Project Design Stage

The Design stage marks the start of our engineering journey. Here, our goal was to rationally design and model biological components that align with our project’s vision before stepping into the Wet Lab. This stage combined two core aspects of synthetic biology design:

• Computational Screening & Modeling: Identifying and optimizing enzyme candidates in silico.
• In Silico Cloning: Constructing plasmid designs virtually to predict feasibility and functionality.

Defining the Design Objective

Our project aimed to engineer a synthetic circuit capable of efficient heavy metal chelation and tolerance. To achieve this, we needed two major functional components:

• A Phytochelatin Synthase (PCS) enzyme optimized for catalytic conversion of glutathione (GSH) into phytochelatins.
• A Metallothionein (MT) protein engineered to bind selectively with target metals (Fe³⁺, Cr³⁺, Al³⁺).

Phytochelatin Synthase (PCS) Screening and Design Pipeline

• Step 1: Database Mining
  1. We began with a database-wide search across NCBI, collecting 6,585 known PCS peptide sequences from diverse organisms.
• Step 2: Functional Filtration
  1. Using literature-backed evidence, we filtered sequences based on experimentally verified activity or structural data, resulting in 389 candidates of potential interest.
• Step 3: Multiple Sequence Alignment
  1. For organisms containing multiple PCS isoforms, we performed multiple sequence alignment (MSA) using Clustal Omega, selecting one representative isoform per organism with the highest conservation score.
• Step 4: Molecular Docking Simulations
  1. We ran two rounds of molecular docking to evaluate substrate-binding affinity with glutathione (GSH):
  2. Round 1: 10 Genetic Algorithm (GA) runs per sequence to explore broad binding conformations.
  3. Round 2: Shortlisted sequences (binding affinity ≤ –7.5 kcal/mol) were re-docked using 50 GA runs for refined accuracy, focusing on the N-terminal catalytic domain.
  4. This screening led to our top 5 PCS candidates, each exhibiting strong predicted GSH-binding and catalytic potential.

Engineering Metallothionein (MT)

For the metal-binding component, we started with a native Metallothionein (MT) sequence from Triticum aestivum (wheat), known for mercury and cadmium binding. To tailor it toward our project’s targets—iron, chromium, and aluminium—we used in silico mutagenesis and binding pocket re-engineering. This optimized MT variant displayed enhanced binding site complementarity toward the new metals and was finalized as our Engineered MT construct (BBa_25YMLSNG). This part was later integrated into our composite circuit and prepared for submission to the iGEM Registry as a new engineered part.

In Silico Cloning: Construct Assembly in SnapGene

Once our protein candidates were finalized, we proceeded to assemble the genetic constructs virtually using SnapGene.
This step allowed us to simulate the cloning strategy, check for restriction sites, and verify construct integrity before lab work. Our in silico cloning pipeline included:

• Designing constructs for both PCS and MT using promoters, RBS, and terminators compatible with E. coli expression systems.
• Verifying open reading frames (ORFs), codon optimization, and restriction enzyme compatibility.
• Assembling composite parts (e.g., Engineered MT + T7 Terminator) and visualizing their map structure.

Integrating Computational & Experimental Design

The insights from docking and structural modeling directly informed our wet lab construct selection. By integrating in silico design with experimental feasibility, we minimized redundant trials and ensured:

• Optimal enzyme-substrate affinity
• Enhanced metal-binding efficiency
• Chassis compatibility
• Ease of modular assembly for DBTL iteration

Our Design stage unified bioinformatics, structural biology, and molecular cloning into a cohesive workflow:

• Screen → Identify potential enzyme candidates.
• Model → Predict structure–function relationships.
• Engineer → Modify and optimize for target metals.
• Assemble → Simulate cloning and composite construction.