The WHHS-Pro-China team is dedicated to developing an efficient and sustainable phosphate recovery and treatment system to address the dual challenges of water eutrophication and phosphate resource depletion. Through innovative genetic engineering and biotechnology, our project contributes to the iGEM community by offering advancements in environmental remediation and resource recovery. The PhosLock 1.0 and PhosRegen 2.0 systems demonstrate the potential of engineered bacteria in wastewater treatment, providing a technical foundation for future iGEM teams to build upon and improve.
Experimentally Validated Parts: The team successfully developed and validated two engineered bacterial strains, utilizing INP (Ice Nucleation Protein) and PelB (signal peptide) surface display systems. These key genetic parts were experimentally proven to function effectively, especially in anchoring phosphate-binding proteins (PBP) to the cell membrane, enhancing phosphate adsorption efficiency in wastewater treatment. This offers future teams stable modules, particularly useful in phosphate recovery applications.
Optimized Adsorption and Desorption Conditions: By adjusting environmental conditions such as pH and temperature, we optimized the phosphate adsorption and desorption process. INP-PBP engineered bacteria demonstrated optimal adsorption at pH 7 and 35°C, while strong acidic and alkaline conditions facilitated efficient phosphate desorption. These experimental parameters can be adapted by other teams.
Scalable System Design: The WHHS-Pro-China team developed the PhosRegen 2.0 system, which utilizes bead adsorption and protein secretion, addressing challenges such as bacterial mortality and operational complexity in traditional systems. The reuse of beads significantly reduces operational costs, making this design feasible for industrial-scale applications.
BBa_K3986004: This part encodes Ice Nucleation Protein (INP) and contributes in the following ways:
INP's Anchoring Function and Surface Display: BBa_K3986004 encodes INP, which has a powerful cell surface anchoring ability. In the PhosLock 1.0 system, INP was fused with the phosphate-binding protein (PBP), successfully displayed on the surface of E. coli. INP’s anchoring function allows PBP to be stably positioned on the outer membrane, facilitating direct interaction with phosphate molecules in wastewater. This significantly enhances adsorption efficiency by increasing the surface area for protein-phosphate interactions compared to intracellular expression systems.
Experimental Validation of INP Anchoring:
Membrane and Cytoplasmic Component Separation: During the experiment, we lysed the cells by sonication, followed by low- and high-speed centrifugation to separate membrane and cytoplasmic components. Phosphate adsorption tests revealed that the membrane fraction had significantly higher adsorption capacity than the cytoplasmic fraction, further verifying that INP successfully anchored PBP to the cell surface.
Methodology: Using ultracentrifugation and differential separation, we obtained two fractions—cell membrane and cytoplasm. Phosphate adsorption tests on these fractions demonstrated that INP-anchored proteins were primarily located on the cell membrane, showing strong adsorption.
Expanding INP Applications:
Environmental Remediation: INP can be used to anchor other functional proteins, expanding its potential for heavy metal removal and dye degradation in wastewater. For example, proteins that bind mercury or lead could be anchored via INP on bacterial surfaces, developing highly effective biotools for metal-contaminated water treatment.
Biosensor Development: INP can also be applied to biosensor development by anchoring sensor proteins on the cell surface. This could enable the real-time detection of specific environmental pollutants, turning engineered bacteria into efficient biosensors for hazardous substances in water.
Drug Delivery and Targeted Therapy: Beyond environmental applications, INP’s anchoring ability can be used in the biomedical field. By fusing INP with drug delivery proteins, targeted drug delivery systems based on engineered cells could be developed, allowing for more precise and localized treatment in medicine.
Experimental Design and Process: The team performed detailed experimental validation, including bacterial growth conditions and phosphate adsorption efficiency tests, providing comprehensive data to support our findings.
Contributed Biological Parts: BBa_K3986004, encoding INP, provides future iGEM teams with a versatile anchoring module that can be fused with other target proteins and displayed on the cell surface. This system is applicable not only in wastewater treatment but also in biosensor development and environmental remediation.
Reproducible Experimental Protocols: The team’s detailed experimental protocols, including membrane separation, anchoring validation, and adsorption tests, provide valuable references for future teams in experimental design and technology validation.
Expanding INP’s Application Scope: Future teams can further develop INP by fusing it with more functional proteins, broadening its use in environmental remediation, biosensors, and biomedicine. Teams could also explore using other bacteria or microorganisms as chassis to diversify the system’s applications.
Optimizing Anchoring Efficiency: Future research could focus on optimizing the efficiency of INP anchoring through mutations or gene editing, improving its stability and adsorption capacity under different environmental conditions.
Through the development and validation of BBa_K3986004, the WHHS-Pro-China team demonstrated the wide-ranging potential of INP as a cell surface anchoring tool. This work provides powerful tools and experimental frameworks for future iGEM teams working on wastewater treatment, environmental remediation, and biosensor development.
