Project Description

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.

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.