Our goal was to create a complete system device that can be used to adsorb heavy metal ions and precious metal ions in industrial wastewater. After the scorching efforts of the human practice team members, we learned that three heavy metal ions, cadmium, copper, and lead, and one precious metal ion, silver, have high adsorption values.
To prove that our proteins and experimental devices can work in the real world, the Human Practice team members conducted in-depth discussions with various stakeholders. In addition, the modeling group, the human practice group, and the hardware group worked together with the experimental group to prove the validity of our concept. To make the final result more perfect. The proof of concept is divided into four parts, corresponding to the experimental group, human practice group, hardware group, and modeling group.
We hope that our project will not only change the existing status quo of wastewater treatment but also open a new path for synthetic biology and enhance the economic benefits of synthetic biology in wastewater applications. Therefore, we endeavor to involve stakeholders in the whole process. Therefore, the project's upgrading direction, implementation method, and other ideas are created and provided by experts. We will use the Reaction timeline to show how the experts have provided input and confirmed that our project is an effective and viable approach to wastewater management. To read more about the advice we received from our stakeholders, we recommend visiting our integrated Human Practice page.
To ensure that the expression of the CsgA-AG4 fusion protein is not affected by the CsgA gene in the genome, we planned to knock out the CsgA and CsgB genes in the genomes of BL21(DE3) and MC4100. Knocking out the CsgB gene facilitates the detachment of curly fiber proteins from the outer membrane of bacterial cells. Figure 1 shows the length of the knockout fragment as verified by DNA agarose gel electrophoresis and qPCR after knocking out the gene.
Figure 1: The agarose gel electrophoresis of the DNA fragment in the genome from MC4100. Sample 1, sample 2, and sample 3 show the strips are 1 kb. Samples 5 and 6 have the highlight strip which are near 1.5kb. The length of the highlight strips is the same as the recombinant fragment which is 1528bp. This result means the CsgB and CsgA have been knocked out.
Figure 2: The agarose gel electrophoresis of the DNA fragment in the genome from BL21(DE3). Sample 1, sample 2, and sample 3 show the strips are 1 kb. Sample 5 has the highlight strip which is near 1.5kb. The length of the highlight strips is the same as the recombinant fragment which is 1528bp. This result means the CsgB and CsgA have been knocked out.
The purpose of the Congo Red Spin Down Assay was to find the optimal IPTG concentration and confirm protein expression for the expressed proteins of JF1, BL21(DE3), and MC4100 strains. The Congo Red reagent could stain amyloid protein to quantitatively measure the expression of the CsgA-AG4 fusion protein. A concentration gradient of IPTG from 0-1.5mM was set up to treat overnight expression cultures.
The relative value of protein expression can be obtained by comparing the OD490 of the culture without introduced plasmid and the OD490 of the tested culture. Figure 1 shows the relative value (OD490) of protein expression of the JF1 strain as a function of IPTG concentration. Figure 2 shows the relative value (OD490) of protein expression of the BL21(DE3) strain as a function of IPTG concentration. Figure 3 shows the relative value (OD490) of protein expression of the MC4100 strain as a function of IPTG concentration.
Figure 3: The graph illustrates the relative protein expression levels of Amyloid-like Curli Fiber Protein produced by the JF1 strain under different IPTG concentrations. The y-axis represents the discrepancy in absorbance values at 490nm between the non-plasmid-transformed JF1 strain subjected to Congo Red assay and the plasmid-transformed JF1 strain induced with various IPTG concentrations to show the relative curli fiber protein expression level. The gradient of IPTG concentrations ranges from 0mM to 1.5mM along the x-axis. The depicted data provide insight into the impact of IPTG concentration on the protein expression of Amyloid-like Curli Fiber Protein in JF1, aiding in the understanding of its protein expression level.
Figure 4: The graph illustrates the relative protein expression levels of Amyloid-like Curli Fiber Protein produced by the BL21(DE3) strain under different IPTG concentrations. The y-axis represents the discrepancy in absorbance values at 490nm between the non-plasmid-transformed BL21(DE3) strain subjected to Congo Red assay and the plasmid-transformed BL21(DE3) strain induced with various IPTG concentrations to show the relative curli fiber protein expression level. The gradient of IPTG concentrations ranges from 0mM to 1.5mM along the x-axis. The depicted data provide insight into the impact of IPTG concentration on the protein expression of Amyloid-like Curli Fiber Protein in BL21(DE3), aiding in the understanding of its protein expression level.
Figure 5: The graph illustrates the relative protein expression levels of Amyloid-like Curli Fiber Protein produced by the MC4100 strain under different IPTG concentrations. The y-axis represents the discrepancy in absorbance values at 490nm between the non-plasmid-transformed MC4100 strain subjected to Congo Red assay and the plasmid-transformed MC4100 strain induced with various IPTG concentrations to show the relative curli fiber protein expression level. The gradient of IPTG concentrations ranges from 0mM to 1.5mM along the x-axis. The depicted data provide insight into the impact of IPTG concentration on the protein expression of Amyloid-like Curli Fiber Protein in MC4100, aiding in the understanding of its protein expression level.
Figure 6: The Congo red staining of bacteria. The red sediment means the bacteria expressed the amyloid.
Respectively, the optimal concentration of IPTG for the three strains is JF1: 0.1mM; BL21(DE3): 0.6mM; MC4100: 0.4mM. For details of the Congo Red Spin Down Assay, please refer to the result part.
Figure 7: The fusion protein CsgA-AG4 has been stained by Congo Red Dye. These photographs show the curli films from JF1 (A), BL21(DE3) (B), and MC4100 (C).
To evaluate the ability of CsgA-AG4 fusion protein for adsorbing silver ions, temperature, time, and silver ion concentration gradients were established.
Members of the experimental team first conducted pre-experiments to estimate the approximate range of time and temperature and then determined the relatively precise optimal temperature and time based on the predictions of the modeling team and the re-experiments of the experimental team.
Figure 8 shows the effect of different environmental temperatures on the adsorption of silver ions by fusion proteins (incubation from three bacterial strains). Figure 9 shows the effect of different adsorption times on the adsorption of silver ions by fusion proteins (incubation from three bacterial strains). Figure 10 shows the relatively accurate optimal adsorption time and temperature based on a large amount of data.
Figure 8: Assessment of optimal temperature. This graph indicates the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at various temperatures over 8 hours. The optimal temperature is found to be 25℃.
Figure 9: Assessment of optimal time. This graph depicts the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at 25°C across varying adsorption times. The figure highlights the optimal adsorption time is found to be 8 hours.
Figure 10: The function image of time-temperature-adsorption efficiency. A represents a three-dimensional graph illustrating the adsorption efficiency as a function of temperature and time, obtained through fitting. The face center composite design (α=1) was carried out using the design expert 13. B displays a contour plot of the same function.
Based on the relatively accurate optimal adsorption time and temperature, members of the experimental team established a series of silver ion concentration gradients to evaluate the adsorption efficiency of the fusion protein under different silver ion concentrations.
Figure 11: Assessment of optimal silver ion concentration. This graph illustrates the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at 25℃ across varying silver ion concentrations during an 8-hour incubation period.
Figure 12: This figure gave a fitting Langmuir curve based on the experiment data by Python.
According to the fitting results which indicate the absorption ability of fusion protein based on the experiment data, a calculated n (The average ion number that can be absorbed by a protein) is obtained. The n is 6.4442, which means that every CsgA-MBP3 can absorb about 6.4442 ions.
Figure 13: This figure describes the needed amount (unit: /mol∙L^(-1)) of CsgA-AG4 when the concentration of decreases below the safe sewage discharge standard.
Also, we built a model about the required amount of CsgA-AG4 when the concentration of decreases below the safe sewage discharge standard. It claims that our system has an obvious absorption ability.
If you want more details, please refer to the result and model part.
To further verify the expression of the amyloid protein CsgA, we observed the fusion protein CsgA-AG4 by SEM. Moreover, we observed the concentration and ratio of each element in the protein sample after adsorbing silver ions. Figure 9 shows the morphology of the CsgA protein at different magnifications by Qingdao Yuance Test Technology Services Co., Ltd..
Figure 14: Scanning Electron Microscopy (SEM) analysis. The experimenter used SEM to observe the fusion protein produced by the MC4100 strain. The part marked in red in the figure is the CsgA-AG4 fusion protein which can be seen clearly. The magnification of A, B, and C are 5 KX, 10 KX, and 20 KX respectively.
Figure 15: SEM electron microscopy shows the image and the percentage of each element in the protein. The experimenters selected the central region of the whole protein for elemental content analysis. This allowed for the intuitively visual observation of the distribution and content of various elements within the target protein. The presence of silver elements can demonstrate the absorption of silver by the target protein.
Figure 16: SEM Elemental Mapping of CsgA-AG4 after absorbing silver ions. (a) Element carbon distribution (red). (b) Element nitrogen distribution (blue). (c) Element oxygen distribution (purple). (d) Element sulfur distribution (dark green). (e) Element silver distribution (yellow). (f) Element phosphorus distribution (light green). Scale bar: 1μm.
If you want more details, please refer to the results part.
Additionally, to further verify that the fusion protein CsgA-AG4 adsorbs silver ions, we used STEM to observe the structure of the CsgA-AG4 fusion protein.
Figure 17: STEM-HAADF imaging of nano silver synthesis on CsgA-AG4 fusion protein (30kV). The magnification of A~G are 10.00KX, 20.00KX, 10.00KX, 20.00KX, 10.00KX, 50.00KX, 50.00KX (done by Yuantest Laboratory).
The idea was to create a fusion protein, SUMO-MSmtA4-CBM-sfGFP, with the potential to efficiently bind to cellulose surfaces, chelate heavy metals, fluoresce, and serve as a versatile tool for various biological and environmental applications.
The scope of this proof of concept was to validate the feasibility and functionality of the SUMO-MSmtA4-CBM-sfGFP fusion protein. Specifically, we aimed to demonstrate the following:
The POC would be considered successful if:
Creation of Fusion Protein: Express and purify the SUMO-MSmtA4-CBM-sfGFP fusion protein following established protocols.
The expression plasmid pET21a-SUMO-MSmtA4-CBM-sfGFP was transformed into TOP10 competent cells for large-scale cultivation.
Subsequently, the pET21a-SUMO-MSmtA4-CBM-sfGFP plasmids were extracted and transformed into the expression host Escherichia coli BL21(DE3) for the expression of recombinant fusion proteins (SUMO-MSmtA4-CBM-sfGFP).
The transformed cells were grown in 500 mL of LB liquid culture medium at 37°C with a final concentration of 100 μg/mL ampicillin.
When the OD600 reached 0.6 ± 0.05, protein expression was induced by adding Isopropyl-β-D-thiogalactoside (IPTG) to the culture medium and incubating at 16°C for 18 hours. After 18 hours of IPTG induction, the cells exhibited noticeable fluorescence compared to the uninduced cells, indicating successful expression of the fusion protein.
The cells were then collected by centrifugation at 12,000 x g for 10 minutes at 4°C and subsequently lysed by sonication in a lysis buffer (50 mM Tris-HCl, pH 7.8, 0.2 mM PMSF) at a five-fold volume ratio (w/w).
The cell lysis was considered complete when the bacterial solution became relatively transparent. After centrifuging the cell lysate at 10,000 x g for 20 minutes, the supernatant was recovered.
Using enzyme-linked immunosorbent assay and observation to demonstrate the expression and function of sfGFP protein
First, fused sfGFP with MSmtA4. Used a flexible linker to ensure that the coding sequence of sfGFP is incorporated into the target protein, allowing it to interact with the target protein.
Observed the localization and distribution of the fused protein within cells under normal illumination. If sfGFP is functioning properly, researchers will observe a green fluorescence signal.
Figure 19: On the left is microcrystalline cellulose that binds to proteins containing CBM-sfGFP, while on the right is the control (Pure microcrystalline cellulose).
Confirmation of the binding between CBM and microcrystalline cellulose using SEM scanning electron microscopy and BCA protein content determination
Binding to Cellulose: Conduct experiments to test the binding affinity of the fusion protein to cellulose surfaces
At a ratio of 1:250, microcrystalline cellulose was added to the supernatant obtained after cell lysis. The mixture was then gently shaken at 100 rpm for 1 hour at room temperature to ensure thorough contact between the protein and microcrystalline cellulose.
After the incubation period, the mixture was centrifuged at 10,000 x g for 5 minutes to remove the supernatant. Subsequently, ultra-pure water was added for washing, followed by another round of centrifugation to eliminate the supernatant and obtain the desired product.
By using the BCA protein measurement method to compare the protein content of the supernatant obtained after centrifugation and before binding with microcrystalline cellulose, a significant decrease in protein content was observed.
This preliminary indicates that microcrystalline cellulose binds to proteins.
Figure 20: Protein concentration measured using the BCA method. After the fusion protein containing CBM was fully contacted with microcrystalline cellulose for 1 hour, the content changes of the protein before and after adsorption were measured by the BCA method.
Then, SEM scanning electron microscopy was used to capture the binding between the fusion protein containing CBM and microcrystalline cellulose by Qingdao Yuance Test Technology Services Co., Ltd.
Figure 21: Scanning Electron Microscopy (SEM) analysis. A represents Microcrystalline cellulose that is not bound to any protein, and B represents Microcrystalline cellulose bound to proteins containing CBM (done by Yuantest Lab).
Chelation of Heavy Metals: Evaluate the fusion protein's ability to chelate heavy metals through ICP-MS
Using ICP-MS technology, the concentrations of heavy metal ions were measured both before adsorption and after adsorption. The difference between these concentrations was observed to determine whether MSmtA4 interacted with the ions.
Figure 22: Wild-type SmtA and docking result. form ionic bonds with Cys14, Cys54, Cys52, and Cys47. Docking score: -6.091kcal/mol
Figure 23: MSmtA and docking result. forms ionic bonds with Cys14, Cys54, Cys52, and Cys47. Docking score: -6. 133kcal/mol.
Figure 24: The removal rate of metal ions by biosorbent (SmtA) and biosorbent (MSmtA4). The x-axis represents , , , and the y-axis represents the metal removal (%). The grey column represents biosorbent (SmtA), and the black column represents biosorbent (MSmtA4). **
Figure 25: Optimization of adsorption time and comparison of the removal efficiency between biosorbent (SmtA) and biosorbent (MSmtA4). The x-axis represents the time interval and the y-axis represents the metal removal (%).
In this device, the protein that adsorbs heavy metals will be placed in the middle of the device. The sewage flow will follow the path of the blue arrow. In this device, the sewage will fully come into contact with the protein, forming an internal circulation within the device. An additional water pump can be used to pump out the heavy metal ion-removed sewage after sufficient reaction.
Figure 26: Heavy metal ion sewage will have an internal circulation in the device to ensure that the protein is fully in contact with it.
Figure 27: This diagram shows the internal model of a protein device that adsorbs heavy metal ions. Due to time issues, we did not 3D print the complete external structure. We plan to complete the external installation in the future.
Based on the preliminary design blueprint, we further planned some functions of the device. We added a waterproof motor and fan blades to the device to drive the internal circulation of sewage. The temperature and humidity sensor is installed at the bottom of the device. In addition, ion-selective electrodes are used to measure the concentration of silver ions.
Figure 28: We designed some unique features on the device that adsorbs silver ions to the protein, including temperature and humidity sensors, ion-selective electrodes and waterproof motors.
Figure 29: Noble metal ion sewage hardware. This model represents that a water proof motor added in the hardware and can ensure the water to circulate clockwise and counterclockwise. The protein is sandwiched between two discs, with multiple discs placed inside the device's core, and sensors and displays are added to monitor conditions within the device in real time.
Figure 30: Application of protein membranes in hardware. As shown in the figure, place the prepared protein membrane with CsgA+AG4 fusion protein in the place where the protein membrane is placed, add 25 degrees 4 micromolar silver nitrate solution to the device, and monitor its concentration, temperature, and pH changes in real-time. We use the internal circulation system to circulate the water alternately clockwise and counterclockwise in the device and incubate for 8 hours to mimic the real wastewater adsorption condition.
The successful completion of this proof of concept will provide valuable evidence of the SUMO-MSmtA4-CBM-sfGFP fusion protein's feasibility and potential, paving the way for its application in diverse fields and addressing challenges related to cellulose modification, heavy metal chelation, and protein research.