In the noble metal project, three bacteria strains were applied, which were JF1, BL21(DE3), and MC4100. We designed three biological pathways based on the different strains' different characteristics. The genetic circuit of pET21d(+)-CsgA-AG4-CsgC-CsgD-CsgE-CsgF-CsgG is designed for JF1. The biological pathway of pET21a(+)-CsgA-AG4 is designed for BL21(DE3). The genetic circuit of pGEX-6P-1-CsgA-AG4 is designed for MC4100. The Wiki of Engineering Success explains the logic of these genetic circuit constructions and exhibits a lot of details.
Figure 1: The genetic circuit of the biosorbent synthesis for noble metal ions adsorption. IPTG induces this biological pathway. T7 promoter is chosen for the JF1 strain. CsgA (BBa_K4968004) and AG4 (BBa_K4968005) link with GSGGSG linker. CsgC, CsgD, CsgE, CsgF, and CsgG are transcribed respectively.
Figure 2: pET-21d(+)-CsgA-AG4-CsgCDEFG plasmid map created by SnapGene. This graph displays the CsgA-AG4-CsgCDEFG biological pathway in the pET21d(+) plasmid.
Figure 3: The genetic circuit of the biosorbent synthesis for noble metal ions adsorption. IPTG induces this biological pathway. T7 promoter is chosen for the BL21(DE3) strain. CsgA and AG4 link with GSGGSG linker.
Figure 4: pET-21a(+)-CsgA-AG4 plasmid map created by SnapGene. This graph displays the CsgA-AG4 biological pathway in the pET21d(+) plasmid.
Figure 5: The genetic circuit of the biosorbent synthesis for noble metal ions adsorption. IPTG induces this biological pathway. Tac promoter is chosen for the MC4100 strain. CsgA and AG4 link with GSGGSG linker.
Figure 6: pGEX-6p-1-CsgA-AG4 plasmid map created by SnapGene. This graph exhibits the biological pathway of CsgA-AG4 in the pGEX-6P-1 plasmid.
We employed a homologous recombination method based on the Lambda Red system, which is grounded in the principles of bacteriophage λ DNA repair, to knock out the CsgA and CsgB genes from the genomes of the MC4100 and BL21(DE3) bacterial strains. This was done to eliminate the interference of curli fiber without the AG4 protein and to abolish the anchoring effect of curli fiber on the outer membrane, as detailed in the Engineering Success.
We constructed a linear DNA fragment, RecomSwapNeo R/Kan R (BBa_K4968006), containing the target editing genes, and integrated it into the same position in the bacterial genome through homologous recombination. The CsgA and CsgB genes in the genome were replaced with antibiotic-resistance genes. To achieve this homologous recombination, a crucial plasmid, pKD46, carrying the required red protein genes of the Lambda Red system, was also essential. These red proteins include Exo, Bet, and Gam.
After the introduction of the linear DNA fragment, Exo, Bet, and Gam proteins collaborated to integrate the antibiotic-resistance genes into the specific location of the target gene. Bet protein facilitated recombination between the sticky ends of the DNA fragment and the host DNA, while Exo protein performed DNA cleavage and repair during this process (Datsenko and Wanner, 2000). The Gam protein was crucial in safeguarding the linear DNA fragment from degradation by bacterial nucleases.
Once successful recombination was achieved, a temperature shift to 37 ℃ was employed to exploit the temperature-sensitive nature of pKD46, leading to the loss of pKD46 and eliminating its influence on subsequent experiments (Doublet et al., 2008).
Figure 7: pUC57-RecomSwapNeo R/Kan R plasmid made by SnapGene. This graph indicates the RecomSwapNeo R/Kan R insert in the pUC57 plasmid.
Figure 8: The strain growth on plates after Lambda red homologous recombination. After lambda red homologous recombination by electroporation, the strain is grown on LB plates containing kanamycin, and the strain with successful knockout will carry kanamycin resistance and grow normally, while the failed knockout will not grow. Strains circled in red are used as subsequent colony PCR steps for further validation.
Figure 9: 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.
Figure 10: 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.
Due to the deletion of the gene of CsgB, the CsgA-AG4 fusion proteins were separated from bacteria conveniently. Refer to Courchesne et al. (2017), the CsgA-AG4 fusion proteins were separated from the bacteria by a vacuum filter. The bacteria solution after overnight expression was incubated with 0.8M guanidinium chloride (GdmCl). After that, filter about 70mL of bacteria solution by vacuum filter onto 47 mm polycarbonate filter membranes with 10 μm pores. Then, the filtered substances were washed with 8M GdmCl, nuclease, and SDS. The purpose of the GdmCl was to lysis the bacteria and disrupt the integrity of the bacterial outer membranes, which caused the debris of bacteria to go through the polycarbonate filter membranes with 10 μm pores.
To verify the curli fiber protein that remained on the polycarbonate filter films was secreted through the secretory channel protein instead of lysis, we stained the CsgA-AG4 protein with Congo red. The CsgA proteins are folded in the channel. If the CsgA proteins fold successfully, they will be secret to the channel. If the CsgA proteins are not through the channels, they will fold unsuccessfully, which are not stained by Congo red. Nuclease with DNase and RNase is used to digest the residual DNA and RNA on the curli fiber protein. SDS was applied to separate the CsgA-AG4 from the polycarbonate filter films scraped by a spatula. Figure 10 presents the process of purified proteins of CsgA-AG4.
Figure 11: The image of the process from plasmid introduced and gene knockout to the CsgA-AG4 filtration and purification created by Biorender. The process is shown in cartoon form.
Congo red can stain the polysaccharides and amyloid proteins. CsgA protein is an amyloid curli fiber protein that can be stained by Congo red. The CsgA-AG4 protein film stained by Congo red appeared red. Based on the characteristic of CsgA-AG4 stained by Cong, we explored the optimal IPTG which induced protein expression by the method of Congo red staining. The higher content of the CsgA-AG4 fusion protein, the supernatant was clearer after staining by Congo red. The red degrees of the supernatant were detected by the plate reader under 490nm. The difference between the OD490 of the 0.0015% Congo red and the supernatant after the bacteria stained by 0.0015% Congo red reflected the CsgA-AG4 fusion protein expression. More details are expressed in the Wiki of the Engineering Success.
Figures 12 to 14 show the protein expression under IPTG gradients induced and expressed by the OD490 of the difference between the Congo red staining of the bacteria without plasmid and with IPTG induced. These results reflected the protein expression and eliminated the interference of polysaccharides on the outer membrane. Figures 15 to 17 show the protein expression under IPTG gradients induced and presented by the OD490 of the difference between the Congo red staining of the bacteria without IPTG and with IPTG induction. These results indicated only the IPTG effect on CsgA-AG4 fusion protein expression. The optimal concentrations of IPTG of JF1, BL21(DE3), and MC4100 were 0.2mM, 0.6mM, and 0.4mM.
Figure 12: 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 13: 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 14: The graph of 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 15: The graph portrays the influence of varying IPTG concentrations on the expression of Amyloid-like Curli Fiber Protein by the JF1 strain. The y-axis delineates the disparity in absorbance values at 490nm between the plasmid-transformed JF1 strain, which remained uninduced by IPTG, and the same strain induced with a gradient of IPTG concentrations ranging from 1mM to 1.5mM. This difference in absorbance values captures the modulation of protein expression levels in response to incremental IPTG concentrations. The x-axis represents the IPTG concentration gradient. These results elucidate the role of IPTG in regulating the expression of Amyloid-like Curli Fiber Protein in JF1, providing critical insights into its production dynamics.
Figure 16: The graph portrays the influence of varying IPTG concentrations on the expression of Amyloid-like Curli Fiber Protein by the BL21(DE3) strain. The y-axis delineates the disparity in absorbance values at 490nm between the plasmid-transformed BL21(DE3) strain, which remained uninduced by IPTG, and the same strain induced with a gradient of IPTG concentrations ranging from 1mM to 1.5mM. This difference in absorbance values captures the modulation of protein expression levels in response to incremental IPTG concentrations. The x-axis represents the IPTG concentration gradient. These results elucidate the role of IPTG in regulating the expression of Amyloid-like Curli Fiber Protein in BL21(DE3), providing critical insights into its production dynamics.
Figure 17: The graph of the influence of varying IPTG concentrations on the expression of Amyloid-like Curli Fiber Protein by the MC4100 strain. The y-axis delineates the disparity in absorbance values at 490nm between the plasmid-transformed MC4100 strain, which remained uninduced by IPTG, and the same strain induced with a gradient of IPTG concentrations ranging from 1mM to 1.5mM. This difference in absorbance values captures the modulation of protein expression levels in response to incremental IPTG concentrations. The x-axis represents the IPTG concentration gradient. These results elucidate the role of IPTG in regulating the expression of Amyloid-like Curli Fiber Protein in MC4100, providing critical insights into its production dynamics.
The method of measuring the adsorption of silver ions by CsgA-AG4 was calculated as the difference between the concentration of AgNO3 before treatment and after being absorbed by CsgA-AG4. The concentrations of the were detected by ICP-MS which was operated by technician Xiaoping Xie. Figure 18 exhibits the adsorption efficiency of silver ions by curli fiber films from different strains at 25℃ for different hours. Figure 19 presents the adsorption efficiency of silver ions by the CsgA-AG4 fusion protein from three strains at different temperatures for 8 hours.
After exploration of the optimal temperature and times, we set the concentration gradients of silver ions to research the effect on protein activity of CsgA-AG4 by improving silver concentration (Figure 20).
Following China's regulatory guidelines for wastewater discharge within the electronics industry, the permissible limit for total silver content is set at 0.3 mg/L (Ministry of Ecology and Environment of the People’s Republic of China, 2023). Following the application of the CsgA-AG4 fusion protein and subsequent treatment, the curli fiber films produced by MC4100 were found to effectively reduce the concentration of to levels that comply with the national emission standard, which is less than or equal to 10 μM . Similarly, the curli fiber films generated by BL21(DE3) demonstrated efficient treatment capabilities, ensuring that the concentration remains at or below 8μM in compliance with the national emission standard. Furthermore, the curli fiber films developed by JF1 reduce the concentration to a maximum of 6μM, meeting the national emission standard requirements. Figure 21 proves that the polycarbonate filter films do not have the ability of silver adsorption.
Figure 18: This graph presents the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at 25℃ on different adsorption times. The maximum adsorption efficiency of CsgA-AG4 is from MC4100. This figure indicates the optimal adsorption time is 8 hours.
Figure 19: This graph of the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at different temperatures for 8 hours. The optimal temperature is 25℃. The CsgA-AG4 protein of MC4100 has the maximum adsorption efficiency. The adsorption efficiency of protein from JF1 is the lowest.
Figure 20: This graph indicates the adsorption efficiency of CsgA-AG4 recombinant protein from three strains at 25℃ on different silver ions concentrations and incubated for 8 hours. The maximum adsorption efficiency of CsgA-AG4 is from MC4100.
Figure 21: This figure illustrates the silver ion adsorption capacity of the control group, the polycarbonate membrane, under different adsorption conditions. 1) One layer of polycarbonate membrane adsorbed for one hour. 2) Three individual polycarbonate membranes adsorbed for 1 hour. 3) Add one layer of polycarbonate membrane adsorbed for one more hour (total 2 hours). 4) Three individual polycarbonate membranes adsorbed for 2 hours. 5) Add one layer of polycarbonate membrane again adsorbed for one more hour (total 3 hours). 6) Three individual polycarbonate membranes adsorbed for 3 hours.
AG4 can reduce the silver ions to nano-silver, which can be observed as the highlight point by STEM. We prepared three samples of the CsgA-AG4 films from MC4100 and JF1 to do SEM and STEM. Two samples of curli fiber on the polycarbonate filters from the MC4100 and JF1 were done SEM. The sample of curli fiber already absorbed silver ions from MC4100 was done STEM.
SEM and STEM were completed by Qingdao Yuance Test Technology Services Co., Ltd. The structures of the CsgA-AG4 were shown in Figure 22 and Figure 23, which were from JF1 and MC4100 severally and done by SEM. The percentage of the silver elements distributed in the CsgA-AG4 films was expressed in Figure 24. The image of element mapping presented the silver element distribution visually (Figure 25). The highlight spots and black spots were nano-slivers observed on the image of STEM-HAADF (Figure 26).
Compared to the silver mapping and the STEM-HAADF, the yellow spots were more than the bright, and black spots, which meant that besides the reduction of silver ions by CsgA-AG4 proteins, there was electrostatic adsorption of silver ions by protein.
Figure 22: Scanning Electron Microscopy (SEM) analysis. The experimenter used SEM to observe the fusion protein produced by the JF1 strain. The part marked in red in the figure is the CsgA-AG4 fusion protein which can be seen clearly. The magnification of A and B are 5 KX and 20 KX respectively (done by Yuantest Laboratory).
Figure 23: 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 (done by Yuantest Laboratory).
Figure 24: 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 (done by Yuantest Laboratory).
Figure 25: 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 (done by Yuantest Laboratory).
Figure 26: 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).
Figure 27: The image of the silver mapping and the STEM-HAADF. The left image is the silver mapping by SEM and the yellow color distribution shows the silver element, including silver elemental substances and silver ions. The middle and right images are the STEM-HAADF which exhibits nano-silver (silver elemental substances).
Finally, it was put into use in hardware to verify the actual adsorption efficiency of the final protein membrane in hardware, and due to the limited production time, a preliminary hardware model was first constructed, which can simulate the real situation of influent, outflow, internal circulation, and real-time monitoring of conditions in the device. We put the protein membrane produced in the laboratory under optimal conditions into hardware and added silver nitrate solution to simulate the sewage environment for testing.
Figure 27: 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.
In the heavy metal project, we plan to use BL21(DE3) as the strain expressing the target protein. At the genetic design level, we designed MSmtA4 derived from four amino acid point mutations. And we used the gene pathway pET21a(+)-SUMO-MSmtA4-CBM-sfGFP to express the fusion protein. More details, please refer to the Engineering Success.
SUMO (Small Ubiquitin-like Modifier) (BBa_K4968007) is a ubiquitin-like protein that is commonly found in eukaryotic organisms. When SUMO is fused to the N-terminus of the target protein, it can improve the folding of the target protein, increase its solubility, and increase protein yield.
Carbohydrate Binding Modules (CBMs) (BBa_K4968002) are components of several enzymes, and their primary function is to bind to specific carbohydrates. In our project, we have chosen a family 2 CBM from endoglucanase A of C.fimi.
Super folded GFP (sfGFP) (BBa_K4968003) is a fluorescent marker that does not induce misfolding when fused to other proteins, thereby increasing the stability of the fusion protein. Under daily visible light irradiation, substances expressing sfGFP will show strong green fluorescence. sfGFP can fold and function faster compared to ordinary GFP.
Figure 1: The genetic circuit of the biosorbent synthesis for heavy metal ions adsorption. IPTG induces this biological pathway. T7 promoter is chosen for the BL21(DE3) strain. SUMO and MSmtA4 link with GSGGSG linker. MSmtA4 and CBM link with GSGGAGGS. CBM and sfGFP link with GGGSPTG.
Firstly, we aimed to acquire the single M-SmtA4（BBa_K4968001) fragment and the composite SUMO-SmtA-CBM-sfGFP （BBa_K4968012) fragment by PCR. Next, the construction of SUMO-SmtA-CBM-sfGFP（BBa_K4968012) in pET-21a(+) plasmid could be achieved by the ligation of enzyme-digested pET-21a(+) with the SUMO-SmtA-CBM-sfGFP（BBa_K4968019) fragment. In addition, used the composite CBM-sfGFP-pUC_GW_Kan R-SUMO（BBa_K4968017) part acquired by PCR, along with SUMO-MSmtA4-CBM-sfGFP（BBa_K4968021) in pUC _GW_kan R plasmid and added Gibson assembly HiFi Master Mix to mixture. The final product SUMO-MSmtA4-CBM-sfGFP（BBa_K4968021) in pET-21a(+) plasmid could be obtained.
After PCR, the following agarose gel electrophoresis showed that the size of the fragments and the enzyme digestion for validation were correct. Figure 3 was the DNA sequencing result of pET-21a(+) -SUMO-MSmtA4-CBM-sfGFP. The sequencing result showed that Lys at positions 8, 22, 45 and Arg at position 26 were successfully mutated into Cys at the same time.
The final product pET-21a(+)-SUMO-MSmtA4-CBM-sfGFP was confirmed by DNA sequencing.
Figure 2: This figure indicated the result of running DNA agarose electrophoresis after PCR reaction, enzyme digestion and Gibson assembly. (A) The PCR product of the M-SmtA4 fragment. (B) The PCR product of the SUMO-pUC_GW_Kan R-sfGFP-CBM (BBa_K4968017) fragment. (C) The double enzyme digestion of SUMO-MSmtA4-CBM-sfGFP (BBa_K4968021) in pUC _GW_kan plasmid after Gibson assembly. (D) The double enzyme digestion of SUMO-SmtA-CBM-sfGFP (BBa_K4968019) and SUMO-MSmtA4-CBM-sfGFP (BBa_K4968021) in pET-21a plasmid.
Figure 3: Sequencing results of site-directed mutagenesis. Amino acid mutations at four positions, changing Lys8, Lys45, Lys22 and Arg26 into Cys.
Since pET-21a(+) plasmid was selected as the backbone, IPTG could be used as the inducer to bind with LacI protein and turn on the lacI promoter, so that our fusion protein would express.
Two plasmids, pET21a(+)-SUMO-SmtA-CBM-sfGFP and pET21a(+)-SUMO-MSmtA4-CBM-sfGFP were transformed into E. coli BL21(DE3) respectively. After obtaining the modified bacteria, we induced the expression of the fusion protein by adding 1mM IPTG and incubated at 16℃.
The modified bacteria were harvested by centrifugation. Importantly, there were significant differences between the color of induced E. coli BL21(DE3) and that of E. coli BL21(DE3) without inducement (Figure. 4A). The strong green fluorescent light of induced was observed under daylight when compared to that of uninduced. To further identify whether the expression was successful or not, the induced bacteria were lysed by sonication and the supernatant obtained by centrifugation was used for SDS-PAGE. The results of SDS-PAGE analysis for the fusion protein in E. coli BL21(DE3) are shown in Fig. 4B. We found that both fusion proteins were strikingly expressed in a soluble form with a molecular weight of 55.8 kDa.
Combining the results of the green fluorescent light with that of the correct molecular weight, we could determine that both recombinant fusion proteins were successfully expressed and in a soluble form.
Figure 4: This figure indicated the expression of the fusion protein. (A) The obvious color change can be observed after inducement. The color of both bacteria precipitates (Right) corresponding to two fusion proteins turns green compared to the uninduced one (Left). (B) The supernatant with fluorescent green after cell lysis. (C) SDS-PAGE of crude extract after expression. Crude extract 1: Containing SUMO-SmtA-CBM-sfGFP. Crude extract 2: SUMO-MSmtA4-CBM-sfGFP.
Figure 5: These figures (from A-E) indicated that the Scanning Electron Microscopy (SEM) observation of the fusion protein SUMO-MSmtA4-CBM-sfGFP. From picture A to E, the magnifications are 1.00KX, 2.00KX, 5.00KX, 10.00KX, 20.00KX(done by Yuantest Laboratory).
The concentration of IPTG used could dramatically influence the expression level of protein. Induction with low concentration might decrease the rate of expression, while induction with high concentration might also cause toxic to the bacteria. Therefore, it was essential to determine the optimal concentration of IPTG for the fusion protein expression. The fluorescence intensity is positively correlated with the concentration of sfGFP, thus the induced expression level by different concentrations of IPTG could be assessed.
The modified bacteria were induced with 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM and 1 mM of IPTG respectively. The unconcerned variable disturbance was removed by keeping other treatments the same. After centrifugation and sonication, the fluorescence intensity of the supernatant at 512 nm was measured by the microplate reader.
The correlation of the fluorescence intensity and IPTG concentration is shown in Figure 5. Notably, the fluorescence intensity of fusion proteins reached peaks at 0.4 mM and 0.8 mM concentration of IPTG respectively.
After the discussion of this situation, we decided to use 0.4mM IPTG to induce the protein expression.
The possible reason for this situation is incomplete cell lysis by ultrasound.
According to the following experiments (see Figure 6 for details), it can be proved that under the induction of 0.4mM concentration of IPTG, two fusion proteins, SUMO-MSmtA4-CBM-sfGFP and SUMO-SmtA-CBM-sfGFP, can bind to microcrystalline cellulose only to a limited extent.
Figure 5: Optimization of the concentration of IPTG for the expression of both fusion proteins.
We aimed to demonstrate the binding ability of both fusion proteins on microcrystalline cellulose. Therefore, the protein supernatants before and after adsorption could be verified by SDS-PAGE to check whether the bands for both fusion proteins disappeared or not.
Supernatants for both fusion proteins were prepared for running the gel. 0.5 mg microcrystalline cellulose was added to 1 mL supernatant. After 1h shaking, the remained supernatants for both fusion proteins were also prepared for running the gel.
Importantly, the color of the prepared biosorbent is extremely distinct with that of the pure microcrystalline (Figure 6A). Lane 2 shows that the band for fusion proteins disappeared, indicating that it was adsorbed on microcrystalline cellulose (Figure 6B). This result showed that both could be purified using microcrystalline cellulose in a single step. Additionally, this result is consistent with previous studies where CBM was observed to bind to cellulosic material (Hong et al., 2007).
To further test the number of fusion proteins binding on the carrier in a quantification manner, BCA assay was used to determine the reduction of fusion proteins in the supernatant. Figure 6C showed that more than 10000 ug/mL of fusion proteins were bound to the carrier after adsorption, and there was a significant difference before and after the adsorption.
The difference between before binding and after binding is significant. However, the difference of the protein which could bind to microcrystalline cellulose between SmtA and MSmtA4 could be ignored.
Figure 6: Assessment of fusion protein binding ability to microcrystalline cellulose. (A) The color difference between the pure microcrystalline cellulose and the biosorbent. (B) SDS-PAGE before and after the addition of the pure microcrystalline cellulose to crude extract. Lane 1: Before addition. Lane 2: After addition. (C) The protein concentration before and after binding.
A significant decrease in protein content was observed by comparing the supernatant obtained after centrifugation with the protein content prior to binding to microcrystalline cellulose. This preliminary observation indicates binding of the protein to microcrystalline cellulose. Scanning electron microscopy (SEM) was used to observe the binding of protein to microcrystalline cellulose to confirm this hypothesis.
Scanning electron microscopy (SEM) was used to observe the binding of protein to microcrystalline cellulose to confirm hypothesis.
Figure 7: Scanning Electron Microscopy (SEM) analysis. The microstalline in the figure A is soomth, representing Microcrystalline cellulose do not bound to any protein. The microstalline in the figure B is rough, representing Microcrystalline cellulose do bind to proteins containing CBM (done by Yuantest Lab).
It was necessary to evaluate the ability of adsorption and removal of target heavy metals in the mixed system. We chose to set two mixed systems with 25 mL heavy metal solutions. The initial concentrations of , and in the first system were all 20 mg/L, and that of the second was 50 mg/L, 20 mg/L and 20 mg/L respectively according to our preliminary experiment.(Mwandira et al., 2020).
The solution was prepared, and we first tested the heavy metal ions adsorption ability of the microcrystalline cellulose by adding 0.1 g microcrystalline cellulose to the 25 mL mixed solution every hour. Then the concentration of the heavy metal ions in the supernatant was determined by ICP-MS. The obtained biosorbents (0.1 g/mL) were added to the corresponding solution (pH = 6.0). The adsorption experiments were carried out at 25 °C. Samples were taken at 4 h. The concentration of , and in the mixed system was determined by ICP-MS, then the removal efficiency of , and by the biosorbents was calculated.
The adsorption of , and by microcrystalline cellulose was not obvious in the mixed system (Figure 7). It indicated that these three kinds of metals were barely adsorbed by the microcrystalline cellulose. As seen in Figure 8, the removal efficiency of the microcrystalline cellulose-SUMO-MSmtA4-CBM- sfGFP for , and is approximately 60%, 70% and 75%, while the result for microcrystalline cellulose-SUMO-SmtA-CBM- sfGFP is approximately 50%, 60% and 70%.For another system that the initial concentration of cadmium cation is 50 mg/L, it is notably that adsorption efficiency of cadmium cation for biosorbent (SmtA) decreases while that for biosorbent (MSmtA4) increases as compared to the results of the first system (Fig. 9). It is suggested that the higher adsorption efficiency of cadmium cation for biosorbent (MSmtA4) achieve at higher concentration of cadmium cation, which is probably due to its higher specific surface area and higher porosity.
After testing in the 25 mL mixed system, it has been demonstrated that the adsorption ability of the microcrystalline cellulose is negligible. Our mutation improvement is successful that enhancing the adsorption ability for all three metals in the mixed solution.
Figure 7: The effect of microcrystalline cellulose in the mixed system. The duration of the adsorption was 3 hours. This figure indicated that microcrystalline cellulose did not adsorb heavy metal ions.
Figure 8: The removal rate of metal ions by biosorbent (SmtA) and biosorbent (M-SmtA4) in the first mixed system. This figure indicated that MSmtA4 had the better ability of adsorbing heavy metal ions than SmtA.
Figure 9: The removal rate of metal ions by biosorbent (SmtA) and biosorbent (MSmtA4) in the second mixed system. The concentration of original solution was 50mg/L. The concentration of original solution was 20mg/L. The concentration of original solution is 20mg/L.
Courchesne, N.M.D. et al. (2017) ‘Scalable Production of Genetically Engineered Nanofibrous Macroscopic Materials via Filtration’ ACS Biomaterials Science & Engineering, 3(5), pp. 733-741. Available at: https://doi.org/10.1021/acsbiomaterials.6b00437
Datsenko, K.A. and Wanner, B.L. (2000) ‘One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products’ PNAS, 97(12), pp. 6640-6645. Available at: https://doi.org/10.1073/pnas.120163297
Hong, J., Ye, X., and Zhang, Y. H. (2007). ‘Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications’ Langmuir, 23(25), pp. 12535-12540. Available at: https://pubs.acs.org/doi/full/10.1021/la7025686
Ministry of Ecology and Environment of the People’s Republic of China (2023) Guideline on available techniques of water pollution prevention and control for electronic industry. Available at: https://www.mee.gov.cn/ywgz/fgbz/bz/bzwb/kxxjszn/202306/t20230621_1034274.shtml
Mwandira, W. et al. (2020) ‘Cellulose-metallothionein biosorbent for removal of Pb(II) and Zn(II) from polluted water’ Chemosphere, 246, 125733. Available at: https://www.sciencedirect.com/science/article/pii/S0045653519329741