Significance: Heavy metal contamination, specifically mercury ($Hg^{2+}$), poses a critical threat due to its persistence and bioaccumulation in living organisms. In industrial and mining regions like Lima, Peru, traditional detection relies on expensive analytical equipment (ICP-MS), which is inaccessible to local communities. This project addresses the urgent need for a low-cost “sense-and-respond” biological system.
Broad Objective: The overall goal of this project is to engineer Pseudomonas putida to serve as a dual-action biosensor and bioremediator that can simultaneously detect mercury concentrations and initiate physical sequestration.
Hypothesis: This project tests the principle that a MerR-regulated genetic circuit can exhibit high sensitivity to $Hg^{2+}$ ions, triggering a proportional expression of Green Fluorescent Protein (GFP) for quantification and SmtA (a bacterial metallothionein) for sequestration.
Specific Aims:
Methods: The technical approach utilizes Golden Gate Assembly to create a bicistronic operon under the control of the mercury-responsive $P_{mer}$ promoter. Detection is measured via fluorometry (488nm/509nm). Remediation is achieved through the expression of cysteine-rich Metallothioneins (MTs). These proteins use thiol-group coordination to bind $Hg^{2+}$ ions with high affinity. To prevent cellular toxicity, these proteins are fused to the Lpp-OmpA system for display on the outer membrane of the cell.
The first aim of my final project is to construct and validate a mercury-responsive genetic circuit in Pseudomonas putida by utilizing the MerR regulatory protein and the $P_{mer}$ promoter. MerR acts as a specialized transcription factor; when $Hg^{2+}$ binds to its conserved cysteine residues, it induces a conformational twist in the DNA, allowing RNA polymerase to initiate transcription of the GFP reporter. This aim will establish the operational detection limit of the biosensor.
The next step following the successful creation of the sensor is to engineer a remediation module by expressing SmtA Metallothioneins. These proteins are highly enriched in Cysteine (Cys) residues, which provide sulfur atoms for stable coordination of heavy metals. To maximize capture efficiency, I will utilize a surface-display strategy (fusing SmtA to the OmpA protein) so the bacteria can sequester mercury directly from the external environment without requiring intracellular transport.
The long-term vision is to democratize environmental monitoring by deploying these microbes in a “Living Filter” system. This involves immobilizing the engineered P. putida in a porous matrix to create a self-regenerating water treatment device. This challenges the current paradigm of centralized waste management by providing local communities with a cost-effective, autonomous method for both monitoring and cleaning their water sources.
The MerR protein is a highly specific metalloregulatory protein. Research by Brown et al. (2003) demonstrates that MerR binds to the $P_{mer}$ operator as a homodimer; upon mercury binding, it rotates the DNA to activate transcription. For sequestration, Metallothioneins (MTs) like SmtA are utilized. As discussed by Romero-Isart and Vasak (2002), these proteins form metal-thiolate clusters, allowing a single protein to bind multiple mercury ions with high stability. Currently, most technologies offer “sensing only” or “remediation only”; an integrated, field-robust system in a resilient chassis like P. putida is a notable gap in current research.
This project is innovative because it integrates detection and remediation into a single, automated “sense-and-respond” circuit.