Proof of Concept

Overview

Our project aims to engineer a biodegradable biofilter system that captures and removes toxic metals from water using metal-binding proteins expressed in E. coli. The system integrates three core elements: engineered peptides (Phytochelatin and Metallothioneins), protein immobilization on biopolymer beads, and a modular filtration prototype.
With successful expression and purification of both peptides, immobilization on an alginate bead matrix, and demonstration of significant metal uptake through controlled filtration tests, we aim to establish a functional proof of concept showing that our engineered biohybrid system can efficiently and selectively remove toxic metals from water in a biodegradable, modular format.
Each subsystem — biological, material, and mechanical — would be validated individually and in combination, confirming that our modular design functions as intended. This would establish a clear proof of concept for a protein–polymer hybrid capable of selective, efficient heavy-metal sequestration.

Protein Engineering: Metal-Binding Peptides

By expressing Phytochelatin Synthase (PCS) and Metallothionein (MT) in E. coli BL21(DE3) using the pET28(b+) expression vector and IPTG induction, and verifying purified proteins through SDS-PAGE and Ni–NTA affinity chromatography, we would establish the foundation for demonstrating successful recombinant protein production.
When subsequently exposed to metal ions like iron, aluminium, mercury, chromium, the recombinant proteins are expected to show high binding efficiency as quantified by ICP-MS, thereby establishing their functional affinity toward toxic metals.
By doing this, we would validate that our engineered biomolecules can act as effective biological metal adsorbents, forming the basis for downstream material integration.

Material Integration: Immobilization on Biopolymer Beads

To translate the protein-level binding into a usable material, we would immobilize PCS and MT on sodium alginate beads through EDC–NHS coupling, forming a covalent yet biocompatible linkage. The biohybrid beads would be expected to retain >90% of metal-binding activity compared to the free proteins. By doing this, we would demonstrate that protein functionality can be preserved post-immobilization, confirming the feasibility of embedding engineered peptides into a biodegradable support suitable for real-world applications.

System Demonstration: Functional Filtration Prototype

By packing the biohybrid beads into a dual-layer concave–convex filtration prototype designed to optimize fluid distribution and surface contact, we would be able to test the system’s performance with simulated industrial effluent under gravity flow. We would expect the prototype to achieve:

1. Quick and effective capture of toxic metals, highlighting the prototype’s practical potential
2. Rapid adsorption kinetics consistent with Langmuir isotherm behaviour
3. Regeneration efficiency >90% after three EDTA wash cycles

Our Accomplishment This Year

Our project establishes a modular, multi-layered approach to biological toxic-metal sequestration, combining rational gene design, material integration, and prototype validation. Through this work, we laid the foundation for future iGEM teams and environmental biotechnology applications.

1. Genetic and Molecular Contributions
   1. Designed and cloned Phytochelatin Synthase (PCS) and Metallothionein (MT) constructs in E. coli BL21(DE3).
   2. Verified gene constructs through restriction digestion and PCR, confirming readiness for downstream expression.
   3. Conducted in silico analyses and molecular docking to evaluate metal-binding potential, providing a computational foundation for selecting and optimizing peptide sequences.
2. Material and Interface Design
   1. Developed a strategy for immobilizing metal-binding peptides on sodium alginate beads via EDC–NHS coupling.
   2. Characterization of bead stability, porosity, and theoretical retention of protein activity, establishing a biohybrid material platform for filtration.
   3. This approach bridges synthetic biology and materials engineering, creating a foundation for reusable, modular biofilters.
3. Prototype Development and Functional Modeling
   1. Construction of a dual-layer concave–convex filtration prototype to optimize flow and surface contact.
   2. Performance of model-based evaluation of adsorption efficiency and regeneration potential, validating the feasibility of the integrated system for selective metal removal.
   3. Integrated computational adsorption modeling (Competitive Langmuir Isotherm) to predict multi-metal uptake, aligning theoretical performance with experimental design.
4. Broader Contributions to Synthetic Biology and iGEM
   1. Establishment of reproducible, scalable methodologies for gene design, material embedding, and prototype testing.
   2. Provided a modular framework that future teams can expand, combining gene selection, peptide–polymer immobilization, and filtration system design.
   3. Demonstrated cross-disciplinary integration, linking computational predictions, molecular engineering, and materials science into a cohesive proof-of-concept system.

Future Prospect

• Enhance the binding efficiency through peptide sequence optimization and linker tuning.
• Expand to multi-protein immobilization for broad-spectrum metal capture.
• Integrate an IoT-based monitoring module to track filtration efficiency in real time.

Scale up to field-deployable prototypes and test with real contaminated water samples from industrial regions of Odisha.