# Systematic evaluation of pigment-based whole-cell lead biosensors: challenges in genetic circuit engineering and critical considerations for background noise control

## TL;DR

This study improves lead detection using engineered bacteria by optimizing genetic circuits and reducing background noise for more accurate and sensitive biosensors.

## Contribution

A systematic evaluation of pbr operons and circuit designs to create high-performance, pigment-based Pb(II) biosensors with sub-nanomolar sensitivity.

## Key findings

- The pKp-DV construct achieved a limit of detection of 0.0008 μM and a dynamic range of 0.0061-50 μM.
- Transcription terminators reduced background noise by 60% and improved detection limits.
- Hybrid circuits and regulator dosage showed limited improvement, suggesting the need for more advanced designs.

## Abstract

Despite advances in whole-cell biosensors for Pb(II) detection, background leakage and limited dynamic range hinder field deployment. This study systematically evaluates four diverse pbr operons and optimizes genetic circuit architectures to develop high-performance pigment-based Pb(II) biosensors.

pbr operons from Cupriavidus metallidurans, Pseudomonas aeruginosa, and Klebsiella pneumoniae were engineered into E. coli TOP10 using decoupled violacein reporter circuits. Five optimization strategies were tested: transcriptional terminators, graded regulator expression (P302-PJ23119 promoters), dual-gene copies, hybrid MerR-PbrR regulators, and circuit architecture variants. All assays used triplicate replicates (n = 3) across 8 time points and 12-15 Pb(II) concentrations.

The pLVPK-derived pKp-DV construct (PbrR.Kp) achieved optimal performance: LOD of 0.0008 μM, dynamic range 0.0061-50 μM, and high Pb(II) specificity. Transcription terminator insertion further reduced LOD to 0.0002 μM and background noise by 60%. However, regulator dosage and hybrid circuits showed minimal improvement, indicating limited synergistic benefits.

PbrR.Kp selection and transcriptional insulation are key strategies for reducing leakage. Hybrid circuits require more sophisticated designs. Future work should focus on chromosomal integration and environmental matrix validation to ensure the development of robust, field-ready sensors.

Flowchart depicting a systematic evaluation process for a sub-nanomolar pigment-based Pb(II) biosensor. It includes three sections: “Input” with four pbr operons and associated bacterial strains, “Optimization” with circuit strategies like terminators and hybrid circuits, and “Output” showing the best performer with specifications like LOD of 0.8 nanomolar and selectivity for Pb(II) and Hg(II). A structural formula of deoxyviolacein is shown at the bottom.