Optimization of Heavy Metal Sensors Based on Transcription Factors and Cell-Free Expression Systems

Abstract

Many bacterial mechanisms for highly specific and sensitive detection of heavy metals and other hazards have been reengineered to serve as sensors. In some cases, these sensors have been implemented in cell-free expression systems, enabling easier design optimization and deployment in low-resource settings through lyophilization. Here, we apply the advantages of cell-free expression systems to optimize sensors based on three separate bacterial response mechanisms for arsenic, cadmium, and mercury. We achieved detection limits below the World Health Organization-recommended levels for arsenic and mercury and below the short-term US Military Exposure Guideline levels for all three. The optimization of each sensor was approached differently, leading to observations useful for the development of future sensors: (1) there can be a strong dependence of specificity on the particular cell-free expression system used, (2) tuning of relative concentrations of the sensing and reporter elements improves sensitivity, and (3) sensor performance can vary significantly with linear vs plasmid DNA. In addition, we show that simply combining DNA for the three sensors into a single reaction enables detection of each target heavy metal without any further optimization. This combined approach could lead to sensors that detect a range of hazards at once, such as a panel of water contaminants or all known variants of a target virus. For low-resource settings, such "all-hazard" sensors in a cheap, easy-to-use format could have high utility.

Transcription Factor-Based Biosensors for Detecting Pathogens

“…Since TFs corresponding to heavy metals have been identified, the cell-free biosensors for detecting heavy metals have been reported by many research groups [ 72 , 73 , 74 ]. Recently, Beabout et al reported heavy metal biosensors based on cell-free expression systems.…”

Large-Data Omics Approaches in Modern Remediation

“…Advancements in synthetic biology may be a precursor to the future of remediation. These may include specifically engineered microorganisms and synthetic communities and synthetic biomarkers (Beabout et al 2021). As advancements in methods continue to improve accuracy and resolution, so do the libraries of known analytes.…”

Biotechnology Applications of Cell-Free Expression Systems

“…Also, cell-free systems do not risk the release of genetically modified organisms into nature, as simple metabolic pathways do not pose the same environmental threat as engineered microbes [23,54]. Without homeostatic concerns, cell-free biosensors can also operate in a diverse range of environments that would otherwise be toxic for cell-based systems, and cell-free systems can achieve higher sensitivities to toxic analytes because of the systems' ability to withstand higher concentrations of toxins [23,54]. Finally, cell-free systems are also not subject to concerns about evolutionary change altering sensor function [23].…”

“…One possible avenue of detection is the utilization of transcription factors for the detection of target ligands. Transcription factor detection involves the expression of a reporter molecule such as green fluorescent protein (GFP) in the presence of a target analyte, where the presence of the analyte will cause an inhibitory molecule, which is bound to the operator controlling the expression of the reporter molecule, to break off, allowing for the expression of the reporter [54,56,57]. Another possible detection method utilizes riboswitches, which are RNA structures that regulate gene expression through the binding of certain metabolites [23].…”