Synthetic biology has advanced for over two decades and during this time, its techniques have been continuously applied to various industries, such as medicine, environmental management, energy, agriculture, materials science, food production, and more. Synthetic biology is well-suited for wastewater treatment. We have listed the following advantages
But synthetic biology is rarely actually practiced for use in wastewater. Because there are these major problems that need to be solved
Complexity: Wastewater contains a wide range of organic and inorganic pollutants, which can vary widely in type and concentration. Designing synthetic biology systems to treat multiple pollutants simultaneously requires overcoming complexity and interactions.
Stability: Synthetic biology systems need to operate stably under varying environmental conditions, and the wastewater treatment environment can be volatile, which may pose a challenge to the stability of synthetic biology systems.
Safety: the use of synthetic biology systems for wastewater treatment requires ensuring the safety of the biological system to prevent uncontrolled releases or biohazards.
Cost of practical application: The potentially high cost of applying synthetic biology systems to wastewater treatment, including designing, constructing, and maintaining the system, maybe a limiting factor.
The advantages of synthetic biology are very suitable for the treatment of wastewater, but its disadvantages also lead to problems that need to be solved to apply synthetic biology to wastewater treatment. XJTLU-CHINA wanted to explore this potential and chose to use synthetic biology to address the metals in industrial wastewater and to overcome these disadvantages with the solutions we created.
Our project has prepared specific solutions for the shortcomings. To address the stability and complexity of the water flow, we created hardware to simulate the real state of the water flow and used a model to describe the process of absorption in hardware. To prevent the uncontrolled release of organisms that could contaminate the external environment, we used a cell-free system using only proteins as membranes, which not only solved the safety issues that could be created by cells but also worked better with the hardware. To solve the cost problem of the initial implementation, we solve the problem with another way of thinking, our project can absorb not only heavy metals, but also precious metals, and conventional methods. For example, chemical method, even if the metal is removed, it is difficult to recycle. Our method can adsorb and recycle these metals to produce a higher economic effect, thus overcoming the initial problem of higher cost.
To compensate for the drawbacks of the current methodologies of purifying the electroplating wastewater, we designed the Claritein applied in the adsorption of heavy and noble metal ions. These biosorbents are placed in the hardware to absorb heavy and noble metal ions simultaneously.
According to Lee et al. (2019), we found AG4, a silver-binding dodecapeptide in which the tyrosine residue can reduce silver ions to nano-silver. In addition, AG4 can interact with nano-silver and adsorb nano-silver to a certain extent. Inspired by this literature, we chose to use AG4 to adsorb silver ions in industrial wastewater.
CsgA is an amyloid coiled fibrin which is the major structure of the curli fiber on the outer membrane of E. coli. Inspired by Courchesne (2017), CsgA protein could filtered by a vacuum filter and form free-standing curli films. Filtered CsgA protein on the polycarbonate filter films could form membranes and be insoluble in water. The curli fiber films provide the platform to link the AG4 short peptide. Depending on the literature from Olmez et al. (2019), we combined CsgA and AG4 into a fusion protein. We used three strains which were JF1, BL21(DE3), and MC4100 to assess the capacity of the protein expression. MC4100 had the powerful ability of protein expression. Based on the different strains and the modeling results, different promoters were chosen. The details of different strains' expressions are exhibited in the Engineering. Figure 1 to 6 depict the biological pathways and plasmid maps of three strains.
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 and AG4 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. Tac promoter is chosen for the MC4100 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. T7 promoter is chosen for the BL21(DE3) strain. CsgA and AG4 link with GSGGSG linker.
Figure 19: 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.
In order to purify the fusion protein better and more efficiently, CsgA and B should be knocked out of the genomes of Chassis bacteria through the method of lambda red (Sawitzke et al., 2011). As shown in the figure, the protein encoded by the CsgB has the function of anchoring the curli fiber encoded by the CsgA on the bacterial cell membrane. Figure 6 exhibits the mechanism of the CsgA protein exit channel. Figure 7 shows the results of the deletion of genomic genes of CsgA and CsgB when the palsmid inserted. According to Courchesne et al. (2017), In the absence of CsgB, the curli fiber encoded by CsgA is detached from the bacterial body. This simplifies the purification steps. Also, we built a model to describe the production of the fusion protein.
Figure 6: The mechanism of the curli biogenesis system. A, B, C, E, F, and G represent CsgA, CsgB, CsgC, CsgE, and CsgF proteins respectively. Nter and Cter represent the N-terminal and C-terminal of the protein. CsgA: major curli, the structure constituting the amyloid fibers. CsgB: binding the CsgA on the cell surface. CsgC: prevent the formation of the pre-amyloid fibers. CsgE, CsgG, CsgF: the polymerase of the secreted protein (Yan et al., 2020).
Figure 7: The description for deleting the genes of CsgA and CsgB from the genome with plasmid transformed. (CsgA, CsgB): The first group indicates that when retaining the genes of CsgA and CsgB, some amyloid protein will be attached to AG4 dodecapeptide, and some will not, all the curli fiber will be anchored on the outer membrane. (ΔCsgA, CsgB): The second group indicates that when knocking out the gene of CsgA and retaining CsgB, all of the amyloid protein will be attached to AG4 dodecapeptide and anchored on the outer membrane. (CsgA, ΔCsgB): The third group indicates that when retaining the gene of CsgA and knocking out CsgB, all of the curli fiber will detach from the outer membrane. However, some amyloid protein will be attached to AG4 dodecapeptide, the others not. (ΔCsgA, ΔCsgB): The final group indicates that when deleting both the genes of CsgA and CsgB, all of the amyloid protein will link with AG4 dodecapeptide and detach from the outer membrane.
After protein expression, it is filtered with a vacuum filter, and a 10μm pore size polycarbonate membrane is used to host the fusion protein. The polycarbonate membrane then should be stained by Congo red dye as below.
When this membrane was incubated in sewage containing silver ions, the silver ions were reduced to AgNPs by the residues of tyrosine in AG4 (Si et al., 2007). Subsequently, the reduced silver nanoparticles will interact with AG4 and be adsorbed (Zhang et al., 2006). According to this principle, we obtained the Claritein with silver affinity. The result of the adsorption of the silver ions by the curli fiber is presented in the wiki of Result.
To compensate for the drawbacks of the current methodologies of purifying the electroplating wastewater, we designed two types of biosorbents applied in the adsorption of heavy and noble metal ions. These biosorbents are placed in the hardware to absorb heavy and noble metal ions simultaneously.
Metallothionein (MT), is a class of metal-binding proteins widely found in living organisms. It is a low molecular weight protein (2-7 kD) rich in cysteine (20%-30%) and devoid of histidine and aromatic amino acids (Feng et al., 2017). MT can be induced by metals, cytokines, hormones, cytotoxic drugs, organic chemicals, and stress. MT plays a crucial role in regulating the concentration of trace elements within the organism and detoxifying heavy metals. Additionally, it is involved in the regulation of hormones, cellular metabolism, control of cell differentiation and proliferation, and participates in UV-induced reactions and free radical scavenging (Haq, 2003).
The cyanobacterial metallothionein SmtA, originating from Synechococcus PCC 7942, is a protein belonging to the metallothionein family. It can sense and bind specific heavy metal ions such as cadmium, lead, zinc, and others, thereby assisting in regulating the cell's tolerance and metabolism of these toxic metals. It prevents these metal ions from entering the cell or transferring them to cellular compartments by forming complexes with them, thus protecting the cell from the toxicity of heavy metals. This is achieved through the thiol groups (SH) found in the cysteine residues within the protein structure (Mwandira, 2020).
To enhance the surface affinity for heavy metal ions and increase the chelation capacity of heavy metal ions near the protein surface, we designed a variant called MSmtA4 based on SmtA. Saffar et al. found that mutating Lys45 in SmtA to Cysteine resulted in a larger surface accessibility area (ASA) and smaller root mean square fluctuation (RMSF) values. Additionally, residues such as Arg 26, Lys 8, and Lys 22 share similar structures with Lys45. Therefore, we chose to mutate positively charged residues such as Arg 26, Lys 8, Lys 22, and Lys 45 to cysteine, thereby increasing the protein's relative electronegativity to enhance electrostatic interactions and increase the accessible surface area.
We will provide evidence for this by predicting the structure of fusion protein with AlphaFold2 and simulating the docking environment with molecular dynamics simulations.
Figure 8: GIF of MSmtA4 mutated protein. The structure of MSmtA4 protein created by AlphaFold2 and PyMol.The pink parts are the mutated sites (the 8th,22nd,26th, and 45th amino acids).
We design a biological pathway that is composed of the small ubiquitin-like modifier (SUMO), MSmtA4, carbohydrate-binding module (CBM), and superfolder GFP (sfGFP) to produce the biosorbent absorbing heavy metal ions. To prevent instability and degradation due to the intracellular overexpressed of MSmtA4 in Escherichia coli (E. coli) BL21 (DE3), SUMO is chosen as a fusion tag and molecular chaperone for recombinant protein expression. Moreover, SUMO can facilitate proper protein folding and contribute to maintaining the stability of the target protein. Fusion expression of SUMO at the N-terminus of the target protein can improve protein folding, enhance solubility, and increase protein yield. (Marblestone et al., 2006).
Carbohydrate Binding Modules (CBMs) are components of several enzymes, and their primary function is to bind to specific carbohydrates. Research by Kevin Aïssa and other experts has provided compelling evidence of the robust affinity that CBMs have for crystalline cellulose.
The sfGFP is a fluorescent labeling tool that, when fused with other proteins, does not induce misfolding, thereby enhancing the stability of the fusion protein. The addition of sfGFP enables the protein to exhibit visible green fluorescence under normal daylight conditions, which facilitates the detection of the connection between CBM and cellulose.
The fusion protein carries sfGFP, allowing clear observation of green fluorescence in natural light, which aids in evaluating the connection between CBM and microcrystalline cellulose. This innovative design paves the way for the development of a novel bioremediation agent, one uniquely equipped to independently adsorb heavy metal ions, thereby holding the promise of significantly enhancing our capacity to address environmental challenges.
Figure 9: The genetic circuit of the biosorbent synthesis for heavy metal ions adsorption. IPTG induces this biological pathway. SUMO, SmtA, CBM, and sfGFP form the fusion protein.
Figure 10: The producer of dealing with the heavy metal ions in the lab. The fusion protein of SUMO-MSmtA4-CBM-sfGFP binds on the microcrystalline cellulose and absorbs heavy metal ions.
The speed of polluted water should be clarified. Whether an over-fast or an over-slow flow may cause incomplete absorption. The former may decrease the efficient contact time between fusion proteins and metal ions, and the latter may shrink the purifying capacity. The simulation of the polluted water flow will be finished on software to keep the whole hardware valid.
Reference:
Aïssa, K. et al. (2019) ‘Functionalizing cellulose nanocrystals with click modifiable carbohydrate-binding modules’, Biomacromolecules, 20(8), pp. 3087–3093. doi:10.1021/acs.biomac.9b00646.
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
Feng, N., Jiang, F. J., and Chun, C. H. (2017) ‘Metallothionein and Their Biological Functions’, Chinese Journal of Biochemistry and Molecular Biology, 33(9), pp. 893–899. doi:10.13865/j.cnki.cjbmb.2017.09.07.
Marblestone, J.G. et al. (2006), ‘Comparison of SUMO fusion technology with traditional gene fusion systems: Enhanced expression and solubility with SUMO’, Protein Science., 15(1), pp. 182-189. Available at: https://doi.org/10.1110/ps.051812706
Mwandira, W. et al. (2020) ‘Biosorption of Pb (II) and Zn (II) from aqueous solution by Oceanobacillus profundus isolated from an abandoned mine’ Scientific Reports, 21189. Available at: https://doi.org/10.1038/s41598-020-78187-4
Olmez, T. T. et al. (2019) ‘Synthetic Genetic Circuits for Self-Actuated Cellular Nanomaterial Fabrication Devices’ ACS Synthetic Biology, 8(9), pp. 2152-2162. Available at: https://doi.org/10.1021/acssynbio.9b00235
Saffar, B. et al. (2015) ‘Improvement of Cd(2+) uptake ability of SmtA protein by Lys/Cys mutation; experimental and theoretical studies.’ Journal of biomolecular structure and dynamics, 33(11), pp. 2347–2359. Available at: https://doi.org/10.1080/07391102.2015.1054431
Sawitzke, J.A. et al. (2013) ‘Recombineering: Using Drug Cassettes toKnock out Genes in vivo’ Methods in Enzymology, 533, pp. 79-102. Available at: https://doi.org/10.1016/B978-0-12-420067-8.00007-6
Si, S. et al. (2007) ‘Tryptophan-Based Peptides to Synthesize Gold and Silver Nanoparticles: A Mechanistic and Kinetic Study’ Chemistry Europe, 13(11), pp. 3160-3168. Available at: https://doi.org/10.1002/chem.200601492
Yan, Z. et al. (2020) ‘Assembly and substrate recognition of curli biogenesis system’ Nature Communications, 241. Available at: https://www.nature.com/articles/s41467-019-14145-7
Zhang, X. et al. (2008) ‘The bio-inspired approach to controllable biomimetic synthesis of silver nanoparticles in organic matrix of chitosan and silver-binding peptide (NPSSLFRYLPSD)’ Materials Science and Engineering: C, 28(2), pp. 237-242. Available at: https://doi.org/10.1016/j.msec.2006.12.007