Project Introduction
seQUESTer — Tackling Toxic Heavy Metal Contamination for a Sustainable Future
Access to safe drinking water is a cornerstone of community health, economic stability, and environmental integrity. Yet, toxic heavy metals (THMs) such as lead (Pb) continue to threaten freshwater systems globally, with no safe exposure threshold for human health1. Lead contamination is particularly insidious: it accumulates in ecosystems, disproportionately impacts vulnerable populations, and perpetuates cycles of inequality.
The Problem
Lead infiltrates freshwater through both industrial activity—including mining, smelting, and effluent discharge—and aging infrastructure, such as legacy lead service lines in Canadian municipalities2,3. Seasonal fluctuations and water treatment practices can exacerbate lead release, further increasing public exposure. These risks are magnified in communities lacking financial resources to retrofit or replace outdated water systems.
Across Ontario and Canada more broadly, the impacts are profound. First Nations communities experience some of the highest burdens, with over a quarter of systems reporting lead levels above national safety standards4. Similarly, Ontario schools face recurring lead contamination, which has been linked to measurable declines in student performance and cognitive outcomes5. The burden is not only biological but also social—childhood lead exposure is associated with long-term neurocognitive impairment, behavioral challenges, and even elevated crime rates later in life6.
Why It Matters
This crisis extends beyond public health. It undermines educational equity, strains local economies, erodes environmental integrity, and compounds existing socioeconomic disparities1. Communities without sufficient resources—particularly low-income and Indigenous populations—are disproportionately affected, creating a cycle of vulnerability and marginalization4.
At the same time, water risks have broad financial and ecological implications. As Sandhu et al. (2021) highlight, sustainable water investments are increasingly critical to ensuring community resilience in the face of climate change, population growth, and industrial pressures7.
Our Solution
iGEM Guelph addresses the challenge of toxic heavy metal (THM) pollution through seQUESTer, a novel bioengineered filtration system that combines genetic sensing with enhanced microbial sequestration. Using a riboswitch-based system in Saccharomyces cerevisiae, we are developing a microbial scaffold that detects and removes lead from contaminated water. The system has 3 main components including a riboswitch allowing for targeted detection of lead, a state-switchable promoter which acts as a genetic memory switch, and metal transporters for sequestration and removal of lead. Coupled with a biofiltration unit designed for scalability and accessibility, our platform provides a cost-effective, sustainable alternative to traditional filtration methods.
Figure 1: Genetic circuit showing riboswitch detection, ΦBT1 memory system, and metal transporter components
MoClo System
Riboswitch
The foundation of the system is a riboswitch sensor specifically designed to bind Pb²⁺. Two aptamer variants, Pb7S and Pb14S, are tested for their ability to undergo conformational change upon lead binding, thereby regulating the translation of downstream genes. These riboswitches are cloned upstream of a GFP reporter under a constitutive TEF2 promoter, allowing fluorescence to serve as a quantifiable indicator of detection efficiency. By comparing responses across varying Pb²⁺ concentrations, we can validate riboswitch performance and identify the optimal construct for sensitive and specific detection8.
State-switchable promoter
To ensure stable and lasting responses, the riboswitch is coupled to the ΦBT1 integrase-based memory system. In this design, the riboswitch regulates the expression of ΦBT1, which mediates inversion of a state-switchable promoter flanked by attP/attB sites. Once flipped, the promoter permanently reorients to drive transcription of downstream genes, effectively "recording" the detection event even after Pb²⁺ is removed. Controls with inactive or correctly oriented lacZ reporters confirm the specificity of the integrase system 9. When combined, the detection and memory modules allow engineered microbes not only to sense transient exposures to lead but also to maintain a durable transcriptional state that continues bioremediation activities beyond the initial stimulus.
Metal Transporters
Downstream of the memory switch lies a gene cluster encoding metal uptake and detoxification proteins including:
- DMT1, a divalent metal transporter that increases Pb²⁺ uptake
- YCF1, a vacuolar transporter that enhances intracellular storage and tolerance
- GSH1, a gene encoding γ-glutamylcysteine synthetase. Increases intracellular glutathione levels providing the substrate required for Ycf1p-mediated Pb²⁺ sequestration10
This section of the design remains as future directions in our project as wet lab experiments are not complete. In our design these genes are expressed in an “operon-like” configuration using 2A peptides, enabling polycistronic expression under a single promoter. By testing constructs encoding DMT1 alone, DMT1+YCF1, and DMT1+GSH1, we can evaluate synergistic effects on both uptake capacity and tolerance. Lead accumulation can be quantified using ICP-OES analysis, while minimum inhibitory concentration assays measure improvements in resistance compared to unmodified yeast11. A GFP reporter is included in all constructs, providing a built-in visualization of system activity to confirm that sequestration modules are being expressed effectively.
These genetic modules are inspired by successful strategies in yeast, where overexpression of hyperaccumulator traits enabled sequestration of metals at levels 10-100x higher than natural thresholds10. By tightly coupling detection, memory, and detoxification, engineered microbes equipped with this system could serve as living biosensors that both identify and neutralize toxic heavy metals.
Hardware
The dry lab team worked to supplement the genetic engineering efforts by designing and developing a functional biofiltration unit to house the genetically modified Saccharomyces cerevisiae. A novel filter medium was designed to integrate the yeast's biological capabilities of detection and sequestration of lead into a contained, engineered platform suitable for real-world water treatment applications. The filter medium is a three-dimensional porous structure that not only anchors yeast cells but also provides them with access to nutrients for growth.
The material used for the filter medium is agarose, a biocompatible polymer, enhanced with adsorbent additives such as zeolite to improve heavy metal capture and biosafety12,13,14. The filter medium is produced using freeze-casting, a method that allows control of pore size and microarchitecture to balance water flow and yeast attachment15. To hold the filter medium and test the performance of the genetically modified yeast, the team developed a 3D-printed filtration apparatus that simulates the conditions of real filtration systems.
Figure 2: Continuous flow filtration system design with porous agarose media
Together, these components create a scalable, sustainable, and environmentally safe biofiltration platform that leverages a genetically modified organism for effective water treatment.
For more information, visit our Hardware page!