Our Contribution to the Field of Synthetic Biology

Our project contributes a multi-layered framework for engineering biological systems capable of efficient metal sequestration, combining rational gene selection, computational design, novel circuit construction, peptide purification, and biopolymer embedding. Beyond tackling heavy metal contamination, these advances expand the synthetic biology toolkit for future iGEM teams and researchers working in environmental biotechnology.

Genetic and Computational Contributions

1. Novel Characterized Parts:
   1. We contributed several new basic and composite parts to the iGEM Registry, including:
   2. Phytochelatin Synthase (PCS) from Polyangium sorediatum
   3. Bifunctional Glutathione Synthase (GSHF) from Streptococcus thermophilus
   4. Metallothionein (MT) from Triticum aestivum (native and rationally engineered)
   5. MerP and MerT transporters from E. coli K12
   6. These parts were assembled into seven rationally designed genetic circuits—covering PCS-only, PCS+GSHF, native and engineered MTs, AtPCS-only, AtPCS+GSHF, and PCS+GSHF+Mer operon constructs—each tested in silico for reading frame, sequence compatibility, and regulatory balance.
   7. This modular architecture can be easily reused or expanded by future iGEM teams for multi-gene assembly or metabolic pathway integration.
2. In Silico Design Pipeline:
   1. We established a systematic computational docking workflow to identify optimal phytochelatin synthase variants. From over 6,500 PCS sequences, candidates were filtered by structure and function, then evaluated through two-stage molecular docking with glutathione.
   2. The Polyangium sorediatum PCS showed the highest predicted substrate-binding affinity (–10.7 kcal/mol) and was selected as the core enzyme for our construct.
   3. This pipeline provides a reproducible computational strategy that future teams can adapt for pre-screening enzymes and reducing experimental load in synthetic pathway design.
3. Genetic Circuit Architecture:
   1. Our in silico-validated circuit assembly, using RBSs, spacers, and strong terminators with IPTG-inducible promoters, represents a standardized model for predictable, modular circuit construction.
   2. The workflow ensures precise temporal control of gene expression and facilitates design reuse in future bioremediation or enzymatic expression systems.

Experimental and Methodological Contributions

• Refined Workflow for Thiol-Rich Peptide Purification:
  • We developed and validated an improved acid-based extraction and monobromobimane (mBBr) derivatization method for isolating and quantifying small thiol-rich peptides such as phytochelatins and metallothioneins.
  • This approach replaces traditional Ni–NTA purification for these low-molecular-weight peptides, enabling accurate recovery and detection without oxidation or metal interference.
  • The workflow—combining sulfosalicylic acid extraction, mBBr labeling, and fluorescence-based HPLC quantitation—offers a reliable, scalable strategy for future teams studying cysteine-rich or thiol-reactive biomolecules.
• Peptide–Biopolymer Interface Design:
  • We introduced the concept of embedding metal-binding peptides on sodium alginate bead surfaces, creating a hybrid material that unites biochemical selectivity with structural robustness.
  • This innovation demonstrates a bridge between synthetic biology and materials engineering and can serve as a basis for future biosorptive or biofilter prototypes.

Broader Contributions to the iGEM and Synthetic Biology

• Integrated In Vivo and In Vitro Design:
  • By comparing cell-based and enzyme-based systems for metal sequestration, our project provides a dual validation model that future teams can apply to assess pathway efficiency, enzyme activity, and metal-binding performance.
• Cross-Kingdom Genetic Integration:
  • Our constructs combine genes from bacteria, plants, and myxobacteria, demonstrating successful cross-domain expression and expanding the diversity of functional bioremediation parts in the iGEM Registry.
• Standardized, Reproducible Methodology:
  • All protocols for peptide purification, derivatization, and embedding were optimized for accessibility and reproducibility, allowing future iGEM teams to replicate or extend our work without specialized infrastructure.

Basic Parts

Composite Parts

Summary Table