iGEM 2023 — Team nanjing-nflsfc

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# Notebook

**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.

![](https://static.igem.wiki/teams/5022/wiki/images/zz1.jpg)

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.

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# Engineering Success

With the large amount of industrial sewage and human life wastewater discharge, heavy metal pollution of water has become a global environmental problem. Heavy metal pollution is not only a great threat to the ecological balance and the safety of drinking water, but also can act together with other pollutants in living organisms, presenting a complex joint effect in the organism, and ultimately causing comprehensive toxicity. Take our country, China, as an example, there has been no breakthrough in the treatment of cyanobacteria in Lake Taihu, which meanly because that the environmental sewage treatment is costly, and some pollution sources are difficult to detect quickly.

At present, heavy metal detection methods mainly include inductively coupled plasma emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), and atomic fluorescence spectrometry (AFS), etc. However, these detection methods are not convenient enough to carry out effective treatment in a short term, especially the basic cost is high, which is not conducive to the use of large-scale or high-frequency. We can also use biosensor technology to carry out simple, rapid, sensitive and accurate in situ dynamic monitoring and analysis of important pollutants in the environment, which has shown great advantages and potential. However, biosensors are too demanding to be used in practical applications. Synthetic biology has reawakened the potential of whole-cell microbial sensors.

![](https://static.igem.wiki/teams/5022/wiki/images/tt1.jpg)

**Figure .1 Schematic diagram of the principle of Heavy Metal Cadmium's detection and adsorption**

When there is a heavy metal cadmium in the environment, it can be combined with the ligand, the non-cell expression system in the paper base (filter paper), the expression of the cadmium binding protein and the green fluorescent protein, both the visual effect and the heavy metal cadmium in the environment, the double target of detection and purification.

**1.Extraction of water environment DNA and amplification of target gene (cadmium binding protein)**

Collect samples of water environment with potential pollution, use the corresponding DNA extraction kit, extract DNA, and gel electrophoresis detection, the results are shown below.

![](https://static.igem.wiki/teams/5022/wiki/images/tt2.jpg)

**Figure.2 Gel electrophoresis profiles of DNA from aquatic environments**

![](https://static.igem.wiki/teams/5022/wiki/images/tt3.jpg)

**Figure.3 Results of cadmium binding protein CBP amplification**

As shown in the above figure, primers were designed for PCR amplification according to the DNA sequence of cadmium-binding protein, and the corresponding target gene sequences were obtained for two of the samples, with a length of 1100bp.

**Amplification and identification of cadmium-binding protein CBP gene fragment**
The amino acid sequence corresponding to the binding protein gene is as follows.

MNIQIGELAKRTACPVVTIRFYEQEGLLPPPGRSRGNFRLYGEEHVERLQFIRHCRSLDMPLSDVRTLLSYRKRPDQDCGEVNMLLDEHIRQVESRIGALLELKHHLVELREACSGARPAQSCGILQGLSDCVCDTRGTTAHPSDGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKLE

According to its gene sequence, primers are designed to amplify from the total DNA.
The gene sequences used for cloning expression are as

follows.CATATGAACATCCAGATTGGCGAACTGGCAAAACGTACCGCATGTCCGGTTGTTACCATTCGTTTTTATGAACAGGAAGGTCTGCTGCCGCCGCCGGGTCGTAGCAGAGGTAATTTTCGTCTGTATGGTGAAGAACATGTTGAACGCCTGCAGTTTATTCGTCATTGTCGTAGTCTGGATATGCCGCTGAGCGATGTTCGCACCCTGCTGAGCTATCGTAAACGCCCGGATCAGGATTGTGGCGAAGTGAATATGCTGCTGGATGAACATATTCGCCAGGTTGAAAGTCGCATTGGTGCACTGCTGGAACTGAAACATCATCTGGTGGAACTGCGCGAAGCATGCAGCGGTGCCCGCCCTGCTCAGAGCTGCGGTATTCTGCAGGGCCTGAGTGATTGTGTTTGTGATACCCGTGGTACCACCGCACATCCGAGTGATGGTGGTGGCGGCAGTGGCGGCGGCGGTAGCATGGTTAGCAAAGGTGAAGAACTGTTTACCGGCGTTGTTCCGATTCTGGTGGAATTAGATGGCGATGTTAATGGTCATAAATTTTCTGTGAGTGGCGAAGGCGAAGGCGATGCCACCTATGGTAAACTGACCCTGAAATTCATTTGTACCACCGGCAAACTGCCGGTTCCGTGGCCGACCCTGGTGACCACCCTGACCTATGGCGTTCAGTGTTTTAGCCGTTATCCGGATCACATGAAACAGCATGATTTCTTTAAAAGTGCAATGCCGGAAGGTTATGTGCAGGAACGTACCATTTTCTTTAAAGATGATGGCAATTACAAGACCCGTGCCGAAGTGAAATTTGAAGGCGATACCCTGGTGAATCGTATTGAACTGAAAGGTATTGATTTCAAAGAGGATGGTAATATTCTGGGTCATAAACTGGAATATAACTATAATAGCCACAACGTTTACATCATGGCAGATAAACAGAAAAATGGTATTAAGGTTAACTTCAAGATCCGCCATAATATTGAAGATGGCAGTGTTCAGCTGGCCGATCATTATCAGCAGAATACCCCGATTGGCGATGGCCCGGTGCTGCTGCCGGATAATCATTATCTGAGTACCCAGAGCGCACTGAGCAAAGATCCGAATGAAAAACGTGATCACATGGTGCTGCTGGAATTTGTGACCGCAGCAGGCATTACCCTGGGTATGGATGAACTGTATAAACTCGAG

**2.Construction of cyclic expression plasmid**

Firstly, HindIII and EcoRI double digestion was performed, then CBP was ligated with it, and the ligand transformed E.coli BL21(DE3) receptor cells, and screened with ampicillin to obtain a certain number of transformants. The plasmid was verified by enzymatic digestion. As shown in the figure below, the size of the enzymatically digested target gene fragment was as expected.

![](https://static.igem.wiki/teams/5022/wiki/images/tt4.jpg)

**Figure .4 Recombinant plasmid zymography 1, Double zymography of target gene; 2, Plasmid.**

![](https://static.igem.wiki/teams/5022/wiki/images/tt5.jpg)

**The expression vector map used in this project**

**3.Expression and detection of target proteins**

For the successful realisation of the cell-free expression system, we first carried out the validation of traditional cellular expression.

![](https://static.igem.wiki/teams/5022/wiki/images/tt6.jpg)

**Figure .5 Expression and validation of cadmium binding protein**

A: Growth of transformed colonies; B: SDS-PAGE to detect the fusion protein; C: Western Blot to analyse the expression of fusion protein.

The above results show that the cadmium binding protein obtained better expression after transforming E. coli and can be used for subsequent experimental research.

**4.Construction and validation of neckloop switch plasmid**

In order to carry out the visual detection of heavy metal cadmium, we used GFP fluorescent protein as a reporter gene, showing yellow-green colour, and 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 the flanking sequences of GFP.The gene fragment of GFP was 780bp, which was detected using agarose gel electrophoresis, and the results showed the same length as expected (shown below).

**Figure .6 Recognition of pET-22b-neck-loop switch-CBP-GFP. M: Marker; 1: pET-22b-neck-loop**

switch-CBP-GFP plasmid with EcoRI and HindIII restriction enzyme digestion, 2: pET-22b neck-loop switch-CBP-GFP plasmid.

The BL21 strain transfected with the plasmid was cultured on a petri dish, and the results showed normal expression of fluorescent proteins, and these bacteria were used as a positive control for the transfection of the neckloop switch plasmid. The expression results are shown below:

![](https://static.igem.wiki/teams/5022/wiki/images/tt7.jpg)

**Figure 7 .Expression of CBP and GFP protein in BL21 strain.** 1: Positive control of BL21 strain containing pET-22b-CBP-GFP plasmid. 2: BL21 strain transfected with pET-22b-clip switch-CBP-GFP plasmid and Cd

The neckloop switch plasmid was designed and constructed based on the sequence of GFP, the sequence of cadmium-binding protein and the sequence of cadmium aptamer, which is capable of producing a yellow-green reporter protein. The plasmid was synthesised by EcoRI-HindIII cloning site and cloned into pET-22b expression vector to detect cadmium.

This neckloop switch plasmid was validated by restriction endonuclease digestion experiments. The insertion sequence was 780bp and this result was verified by agarose gel electrophoresis.

**5.Induction of neckloop plasmid with chromium ions to express fluorescent reporter gene in BL21 strain**

The pET-22b-neckloop switch-CBP-GFP plasmid was transfected into BL21 strain (below). In the absence of cadmium ion induction, the expression of the GFP gene was not triggered in the BL21 strain, thus failing to express the GFP protein or the amount of GFP protein expression, indicating that the neckloop switch was effective. Subsequently, cadmium ions were transfected into BL21 strains containing the pET-22b-neckloop

switch-CBP-GFP plasmid. It was found that some clones successfully transfected with cadmium ions showed yellow-green colour.

![](https://static.igem.wiki/teams/5022/wiki/images/tt8.jpg)

**Figure .8 GFP expression in BL21 strains.** A: strain without transfection of cadmium ion; B: BL21 strain transfected with both cadmium ion and switch plasmid.

**6.Exploration of the sensitivity of cadmium ion detection in the expression system**

After transfecting the neck-ring switch plasmid in BL21 strain, we explored the culture conditions of the strain and optimised the concentration of cadmium ions and the incubation time.

Since the reporter gene contains colour, we can optimize the reaction conditions by colour. Considering the detection sensitivity and the later paper-based detection system, it is crucial to find the lowest chromium ion detection concentration, and we mainly explored the detection concentration. The corresponding experimental results are shown below.

![](https://static.igem.wiki/teams/5022/wiki/images/tt9.jpg)

**Figure 9 Optimization comparison of different Cd2+ concentrations and cultivation times.** A, Exploration of Cd detection limits; B, Exploration of the linear range of Cd detection.

From the above graph, it can be seen that with the increase of Cd2+ concentration, the expression of fluorescent protein became more and more obvious, and there was a good linear correlation between the concentration of 0.02mg/L and 1mg/L.

**7.Exploration of reaction time of cell-free expression system**
After identifying the optimal detection concentration and related cultivation conditions, we utilized ultrasonic treatment to obtain cell extract. This extract, mixed with ATP, phosphoenolpyruvate (PEP), amino acids, and other components, was used for inducing the expression of target proteins in the presence of cadmium ions. We investigated protein expression at various reaction times and found that 1 hour was the most suitable reaction time.

![](https://static.igem.wiki/teams/5022/wiki/images/tt10.jpg)

**Figure 10 Exploration of the expression time of fluorescent proteins in the cell-free expression system.**
A: Expression of paper-based fluorescent proteins; B: Analysis of fluorescence intensity at different times

![](https://static.igem.wiki/teams/5022/wiki/images/tt11.jpg)

**8.Research on paper-based detection sensor based on cell-free expression system**
Filter paper is an easily accessible, cost-effective, and convenient biological analysis material. After soaking filter paper in bovine serum albumin and drying it, we applied a cell-free reaction system to the filter paper. The paper was then freeze-dried to create paper-based sensors, which can be stored for an extended period. During testing, we added cadmium ion solutions to filter paper sensors containing neck-loop switch plasmids and incubated them at 37°C for 1 hour, allowing the proteins to be expressed.

![](https://static.igem.wiki/teams/5022/wiki/images/tt12.jpg)

**Figure 11 Expression of the reporter gene GFP induced by different cadmium ion concentrations**
The results demonstrate that the expression of the reporter gene GFP can be activated with a cadmium solution of 0.02 mg/L, making it suitable for rapid detection of cadmium contamination in typical aquatic environments.

**9.Testing of Real Water Pollution Samples**
To further validate the practical usability of our prepared sensors, we collected some polluted water samples from the environment. We conducted tests using paper-based sensors and observed faint expression of fluorescent proteins (as shown in the figure below), indicating the potential applicability of these sensors for broader use.

![](https://static.igem.wiki/teams/5022/wiki/images/tt13.jpg)

**Figure .12 Detection using paper-based sensors in real water pollution samples**

# In Summary:

In this study, we have established a paper-based biosensor for the detection of cadmium in aquatic environments using cadmium aptamers. Our results demonstrate that we can detect cadmium solutions with concentrations as low as 0.02mg/L using these paper-based sensors, which exhibit sensitivity comparable to traditional methods for analyzing polluted water. This approach is simple and convenient and holds significant potential for the detection and potential remediation of heavy metal pollution in aquatic environments.

# Reference

[1] GENG H, XU Y, ZHENG L, et al. An overview of removing heavy metals from sewage sludge: Achievements and perspectives [J]. Environmental Pollution, 2020, 266: 115375.

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# Experiments

Overview

In our project, we established a platform for the detection of heavy metal pollution in aquatic environments, where the expression of fluorescent proteins is initiated by nucleic acid aptamer sequences of heavy metals for visualization and detection. We developed a cell-free protein expression system based on stem-loop switch to achieve rapid detection by paper-based microarrays, and at the same time, heavy metal adsorption proteins can be expressed, and heavy metal adsorption can be realized after detection, which is expected to realize the harmless treatment of contaminated water.

Extraction of DNA from polluted water environment

Procedure:

Take 500mL of sewage water and filter it utilizing a 0.45 micron aqueous filter membrane, collect the filter membrane and use Water DNA kit for total DNA extraction. First the filter membrane was added to the Powerbead Tubes, then vortexed and shaken to mix;

Add 60μL of solution C1 and mix upside down several times, then shake vigorously with a vortex shaker for about 5-10 minutes;

In room temperature, centrifuge the solution at 10000g for 30 seconds, transfer the supernatant to another uncleaned collection tube, add 250μL solution C2, vortex and mix well, 4°C for 5 minutes, centrifuge at 10000g for 1 minute at room temperature;

Transfer the supernatant to another collection tube, add 200μL solution C3, 4°C for 5 minutes, centrifuge at 10000g for 1 minute, transfer the supernatant to a new collection tube, add 1200μL solution C4, vortex mix for 5 seconds;

Then gradually transfer the solution (about 675μL each time) to Spin Filter, centrifuge at 10000g for 1 min at room temperature, discard the filtrate, and continue to transfer the supernatant to the filter membrane until all the transfer is completed;

Add 500μL of solution C5 to the Spin Filter, centrifuge at 10000g for 30 seconds at room temperature, discard the supernatant and centrifuge at 10000g for 1 minute;

Carefully transfer the Spin Filter to a two ml collection tube, add 100μL solution C6 for DNA dissolution, centrifuge at 10000g for 30 seconds at room temperature for 2 minutes, the collection solution is the DNA solution, UV spectrophotometer quality control for backup.

Extraction and purification of Escherichia coli plasmid DNA: alkaline cleavage method

PCR amplification of target DNA fragments

Product recovery and purification (QIAGEN)

Purification and recovery of the product according to the steps of the manual

PCR product ligation and transformation

According to the pre-set digestion site for the vector and ligation of PCR products for digestion and ligation. Firstly, Hind III and Nde I were double digested, then CBP was ligated, and the ligand was transformed into E.coli BL21(DE3) receptor cells, and a certain number of transformants were obtained by screening with benzylpenicillin.

Screening and identification of recombinants

Pick the colonies growing on the resistant plate, inoculate them into 4 ml of LB liquid medium respectively (add the corresponding antibiotics to a certain concentration), and incubate them overnight at 37℃ with oscillation.

Plasmids were extracted by alkaline lysis.

Restriction endonuclease digestion was performed for identification.

For related steps of cloning subclones, see Molecular Cloning Laboratory Guide, 2nd edition p55-56

Expression of target protein

Transformation: the recombinant plasmid was transferred into BL21(DE3) E. coli receptor cells, heat-excited at 42°C and then coated on plates containing 50µg/mL ampicillin and cultured at 37°C;

Activation: monoclonal colonies were picked into liquid medium containing 50µg/mL ampicillin and incubated at 37°C;

Induction: when the OD value reaches 0.6, add 0.5 mM inducer IPTG and continue to incubate, respectively, overnight at 20°C and 6 h at 37°C. The one without inducer is the negative control;

Collection of organisms: centrifuge at 4000 rpm for 10 min, discard the supernatant and collect the organisms;

Expression assay: add buffer A suspension to the collected bacilli, and use ultrasonic crusher to make them fully dissolved. Centrifuge to collect the supernatant and precipitate, precipitate using buffer B for dissolution, respectively, the supernatant and precipitated proteins for sampling, ready for gelatinization detection.

Detection of expression products

SDS-PAGE: Protein samples were processed, samples were prepared, 12% separator gel, 5% concentrate gel, gel was run and molecular weight was detected.

Western Blot validation: protein samples were processed, samples were prepared. 5% gel concentrate, 12% separator gel. Primary antibody was mouse anti-His tag, secondary antibody was sheep anti-mouse, TMB color development was used, and labeled antibody was used for validation.

Preparation of E. coli BL21 cell lysate

A single colony of the engineered bacteria constructed above was selected and inoculated in 5 ml of LB medium and incubated overnight at 37°C in a shaker at 220 rpm.

On the next day, the above medium was transferred to 300 mL of 2 × YTPG medium and incubated at 37°C on a shaker at 220 rpm.

Bacteria were collected when the 600 OD value of the bacterial concentration increased in the late logarithmic cycle. Centrifuge 10,000 g

Construction of 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, 1 mM putrescine, 1.5 mM spermidine, 4 mM oxalic acid, 33 mM sodium pyruvate.

Cell-free reaction system (15 μL) was prepared: 6 μL 11 mixture, 1.2 mM ATP, 0.86 mM GTP, CTP and UTP, 5% (V/V) PEG-8000, 0.1 mM phosphoenolpyruvic acid (PEP), 0.27 U/μL RNase inhibitor, 2 mM 20 amino acids, 25% (V/V) E. coli BL2121, 0.1 mM PEP, 0.27 U/μL RNase inhibitor, 2 mM 20 amino acids, 25% (V/V) E. coli PEP. V/V) E. coli BL21 Δ Lac Z cell lysate extract, 5% (V/V) 20 mg/mL X-gal chromogenic substrate.

Add 5 nM pET-22b(+)-CBP-GFP- Neck-loop-switch plasmid

Incubate at 37°C for 1 hour and record the colour change of the solution.

Note: Use coloured proteins as indicators, add the appropriate toe-switch plasmid and do not add the X-gal substrate.

Preparation of paper-based sensors based on cell-free expression system

Strips of Whatmann filter paper (0.6 × 4 cm) were placed in petri dishes, 5% bovine serum protein solution was added to cover the paper completely, and closed at 4°C overnight.

The next day, discard the 5% bovine serum protein solution from the petri dish, add ddH2O to wet the filter paper, place the petri dish in a decolourisation shaker, incubate for 5 min and discard the ddH2O,

Repeat step 2 and wash the filter paper 5 times.

Open the lid of the petri dish and place it in an electric thermostatic drying oven. Wipe the filter paper dry for use.

Spot a dotted volume of 7 μL E. coli BL21 Δ LacZ cell lysate extract on a strip of Whatmann filter paper on the spots pre-marked with a pencil

Placed in an ultra-low temperature refrigerator at -80°C for 6h.

After pre-cooling in a freeze dryer, the paper sheets were sealed with cling film and the cling film was perforated and vented to freeze dry overnight.

Store filter paper induction strips at 4°C until use.

Additional experimental steps:

The PCR products cleanup and fragment recovery from gel using QIAGEN kit according to manual.

Qiagen MinElute® Reaction Cleanup kit

Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).

All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a

conventional tabletop microcentrifuge at room temperature.

Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 μl pH indicator I to

30 ml Buffer PB or add 600 μl pH indicator I to 150 ml Buffer PB). The yellow

color of Buffer PB with pH indicator I indicates a pH of 7.5.

Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer aliquots.

If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I.

Procedure

1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. It is not necessary to remove mineral oil or kerosene. For example, add 250 μl of Buffer PB to 50 μl PCR reaction (not including oil).

2. If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.

3. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.

4. To bind DNA, apply the sample to the MinElute column and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column.

5. Discard flow-through. Place the MinElute column back into the same tube.

6. To wash, add 750 μl Buffer PE to the MinElute column and centrifuge for 1 min.

7. Discard flow-through and place the MinElute column back in the same tube. Centrifuge the column for an additional 1 min at maximum speed.

IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.

8. Place the MinElute column in a clean 1.5 ml microcentrifuge tube.

9. To elute DNA, add 10 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.

10. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

QIAGEN MinElute® Gel Extraction Kit

This protocol is designed to extract and purify DNA of 70 bp to 4 kb from standard or low-melt agarose gels in TAE or TBE buffer resulting in high end-concentrations of DNA. Up to 400 mg agarose can be processed per MinElute column.Important points before starting

The yellow color of Buffer QG indicates a pH ≤7.5.

Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).

All centrifugation steps are carried out at ≥10,000 x g in a conventional table-top microcentrifuge at room temperature.

Procedure

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg or approximately 100 μl). For example, add 300 μl of Buffer QG to each 100 mg of gel. For >2% agarose gels, add 6 volumes of Buffer QG. The maximum amount of gel slice per spin column is 400 mg; for gel slices >400 mg use more than one MinElute column.

3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. IMPORTANT: Solubilize agarose completely. For >2% gels, increase incubation time.

4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). Note: If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow. The adsorption of DNA to the membrane is efficient only at pH ≤7.5. Buffer QG contains a pH indicator which is yellow at pH ≤7.5 and orange or violet at higher pH, allowing easy determination of the optimal pH for DNA binding.

5. Add 1 gel volume of isopropanol to the sample and mix by inverting the tube several times. For example, if the agarose gel slice is 100 mg, add 100 μl isopropanol. Do not centrifuge the sample at this stage.

6. Place a MinElute column in a provided 2 ml collection tube in a suitable rack.

7. To bind DNA, apply the sample to the MinElute column, and centrifuge for 1 min. For maximum recovery, transfer all traces of sample to the column. The maximum volume of the column reservoir is 800 μl. For sample volumes of more than 800 μl, simply load and spin again.

8. Discard the flow-through and place the MinElute column back in the same collection tube.

9. Add 500 μl of Buffer QG to the spin column and centrifuge for 1 min.

10. Discard the flow-through and place the MinElute column back in the same collection tube.

11. To wash, add 750 μl of Buffer PE to the MinElute column and centrifuge for 1 min.

12. Discard the flow-through and centrifuge the MinElute column for an additional 1 min at ≥10,000 x g.

13. Place the MinElute column into a clean 1.5 ml microcentrifuge tube.

14. To elute DNA, add 10 μl of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the membrane, let the column stand for 1 min, and then centrifuge for 1 min.

15. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Enzyme digestion reaction and reaction of ligation between target gene and plasmid

1) Enzyme digestion

![](https://static.igem.wiki/teams/5022/wiki/images/experiments-1.jpg)

Incubate the tube at 37℃ for about 2hours, then cleanup according to the previous approaches.

2) ligation

![](https://static.igem.wiki/teams/5022/wiki/images/experiments-2.jpg)

Mixed thoroughly according to above table, and incubate the tube at 16℃ over night. The ligation products were also cleaned according to the previous approaches.

Preparation competent cell of Escherichia coli

After culture the E.coli BL21(DE3) on LB medium, collect the cells by centrifuge. Resuspend the cell in 0.1M CaCl2, mixed thoroughly, centrifuge again, remove the culture medium, resuspend the cell 0.1M CaCl2 at 4℃, prepared for the next step. Unusually, the cells from 50mL LB medium could be dissolved in 2mL CaCl2 solution.

Transform into Escherichia coli

Thaw competent cells rapidly by immersing frozen tubes in a 37℃ water bath after remove from 70℃ refrigerator. Draw about 50uL of the competent cells in a clean tube and add 5uL recombined plasmid, throw on ice for about 30 min. then put them in 42℃ for 90 second, immediately turn on ice for 2min. add 900uL LB medium and incubate in a roller drum at 37℃ fro 1hours. Took 100ul LB medium contain ampicillin, upside down when dried, put in 37℃ incubator over night. Validation by agarose electrophoresis after the plasmid DNA was extracted from transforming Escherichia coli.

Sample preparation for SDS-PAGE

5mL of induced products was centrifuged with 10000rpm for 10 min under 4℃, collecting the cells.

Resuspend the precipitate with 2mL 0.5M Tris-HCl (pH 6.8), then centrifuged with 10000rpm for 5 min, repeat this step twice.

Resuspend the precipitate with 5mL buffer for ultrasonication under 200W, 1second, with 2 second interval, 30 cycles, total times with 6 min. then centrifuged with 12000rpm for 25 min, collected the supernatant and precipitation respectively.

Resuspend the precipitate with 80µL ddw, add 20µL 5×loading buffer and 5µL DTT, mixed thoroughly. Prepare another tube with the supernatant with the same procedure. Then boiling in 100℃ for 6min for SDS-PAGE detection.

Procedure of PAGE

Preparation of the separation and concentration gels

![](https://static.igem.wiki/teams/5022/wiki/images/experiments-3.jpg)

AP and TEMED should be the last to added. When it was added, mixed thoroughly immediately, and after the gel polymerization, pull out the comb and wash the sample hole with double distilled water.

1)Load 5μL samples, Start electrophoresis under 80V until the bromophenol blue moving into separation gel, then increase the voltage to 100V.

2)After electrophoresis, strip the gel and immerge in the fixed solution until the bromophenol blue change to faint yellow; replace the fixed solution with staining solution, then dyeing and decolorizing for 4 hours.

3)Removing the staining solution, following another decolonization step, it could be watch and took photo after the band of protein was complete clear. The gel could be stored in distilled water.

Western Blotting

Apparatus: Apparatus of SDS-PAGE, Electroblotting Apparatus, Power supply, PVDF membrane（Millipore Immobion-P #IPVH 000 10）, Whatman 3MM paper, Additional Tools: Forceps, sponge pad, scissor, gloves, small plastic or glass container, Shallow tray.

Cell extracts were prepared by homogenizing cells or tissues in the lysis buffer (50 mM Tris–HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 0.5% NP-40) for 45 min. The soluble protein concentration was measured using Bio-Rad Protein Assay (Bio-Rad). The lysates (50 lg) were separated by 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel (SDS–PAGE) and transferred to PVDF membranes for immunoblotting assay. The membrane was blocked in 5% fat-free dry milk, and probed with antibodies against the interest proteins. The blots were visualized using the enhanced ECL reagent. The levels of protein expression were semi-quantified by optical densitometry using Image J software.

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# Results

**Overview**
Detection of heavy metal contamination and remediation of the corresponding environments remain major challenges today. In this study, we present a novel and portable filter paper-based biosensor that can directly detect heavy metals such as cadmium in polluted water. The sensor consists of a stem-loop structure containing a cadmium aptamer nucleic acid and a fluorescent protein expression and regulatory gene. The DNA sequence of the stem-loop structure serves as a switch for fluorescent protein expression, and when cadmium is present, the fluorescent protein is expressed, and the amount of expression can react to a certain extent to the amount of pollutants, and the whole detection and protein expression system is on a strip of filter paper, which allows rapid detection and adsorption and purification of polluted water. Lay the foundation for further pollutant treatment.

**Extraction of water environment DNA and amplification of target gene (cadmium binding protein)**
Collect samples of water environment with potential pollution, use the corresponding DNA extraction kit, extract DNA, and gel electrophoresis detection, the results are shown below.

![](https://static.igem.wiki/teams/5022/wiki/images/vv1.jpg)

**Figure.1 Gel electrophoresis profiles of DNA from aquatic environments.**

![](https://static.igem.wiki/teams/5022/wiki/images/vv2.jpg)

**Figure.2 Results of cadmium binding protein CBP amplification.**
As shown in the above figure, primers were designed for PCR amplification according to the DNA sequence of cadmium-binding protein, and the corresponding target gene sequences were obtained for two of the samples, with a length of 1100 bp.

**Construction of cyclic expression plasmid**
Firstly, HindIII and EcoRI double digestion was performed, then CBP was ligated with it, and the ligand transformed E.coli BL21(DE3) receptor cells, and screened with ampicillin to obtain a certain number of transformants. The plasmid was verified by enzymatic digestion. As shown in the figure below, the size of the enzymatically digested target gene fragment was as expected.

![](https://static.igem.wiki/teams/5022/wiki/images/vv3.jpg)

**Figure.3 Recombinant plasmid zymography. 1, Double zymography of target gene; 2, Plasmid.**

**1.Expression and detection of target proteins**
For the successful realisation of the cell-free expression system, we first carried out the validation of traditional cellular expression.

![](https://static.igem.wiki/teams/5022/wiki/images/vv4.jpg)

**Figure.4 Expression and validation of cadmium binding protein.** A, Growth of transformed colonies; B, SDS-PAGE to detect the fusion protein; C, Western Blot to analyse the expression of fusion protein.

The above results show that the cadmium binding protein obtained better expression after transforming E. coli and can be used for subsequent experimental research.

**Construction and validation of neck loop switch plasmid**
In order to carry out the visual detection of heavy metal cadmium, we used GFP fluorescent protein as a reporter gene, showing yellow-green colour, and 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 the flanking sequences of GFP.The gene fragment of GFP was 780bp, which was detected using agarose gel electrophoresis, and the results showed the same length as expected (shown below).

![](https://static.igem.wiki/teams/5022/wiki/images/vv5.jpg)

**Figure.5 Construction and validation of linear plasmids by PCR and restriction enzyme digestion.** a, double digestion neck loop switch plasmid; b, pET-22b(+)-neck loop switch-CBP-GFP plasmid; M, molecular weight Marker

The BL21 strain transfected with the plasmid was cultured on a petri dish, and the results showed normal expression of fluorescent proteins, and these bacteria were used as a positive control for the transfection of the neckloop switch plasmid. The expression results are shown below.

![](https://static.igem.wiki/teams/5022/wiki/images/vv7.jpg)

**Figure.6 Expression of fluorescent protein in BL21 bacteria transfected with pET-22b(+)-CBP-GFP plasmid.**

The neck loop switch plasmid was designed and constructed based on the sequence of GFP, the sequence of cadmium-binding protein and the sequence of cadmium aptamer, which is capable of producing a yellow-green reporter protein. The plasmid was synthesised by EcoRI-HindIII cloning site and cloned into pET-22b (+) expression vector to detect cadmium.
This neck loop switch plasmid was validated by restriction endonuclease digestion experiments. The insertion sequence was 780bp and this result was verified by agarose gel electrophoresis.

**Induction of neck loop plasmid with chromium ions to express fluorescent reporter gene in BL21 strain**

The pET-22b(+)-neck loop switch-CBP-GFP plasmid was transfected into BL21 strain (below). In the absence of cadmium ion induction, the expression of the GFP gene was not triggered in the BL21 strain, thus failing to express the GFP protein or the amount of GFP protein expression, indicating that the neck loop switch was effective. Subsequently, cadmium ions were transfected into BL21 strains containing the pET-22b(+)-neckloop switch-CBP-GFP plasmid. It was found that some clones successfully transfected with cadmium ions showed yellow-green colour.

![](https://static.igem.wiki/teams/5022/wiki/images/vv8.jpg)

**Figure.7 GFP expression in BL21 strains.A: strain without transfection of cadmium ion; B: BL21 strain transfected with both cadmium ion and switch plasmid.**

**Exploration of the sensitivity of cadmium ion detection in the expression system**

After transfecting the neck-ring switch plasmid in BL21 strain, we explored the culture conditions of the strain and optimised the concentration of cadmium ions and the incubation time.
Since the reporter gene contains colour, we can optimize the reaction conditions by colour. Considering the detection sensitivity and the later paper-based detection system, it is crucial to find the lowest chromium ion detection concentration, and we mainly explored the detection concentration. The corresponding experimental results are shown below.

![](https://static.igem.wiki/teams/5022/wiki/images/vv9.jpg)

**Figure.8 A, Effect of chromium ion concentration on fluorescent protein expression in BL21 strain transfected with neck loop switch plasmid, transfection concentrations from left to right are: 0.01mg/L, 0.02mg/L, 0.05mg/L, 0.1mg/L, 0.5mg/L, 1mg/L; B, linear correlation plots.**

From the above graph, it can be seen that with the increase of Cd2+ concentration, the expression of fluorescent protein became more and more obvious, and there was a good linear correlation between the concentration of 0.02 mg/L and 1mg/L.

**Exploration of reaction time of cell-free expression system**

After clarifying the optimal detection concentration and related culture conditions, the cell extracts were treated with ultrasonic waves to obtain the extracts, which were mixed with ATP, PEP, amino acids, etc., and cadmium ions were added for the induction of the expression of the target proteins, and the expression under different reaction times was explored. The results showed that there was basically no significant change in the fluorescence signal after 1 hour, so 1 hour was determined as the most suitable reaction time.

![](https://static.igem.wiki/teams/5022/wiki/images/vv10.jpg)

**Figure.9 Exploration of fluorescent protein expression time in cell-free expression system.**
A, Expression of paper-based fluorescent protein; B, Analysis of fluorescence intensity at different times.

**Research on paper-based detection sensor based on cell-free expression system**
Filter paper is a bioanalytical material that is easily accessible, inexpensive, and convenient for transport and storage. The filter paper is soaked in bovine serum albumin, dried, and then the cell-free reaction system is added dropwise to the filter paper, freeze-dried to form a paper-based sensor, which can be stored for a long time. For detection, an aqueous solution containing cadmium ions is added dropwise to the filter paper sensor containing the neck loop switch plasmid, and the protein is expressed by reacting at 37°C for 1 hour.

![](https://static.igem.wiki/teams/5022/wiki/images/vv11.jpg)

**Figure.10 Expression of reporter gene GFP induced by different concentrations of cadmium ions.**
The results show that the expression of reporter gene GFP can be activated with 0.02mg/L cadmium aqueous solution, which can be used for rapid detection of cadmium pollution in routine water environment.

**Detection of actual water environment pollution samples**
In order to further verify the practical usability of our prepared sensor, we collected some contaminated water samples from the environment and detected them using the paper-based sensor, which showed that the expression of some fluorescent proteins could be seen vaguely (shown in the following figure). It illustrates the possibility of the sensor's extended application.

![](https://static.igem.wiki/teams/5022/wiki/images/vv12.jpg)

**Figure.11 Paper-based sensor detection in real water environment pollution samples.**

# In Summary

We established a paper-based biosensor in this study to detect cadmium contamination in aqueous environments through cadmium aptamers. The results showed that we were able to detect cadmium in aqueous solutions as low as 0.02 mg/L using the paper-based sensor, which is comparable to the sensitivity of conventional methods for detecting contaminated water, and can satisfy the detection of routinely contaminated water.
This method is simple and convenient, and is of great significance and potential promotion value for the pollution detection and potential treatment of heavy metals in the water environment.

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