Design and Prototyping of Genetically Encoded Arsenic Biosensors Based on Transcriptional Regulator AfArsR

Salma Saeed Khan,Yi Shen,Robert E Campbell,Habib Bokhari,Muhammad Qaiser Fatmi

doi:10.3390/biom11091276

Salma Saeed Khan, Yi Shen + Show 3 more

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https://doi.org/10.3390/biom11091276

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Abstract

Genetically encoded biosensors based on engineered fluorescent proteins (FPs) are essential tools for monitoring the dynamics of specific ions and molecules in biological systems. Arsenic ion in the +3 oxidation state (As3+) is highly toxic to cells due to its ability to bind to protein thiol groups, leading to inhibition of protein function, disruption of protein–protein interactions, and eventually to cell death. A genetically encoded biosensor for the detection of As3+ could potentially facilitate the investigation of such toxicity both in vitro and in vivo. Here, we designed and developed two prototype genetically encoded arsenic biosensors (GEARs), based on a bacterial As3+ responsive transcriptional factor AfArsR from Acidithiobacillus ferrooxidans. We constructed FRET-based GEAR biosensors by insertion of AfArsR between FP acceptor/donor FRET pairs. We further designed and engineered single FP-based GEAR biosensors by insertion of AfArsR into GFP. These constructs represent prototypes for a new family of biosensors based on the ArsR transcriptional factor scaffold. Further improvements of the GEAR biosensor family could lead to variants with suitable performance for detection of As3+ in various biological and environmental systems.

Highlights

Arsenic and arsenic compounds ubiquitously exist in the natural environment in different forms including organic, inorganic and arsine gas
We report our efforts to design, construct, and characterize prototypes of genetically encoded arsenic biosensors (GEARs) based on the Acidithiobacillus ferrooxidans As3+ responsive transcription factor, AfArsR
Development of Genetically Encoded As3+ Biosensors Based on Förster resonance energy transfer (FRET)

Summary

Introduction

Arsenic and arsenic compounds ubiquitously exist in the natural environment in different forms including organic, inorganic and arsine gas. Common organic arsenic compounds include arsanilic acid (C6 H8 AsNO3 ), methylarsonic acid (CH5 AsO3 ), dimethylarsinic acid (cacodylic acid, C2 H7 AsO2 ), and arsenobetaine (C5 H11 AsO2 ). The inorganic compounds, which are the most toxic, include trivalent and pentavalent compounds. Arsenic trioxide (As2 O3 ), sodium arsenite (NaAsO2 ), and arsenic trichloride (AsCl3 ) are the most common trivalent compounds, and arsenic pentoxide (As2 O5 ), arsenic acid (H3 AsO4 ), and arsenates (e.g., lead arsenate PbHAsO4 and calcium arsenate Ca3 (AsO4 )2 ) are the most common pentavalent compounds [1]. High levels of arsenic exposure lead to health problems in humans, including many arsenicosis diseases such as skin disease, respiratory disorders, cardiovascular disorders, developmental neurotoxicity, and various cancers [3–5]

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