Abstract

Toxic metal ion contamination in water poses a significant environmental and public health challenge. Conventional remediation techniques are often costly, inefficient, or environmentally unsustainable. Engineered microorganisms provide a promising alternative for metal ion sequestration, but efficient biofilter design requires precise and robust expression of detoxification genes. In this project, we aim to enhance the capacity of Escherichia coli to sequester metals through co-expression of phytochelatin synthase (PCS), bifunctional glutathione synthetase-fused enzyme (GSHF), and metallothioneins (MTs).

Computational Design of Regulatory Elements

To optimize gene expression, we developed a computational pipeline that integrates transcriptomic data and literature on metal stress response genes in E. coli. Genes were computationally analyzed and regulatory elements—promoters and ribosome-binding sites—were selected based on high mean expression and low variance. This approach ensures predictable and efficient production of metal-binding proteins, minimizing variability in biological filter performance.

Construct Assembly and Modular Design

Selected genes and regulatory elements were assembled into modular constructs using iGEM-compatible cloning strategies. PCS+GSHF and MT modules were designed with optimized spacing and regulatory sequences to enable coordinated co-expression. The modular framework allows iterative addition or modification of parts, supporting future refinement and expansion of the biofilter system.

Significance and Applications

These constructs establish a toolkit for enhanced metal ion sequestration in E. coli, enabling the development of sustainable, efficient biofilters. The workflow demonstrates the integration of computational omics analyses with synthetic biology, providing a scalable framework for rational construct design. Beyond immediate environmental applications, the toolkit can serve as a resource for future iGEM teams and other microbial biotechnology projects targeting metal remediation.

Future Directions

The methodology can be extended to include inducible or environment-responsive regulatory elements, enabling adaptive expression under fluctuating metal concentrations. Field-scale deployment and performance testing will further validate the efficacy of these engineered systems. Overall, this work contributes to sustainable bioremediation strategies while offering standardized tools for synthetic biology applications in environmental engineering.

Motivation

Metal ion contamination in aquatic environments poses a critical ecological, environmental, social and human health risk, particularly in industrial and mining zones such as Sukinda, Odisha. Continuous discharge of effluents rich in cadmium, lead, and mercury has led to persistent contamination of local water sources. Existing remediation strategies, including chemical precipitation, activated carbon, and ion-exchange filters, are often costly, energy-intensive, and generate secondary waste, making them unsuitable for long-term use in resource-limited settings.
Through field engagement and discussions with local stakeholders, environmental scientists, and community members, it became clear that an effective solution must be both biodegradable and affordable, while capable of operating under varying contamination levels. These interactions motivated us to explore a synthetic biology-based filtration system that leverages the natural metal-binding potential of biological molecules.
Our approach builds on the ability of Escherichia coli to produce metal-chelating peptides through engineered biosynthetic pathways. We designed constructs expressing phytochelatin synthase (PCS), a bifunctional glutathione synthetase–fused enzyme (GSHF), and metallothioneins (MTs)—proteins known for their strong affinity to toxic metal ions. These modules were optimized for balanced co-expression to maximize intracellular peptide yield. However, we aimed to move beyond peptide synthesis and purification alone.
Recognizing the need for a deployable, scalable format, we integrated the purified metal-binding peptides into a biopolymer matrix composed of alginate, forming biodegradable hydrogel beads. These beads serve as the active core of our filter prototype, designed to capture and immobilize metal ions from contaminated water. By embedding engineered biological components into a reusable, natural matrix, the system combines the efficiency of biological sequestration with the practicality of physical filtration.
Our literature review revealed that most previous biological remediation studies focused either on in vivo sequestration or on purified biomolecule binding assays, rarely extending to real-world deployable devices. Addressing this gap, our project bridges molecular engineering and applied design by creating an end-to-end workflow—from gene circuit construction and protein expression to bead synthesis and filter modeling. This integrated strategy represents a step toward translating synthetic biology from the lab to the field, advancing sustainable and accessible solutions for heavy-metal remediation.

Design Strategy

To achieve our goal of developing a functional, biodegradable biofilter for metal ion remediation, we began by designing a robust molecular framework for efficient peptide biosynthesis and integration into a deployable matrix. Our first step was to identify suitable genetic constructs capable of high-yield expression of metal-binding proteins in Escherichia coli. For this, we focused on three core modules: phytochelatin synthase (PCS), a bifunctional glutathione synthetase–fused enzyme (GSHF), and metallothioneins (MTs). Together, these enzymes enable E. coli to biosynthesize thiol-rich peptides with high affinity toward toxic metal ions such as Cd²⁺, Pb²⁺, and Hg²⁺.
The design process required precise tuning of gene expression to ensure coordinated peptide production without imposing excessive metabolic burden. To guide this, we built a computational and literature-driven analysis pipeline focused on identifying optimal promoters, ribosome binding sites (RBSs), and spacer sequences compatible with the E. coli DH5α chassis. Drawing upon transcriptomic datasets and existing stress-response studies, we analyzed expression trends of metal-resistance and detoxification genes (such as zntA, copA, merT, and mntH) under heavy-metal exposure conditions. These profiles provided a comparative basis to select regulatory elements that remain stable during metal-induced stress, a critical requirement for sustained biosynthesis during filtration.
Our approach emphasized quantitative evaluation of promoter behavior—assessing mean expression strength and variance across conditions to predict reliability and expression stability. Promoters exhibiting high mean expression with low variance were considered ideal candidates, balancing strong transcriptional output with robustness under stress. This methodology, inspired by prior transcriptomic promoter mining studies, allowed us to systematically shortlist constitutive and inducible promoters suitable for our constructs.
Once the candidate regulatory sequences were identified, we assembled gene cassettes using a modular cloning (MoClo) approach compatible with iGEM standards. Each construct was designed with optimized ribosome binding sites and intergenic spacers to ensure efficient translation of multi-gene assemblies. The PCS+GSHF and MT modules were constructed as independent expression units, allowing flexibility in combinatorial design and downstream characterization.
Following in silico validation, we extended the workflow beyond molecular design to material integration and prototype modeling. Purified peptides produced from induced E. coli cultures were immobilized within a chitosan–alginate hydrogel matrix, forming biocompatible beads capable of capturing and sequestering dissolved metal ions. These beads were further incorporated into a filter column prototype, providing a scalable proof-of-concept for field deployment. This final stage marked a significant step from gene-level engineering to a functional, application-ready biosystem, bridging computational design, molecular biology, and materials engineering into one cohesive workflow.

Genomics, Proteomics, and Metabolomics in Our Metal-Sequestration Project

Our project integrates multiple layers of biological understanding—genomics, proteomics, and metabolomics—to design, express, and validate a complete biological system for heavy metal sequestration. Each omic dimension contributed uniquely to rational gene selection, expression design, and functional evaluation, linking molecular components to the system-level phenotype of metal removal.

Genomics

The genomic layer guided the foundational design and construction of our synthetic modules. We applied bioinformatic and in silico approaches to identify, evaluate, and optimize genes involved in phytochelatin and metallothionein-mediated metal binding.

• Gene Selection and Optimization:
  Using a large-scale genomic mining and molecular docking pipeline, we screened over 6,500 phytochelatin synthase (PCS) sequences to identify the variant with the strongest substrate-binding affinity. The Polyangium sorediatum PCS emerged as the top candidate based on docking scores and biological context.
• Design of Genetic Parts and Circuits:
  The selected genes—PCS, GSHF, MT (native and engineered), and MerP/T—were modularly assembled into expression units with standardized promoters, RBSs, spacers, and terminators. Each element was computationally validated (in silico cloning) for reading frame integrity and compatibility, following the principles of modularity and reusability central to synthetic genomics.
• Registry-Level Contribution:
  These parts, deposited as basic and composite entries in the iGEM Registry, provide a flexible genetic toolkit for future teams designing multi-gene systems or metabolic pathways focused on detoxification and biosorption.

Proteomics

The proteomic dimension of our project focuses on engineering, expression, and purification of thiol-rich peptides and proteins responsible for direct metal sequestration.

• Protein Expression and Engineering:
  • The project explores both natural and rationally engineered peptides, including phytochelatin synthases (AtPCS and PsPCS) and metallothioneins (MTs).
  • Site-directed cysteine modifications and the addition of flexible linkers in engineered MTs were designed to tune metal selectivity, particularly enhancing affinity for hard-acid metals such as Al³⁺.
• Protein and Peptide Production:
  • Larger recombinant proteins such as PCS and MT were expressed in E. coli BL21(DE3) using the T7-inducible system and purified via Ni–NTA affinity chromatography under native conditions.
  • For smaller thiol-rich peptides (phytochelatins), we proposed an acid extraction and monobromobimane (mBBr) derivatization workflow, which enables clean isolation, stabilization, and quantification without oxidation or metal interference.
  • This combination of techniques spans the proteomic spectrum—from full-length enzymes to small thiol polymers—providing a unified expression–purification framework for metal-binding proteins.
• Functional Embedding:
  • The purified proteins and peptides were then conceptualized for surface immobilization on sodium alginate–chitosan matrices, linking proteomic expression to material-level functionality.
  • This approach demonstrates the potential of synthetic proteins as modular biomaterials for sustainable, biodegradable metal-capture systems.

Metabolomics

At the metabolomic level, our work investigates how engineered E. coli strains channel metabolic flux toward glutathione and phytochelatin biosynthesis during metal exposure.

• Intracellular Pathway Engineering:
  • Co-expression of PCS with the bifunctional GSHF enzyme from S. thermophilus created a self-sufficient in vivo biosynthetic system capable of de novo glutathione production and subsequent polymerization into phytochelatins.
  • This closed-loop system eliminates the need for external substrate supplementation, ensuring that the metal sequestration response is driven entirely by internal metabolic capacity.
• Metal–Metabolite Interaction and Homeostasis:
  • The in vivo modules were further enhanced with the merP/merT transporter pair to modulate intracellular metal ion availability, enabling systematic evaluation of how metal uptake and metabolite flux balance influence chelate formation.
  • The outcome links cellular metabolite dynamics—glutathione pools, thiol turnover, and oxidative stress buffering—to the overall metal-binding efficiency of the engineered strain.
• Metabolomic Insight for Optimization:
  • This integrated design demonstrates how metabolite availability, enzymatic activity, and transport can be harmonized to create synthetic metabolic states optimized for detoxification, providing a foundation for future metabolomics-driven engineering of biosorption systems.

Integrative Perspective

• Together, the genomic, proteomic, and metabolomic layers of our project connect sequence-level rational design to system-level function:
• Genomics defined what to express — through rational gene selection, modular circuit design, and composite part construction.
• Proteomics realized how those sequences function — via expression, engineering, purification, and functional peptide embedding.
• Metabolomics revealed how the cell’s internal chemistry supports and regulates these pathways — coupling enzyme activity with glutathione metabolism and metal availability.