of water pollution detection. This simple and convenient method holds significant importance and potential for widespread application in the monitoring and potential remediation of heavy metal pollution in water environments.

References
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[3] Alexander A. Green,1 Pamela A. Silver,1,2 James J. Collins,1,3 and Peng Yin. Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell 159, 925–939, November 6, 2014. http://dx.doi.org/10.1016/j.cell.2014.10.002
[4] XU J, JIANG R, FENG Y, et al. Functional nucleic acid-based fluorescent probes for metal ion detection [J]. Coordination Chemistry Reviews, 2022, 459: 214453.