Difference between revisions of "Part:BBa K5477042"

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Biosensor device II for detection of PAHs, dioxin or dioxin-like PCBs

Summary

This system is designed to detect environmental toxins utilizing the pRAD27-AhR receptor module. The AhR pathway detects toxic compounds. Upon activation by PAHs or PCBs, AhR binds to ARNT (whose expression is regulated by the pRET2 promoter) and forms a transcriptional complex. NCOA, expressed via the pRET2 promoter, further amplifies the transcriptional response by enhancing the activity of the AhR-ARNT complex. This complex binds to the XRE sequence in the reporter module, activating the pMEL1 promoter and triggering the expression of NanoLuc. The resulting bioluminescent signal serves as a direct indicator of the presence and concentration of environmental pollutants like PAH’s, dioxin-like PCB’s and dioxin.

Usage and Biology

In this biosensor system, the combination of receptor modules and a reporter module is designed to detect PAHs, dioxins and PCBs and provide a measurable bioluminescent output in response. This setup integrates the DNA damage-associated pRAD27 promoter with the AhR pathway, creating a sensitive system for monitoring toxins.

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The illustration above depicts the mechanism of the AhR biosensor device without the contaminants. 1) The proteins AhR, ARNT, and NCOA are expressed, with AhR remaining in the cytoplasm. 2) In the absence of contaminants, HSP90 (Heat Shock Protein 90) binds to AhR, preventing its translocation. 3) As a result, an AhR-HSP90 complex forms within the cytoplasm. 4) Consequently, no signal is generated because AhR is not transported to the nucleus to form a complex with ARNT and NCOA, which is essential for activating the xenobiotic response element.

The illustration demonstrates the AhR biosensor mechanism in the presence of a contaminant (such as dioxin or PCB). 1) AhR, ARNT, and NCOA are expressed within the cell, and AhR is initially located in the cytoplasm. 2) Upon binding with a contaminant (e.g., dioxin or PCB), AhR undergoes a conformational change. 3) The AhR-contaminant complex is translocated into the nucleus. 4) In the nucleus, the AhR-contaminant complex interacts with ARNT and NCOA. 5) This complex binds to the xenobiotic response element (XRE) in the DNA, triggering the transcription of target genes and generating a signal output in the form of NanoLuc.

Why do we want to detect PAHs, PCBs and dioxins in breast milk?

Detecting PAHs, dioxins, and dioxin-like PCBs in breast milk is crucial because these toxic environmental pollutants pose significant health risks to infants. PAHs (polycyclic aromatic hydrocarbons), dioxins, and PCBs (polychlorinated biphenyls) are known to accumulate in fatty tissues and are passed to infants through breast milk, potentially exposing them to harmful effects during a critical stage of development. These compounds have been linked to immune system suppression, hormonal disruption, developmental delays, and an increased risk of cancer. Early detection in breast milk is vital for protecting infant health and ensuring safe breastfeeding practices in contaminated environments. Below is a figure of the whole device consisting of our composites.

Receptor Modules

1. pRAD27-AhR | BBa_K5477024: The pRAD27 promoter drives the expression of the Aryl Hydrocarbon Receptor (AhR), which is responsible for detecting environmental toxins, such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). When AhR is activated by toxins, it translocates to the nucleus and dimerizes with ARNT, triggering a transcriptional response.

2. pRET2-ARNT | BBa_K5477025: ARNT (Aryl Hydrocarbon Receptor Nuclear Translocator) is the partner protein that dimerizes with AhR when it is activated by environmental toxins. The pRET2 promoter controls ARNT expression. Once AhR binds a ligand, such as PAHs or PCBs, it pairs with ARNT to form a functional transcription factor complex that activates detoxification-related genes by binding to XRE sequences.

3. pRET2-NCOA | BBa_K5477026: NCOA (Nuclear Receptor Coactivator), expressed under the control of the pRET2 promoter, enhances transcriptional activation by the AhR-ARNT complex.

Reporter Module

1.XRE-pMEL1-NanoLuc | BBa_K5477030: The XRE (Xenobiotic Response Element) is the DNA sequence that the AhR-ARNT complex binds to upon activation. Once the receptor complex binds to the XRE site, the pMEL1 promoter drives the expression of the NanoLuc reporter gene. NanoLuc is a luciferase enzyme that produces a bioluminescent signal in the presence of its substrate, providing a sensitive and quantifiable readout of AhR activation. The bioluminescence intensity directly correlates with the level of toxic ligand binding to AhR, making it an effective and rapid sensor for environmental toxins like PAHs and PCBs.

Results

Promoter Selection and Optimization for Enhanced Sensitivity

Response of pRAD27-AhR biosensor to BPA vs. Aroclor in milk

Response of pRAD27-AhR biosensor to BPA vs. Aroclor

pRAD27-AhR biosensor PCB chlorination preference

pH Sensitivity Optimization of the AhR Biosensor System

Response of pRAD27-AhR Biosensor compared to pSTE12-AhR Biosensor (2.5 µM PAH)

pRAD27-AhR biosensor incubation Overnight OD = 0.5 vs. 3-hours OD = 0.5

Response of pRAD27-AhR Biosensor to PCB3

Response of pRAD27-AhR Biosensor to PAH

Response of pRAD27-AhR Biosensor to Aroclor 1260

Sequence and Features

References