DBTL Cycle Documentation: Bioengineered Metal-Sequestration Filter

Design Phase (Dry Lab)

Our project began with computational design and in silico screening of metal-binding components:

Phytochelatin Synthase (PCS):

• Collected 6,585 known PCS sequences from NCBI.
• Filtered sequences based on literature-reported functional and structural relevance.
• Grouped sequences by organism; performed multiple sequence alignment to select representative isoforms.
• Refined library reduced to 389 candidates.
• Conducted two rounds of molecular docking simulations with glutathione (GSH):
• Round 1: 10 GA runs per sequence; shortlisted sequences with predicted binding affinity \< –7.5 kcal/mol.
• Round 2: 50 GA runs focusing on the N-terminal catalytic domain.
• Selected top 5 PCS candidates with highest predicted GSH-binding efficiency.

Metallothionein (MT):

• Selected native wheat (Triticum aestivum) MT known to bind mercury and cadmium.
• Engineered its binding pocket to shift specificity toward iron, chromium, and aluminium, creating a new composite part for the iGEM Registry.

Build Phase (Wet Lab)

The Build phase involved protein expression and in vitro/in vivo synthesis of metal-sequestering molecules:

• PCS Production:
  • Selected PCS from Polyangium sorediatum, known for high catalytic robustness.
  • Expressed in E. coli; purified enzyme added to GSH and trace metals to produce phytochelatins in vitro.
• In vivo Systems:
  • Two engineered setups in E. coli:
    1. Engineered Metallothionein (MT) optimized for Al³⁺ and Fe³⁺.
    2. PCS + GSH-synthesizing gene from Streptococcus thermophilus for endogenous phytochelatin production.
  • Added merP and merT transporters to enhance mercury uptake.
  • Purification employed acid-based centrifugation, suitable for small thiol-rich peptides.

System Integration & Test Phase

Alginate Bead Formation

Sodium alginate, a biodegradable polysaccharide, was chosen for its ability to form porous, water-permeable gels in the presence of Ca²⁺.

Bead fabrication:
• Alginate solution dropped into a CaCl₂ bath, forming beads 1–2 mm in diameter.
• Beads were soft, porous, and structurally suitable for protein attachment.

Peptide Immobilization via EDC/NHS Coupling

Mechanism:

• EDC activates alginate’s carboxyl groups to form unstable O-acylisourea intermediates.
• NHS stabilizes this intermediate as an NHS ester.
• Free amines on PCS or MT react with the ester to form stable covalent amide bonds, anchoring peptides on the bead surface.

• Outcome: Beads function as metal-binding micro-reactors, with peptides exposed for efficient ion capture.

• Metal adsorption modeling:
  • Used competitive Langmuir isotherms to predict adsorption of multiple metals and account for competition between ions.
  • Measured pre- and post-filtration concentrations to determine metal uptake (Mᵢ), filter capacity, and efficiency.
• Mass transfer considerations:
  • Key resistances include:
    1. Bulk liquid to particle boundary layer
    2. Diffusion through boundary layer
    3. Internal pore diffusion
    4. Adsorption at internal sites
    5. Surface diffusion along pores
  • External and internal resistances influence breakthrough curves in fixed-bed columns.
  • System optimization relies on enhancing diffusion rates and minimizing mass-transfer barriers.

Prototype Development

• Dual-layer concave–convex module for even water flow distribution.
• Alginate beads coated with PCS and MT packed into nylon/polypropylene mesh sheets.
• Modular, reusable, and scalable structure ensures high surface area and durability.

Advantages

• Bead-based format prevents water accumulation and swelling seen in bulk hydrogels.
• High porosity and surface exposure enable efficient metal capture.
• Filter design balances mechanical strength, flow dynamics, and ease of deployment.

Learn Phase

Iterative data-driven optimization informs:

• Bead size, packing density, and flow rate adjustments.
• Peptide density and immobilization efficiency.
• Multi-metal adsorption efficiency and filter longevity.

Interdisciplinary Integration

1. Synthetic biology: PCS and MT design, protein engineering.
2. Chemical engineering: Mass transfer, Langmuir isotherms, fixed-bed adsorption modeling.
3. Material science: Alginate bead formation, surface functionalization, prototype design.
4. Environmental engineering: Practical deployment and water quality assessment.

DBTL Cycle Flow:

Design → Build → Test → Learn

1. Design: PCS docking, MT engineering
2. Build: Protein expression, purification, in vivo and in vitro setups
3. Test: Alginate bead immobilization, Langmuir adsorption, breakthrough analysis
4. Learn: System optimization for capacity, efficiency, and durability