Difference between revisions of "Part:BBa K5477041"

Revision as of 17:39, 30 September 2024

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

Summary

This system is designed to detect the presence of toxic environmental compounds such as PAHs, dioxins and PCBs. The pSTE12-AhR receptor module allows for the detection of these compounds, while the pRET2-ARNT and pRET2-NCOA modules support and amplify the receptor activity. Once activated by these toxins, the AhR-ARNT complex binds to the XRE in the reporter module, triggering NanoLuc expression and producing a luminescent signal which can be quantified.

Usage and Biology

The biosensor device integrates both receptor modules and a reporter module, forming a functional framework to detect PAHs, dioxins and PCBs. Each module plays a specific role in the system, with receptor modules responsible for detecting external signals and the reporter module translating those signals into a measurable output. Below are figures that show how our device works with and without the contaminants.

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. pSTE12-AhR BBa_K5477023: This receptor module uses the pSTE12 promoter to drive the expression of the Aryl Hydrocarbon Receptor (AhR). AhR is a transcription factor that becomes activated upon binding to toxic environmental compounds such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). When activated, AhR translocates to the nucleus and dimerizes with ARNT, binding to xenobiotic response elements (XREs) to activate downstream gene expression.

2. pRET2-ARNT BBa_K5477025: ARNT (Aryl Hydrocarbon Receptor Nuclear Translocator) is a key dimerization partner for AhR. The pRET2 promoter controls ARNT’s expression, ensuring it is available to form a complex with AhR when the latter is activated by its ligands. Once AhR binds to environmental toxins, it partners with ARNT to regulate gene expression through XREs, facilitating a response to the presence of these harmful compounds.

3. pRET2-NCOA BBa_K5477026: NCOA (Nuclear Receptor Coactivator), under the control of the pRET2 promoter, enhances the transcriptional activity of nuclear receptors such as AhR. NCOA interacts with the AhR-ARNT complex, acting as a coactivator to recruit chromatin remodeling factors and transcriptional machinery, boosting the expression of detoxification genes. This enhances the overall sensitivity and robustness of the biosensor.

Reporter Module

1.XRE-pMEL1-NanoLuc BBa_K5477030: The XRE (Xenobiotic Response Element) serves as the DNA binding site for the activated AhR-ARNT complex. Once AhR binds to a ligand (such as PAHs or PCBs), it dimerizes with ARNT and NCOA which then binds to the XRE sequence. This binding activates the downstream pMEL1 promoter, driving the expression of the NanoLuc reporter gene. NanoLuc is a highly sensitive luciferase that produces bioluminescence in the presence of its substrate, providing a measurable output that correlates with the level of AhR activation.

Results

Promoter Selection and Optimization for Enhanced Sensitivity

pH Sensitivity Optimization of the AhR Biosensor System

Response of pSTE12-AhR Biosensor to 2.5 µM PAH

Response of pSTE12-AhR Biosensor to Bisphenol A (BPA)

Detection of Aroclor 1260 by STE12-AhR Biosensor

Sequence and Features

References