Engineering

Our project integrates molecular, genetic, and material-level engineering to build a complete, functional system for heavy metal sequestration. Each design layer — from engineered peptides to bioreactor assembly — was developed using standardized synthetic biology principles and guided by modeling and literature-supported frameworks.
Our engineering strategy for metallothionein (MT) focused on enhancing its ability to selectively bind metal ions such as Al³⁺, Fe²⁺/Fe³⁺, and Cr⁶⁺. We applied a Design–Build–Test–Learn (DBTL) cycle combining protein engineering principles with molecular docking and molecular dynamics (MD) simulations to rationally guide wet lab construct selection.

Peptide Engineering

At the foundation of our system lies the design of thiol-rich peptides and proteins capable of binding metal ions. We began by modifying metallothionein (MT) sequences and doing amino acid substitution to adjust coordination preference toward hard metals. Complementary phytochelatin synthesis pathways were included to generate shorter thiol polymers in vivo and in vivo. Sequence edits focused on optimizing maintaining protein solubility, and preserving proper folding in E. coli. Together, these engineered peptides form the active biological component of our sequestration system.

Synthetic Circuit Design

To express these components efficiently, we followed a modular synthetic biology workflow. Genes encoding PCS (Polyangium sorediatum), bifunctional GSHF (Streptococcus thermophilus), native and engineered MTs, and MerP/T transporters were assembled into standardized IPTG-inducible circuits using defined promoters, RBSs, spacers, and terminators. Each construct was verified in silico for frame integrity and codon compatibility. The modular design allows flexible substitution of parts and facilitates future optimization by other teams. Docking-based bioinformatic screening helped identify high-affinity PCS variants, ensuring rational enzyme selection before wet-lab cloning.

Expression & Purification

All constructs were cloned into the pET-28b(+) vector and expressed in E. coli BL21(DE3), chosen for its robust T7 system and reduced proteolytic activity. Large proteins such as PCS and MTs were purified using Ni–NTA affinity chromatography, while small thiol-rich peptides (phytochelatins) were extracted via acid precipitation and quantified through monobromobimane derivatization followed by fluorescence HPLC. This two-track purification workflow ensured recovery of both enzymatic components for in-vitro catalysis of our peptide synthesis and the extraction of small thiol peptides that are produced inside the chassis in the in-vivo approach, essential to our system’s functionality.

Material & Bead Engineering

The purified peptides were incorporated into sodium alginate beads designed for mechanical strength and controlled porosity. Using a literature-supported GDL internal-gelation approach, we created beads with uniform crosslinking and internal microchannels that facilitate water flow and reduce diffusion resistance. Rheology parameters guided the optimization of alginate concentration and crosslink density to prevent swelling and withstand shear stress under flow.
Peptides were covalently immobilized onto the bead surface using EDC–NHS coupling chemistry, which activates the carboxyl groups of alginate to form stable amide bonds with primary amines on the peptides. This created a durable bio-hybrid matrix with strong peptide retention and accessible binding sites for metal capture, ensuring consistent performance under continuous flow conditions.

System Integration & Performance Modeling

These peptide-loaded beads were packed into modular mesh cartridges, which were then arranged in alternating concave and convex layers to enhance flow distribution within a fixed-bed adsorber. The reactor design was modeled using classical mass-transfer and Langmuir isotherm equations to predict adsorption performance and breakthrough behavior. Flow rate, bed height, and bead diameter can be tuned to achieve effective contact time without excessive pressure drop. A colorimetric or ion-selective sensor placed at the outlet provides real-time monitoring of effluent metal concentrations, enabling feedback-based control and performance tracking.
Through this multi-layered engineering process — from molecular design and modular circuit construction to material optimization and reactor modeling — we developed a reproducible, biodegradable system for selective heavy metal sequestration. Each component was designed to be modular, measurable, and reusable, ensuring that future iGEM teams can build upon our platform to engineer new biosorptive and remediation systems.

Demonstrating Engineering Success: Engineering Metallothionein for Hard Metal Sequestration

To demonstrate engineering success in our project, we performed a complete Design–Build–Test–Learn (DBTL) iteration on metallothionein (MT), repurposing it from a soft-metal binding protein into a flexible scaffold capable of sequestering hard metal ions such as Al³⁺, Fe²⁺/Fe³⁺, and Cr⁶⁺.

Design

Native Triticum aestivum metallothionein is highly cysteine-rich and evolved to bind soft metals (e.g., Zn²⁺, Cu²⁺) through thiol coordination. Guided by Pearson’s Hard–Soft Acid–Base (HSAB) theory, we redesigned the MT to enhance binding to hard acids, which prefer oxygen donors rather than sulfur. Cysteine residues were substituted with alanine to eliminate thiol coordination, and a custom carboxylate-rich metal-binding peptide (EDEGEDEGEDDG) was fused at the C-terminus through a flexible (GGGGS)₂ linker. The resulting engineered sequence (below given) was designed to present multiple glutamate/aspartate residues as hard-base ligands.

Build

The modified MT gene was codon-optimized for E. coli and modeled using AlphaFold 3 for tertiary structure prediction. Structural quality was assessed through Ramachandran plot analysis, which showed a reduction in outliers compared to the wild-type model, confirming improved stereochemical quality. The engineered construct was assembled in pET-28b(+) for T7-driven expression, providing the foundation for downstream in-vitro validation.

Test

Binding affinities were first estimated by molecular docking (AutoDock4) against Al³⁺, Fe²⁺/Fe³⁺, and Cr⁶⁺ ions. Compared to wild-type MT, the engineered variant displayed enhanced affinities:

• Cr⁶⁺: –20.64 kcal/mol (vs –16.8 kcal/mol)
• Fe³⁺: –19.52 kcal/mol (vs –16.4 kcal/mol)
• Fe²⁺: –11.90 kcal/mol (vs –10.2 kcal/mol)
• Al³⁺: –6.00 kcal/mol (vs –4.0 kcal/mol)

To validate the docking predictions, molecular dynamics (MD) simulations were performed using YASARA Structure (AMBER14 force field) over 10 ns in a solvated environment. Key observations included:

• Stable RMSD trajectories with no significant unfolding, confirming conformational stability.
• Persistent coordination of Al³⁺, Fe³⁺, and Cr⁶⁺ ions throughout the trajectory, with no metal dissociation.
• Increased intramolecular hydrogen bonding in the engineered MT, enhancing complex rigidity and retention.
• ΔEₚₒₜ (binding energy) decreased drastically from +260,586 kJ/mol in the wild type to –2,087 to –3,178 kJ/mol in the engineered variant, signifying a thermodynamically favorable complex formation.
• Radius of Gyration (Rg) values remained consistent post-binding, suggesting compact and stable conformations.

Together, these analyses confirmed a successful shift in metal-binding preference and improved structural robustness.

Learn

Through this engineering cycle, we demonstrated that rational residue substitution and carboxylate-rich peptide fusion can effectively reprogram metallothionein’s selectivity from soft to hard metal ions. The engineered variant maintained structural integrity while achieving higher binding affinity and dynamic stability, validating the HSAB-based redesign strategy. Insights from RMSD, Rg, and ΔEₚₒₜ analyses guided subsequent immobilization and bead optimization experiments, where this engineered MT was used as the core bioactive component in our sequestration system.

Conclusion

This iterative engineering of metallothionein demonstrates clear engineering success—a measurable improvement in structure, function, and metal-binding capacity through rational design, computational modeling, and feedback-driven optimization. The insights gained from this cycle directly informed downstream wet-lab design and material integration in the POSEIDON system.

Basic Parts

During the course of the project, and throughout the iterative process of engineering and the DBTL (Design-Build-Test-Learn) cycle, we utilized the parts listed below and formulated composite parts by combining multiple functional modules. This approach allowed us to optimize performance, integrate complementary functionalities, and tailor constructs for our specific application context.

Composite Parts

PCS + GSHF Composite Part

Mer Operon Composite Part

MT Gene Circuit Composite Part

PCS Gene Circuit Composite Part