Part:BBa_K5477041
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 (1) (2) (3) (4). 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, inducing NanoLuc expression and producing a luminescent signal which can be quantified (1) (2) (3) (4).
Usage and Biology
The biosensor device integrates both receptor modules and a reporter module forming a device 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 the 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 (1). 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, inducing the transcription of NanoLuc.
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 (5) (6). 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 (2). 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 (7).
Results
Promoter optimization
Objective: To identify the optimal promoter for the AhR biosensor by evaluating the performance of two different constructs.
Methodology: To determine which AhR biosensor system exhibits the highest sensitivity to polycyclic aromatic hydrocarbons (PAH), we conducted a comparative analysis of two strains using a luminescent endpoint bioassay. Overnight cultures of both systems were prepared and diluted to an optical density (OD) of 3. In a 96-well plate, triplicates of each strain (100 μL per well) were dispensed. A PAH dilution series, prepared in DMSO, was added to each well at a volume of 1 μL. The plate was incubated for 3 hours at 28°C in a shaking incubator.
Response of pSTE12-AhR Biosensor to 2.5 µM PAH under different pH conditions
Response of pSTE12-AhR Biosensor to Bisphenol A (BPA)
Detection of Aroclor 1260 by STE12-AhR Biosensor
Sequence and Features
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Illegal PstI site found at 5228 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 927
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Illegal PstI site found at 5228 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 927
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Illegal BglII site found at 1742
Illegal BamHI site found at 6983
Illegal BamHI site found at 8977 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 927
Illegal EcoRI site found at 2581
Illegal EcoRI site found at 3687
Illegal EcoRI site found at 5719
Illegal EcoRI site found at 6341
Illegal EcoRI site found at 9033
Illegal XbaI site found at 55
Illegal SpeI site found at 1759
Illegal SpeI site found at 3531
Illegal SpeI site found at 3922
Illegal SpeI site found at 5168
Illegal SpeI site found at 5469
Illegal SpeI site found at 6536
Illegal PstI site found at 5228 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 927
Illegal EcoRI site found at 2581
Illegal EcoRI site found at 3687
Illegal EcoRI site found at 5719
Illegal EcoRI site found at 6341
Illegal EcoRI site found at 9033
Illegal XbaI site found at 55
Illegal SpeI site found at 1759
Illegal SpeI site found at 3531
Illegal SpeI site found at 3922
Illegal SpeI site found at 5168
Illegal SpeI site found at 5469
Illegal SpeI site found at 6536
Illegal PstI site found at 5228
Illegal AgeI site found at 1808
Illegal AgeI site found at 3971 - 1000COMPATIBLE WITH RFC[1000]
References
1. Carambia, A., Schuran, F.A. The aryl hydrocarbon receptor in liver inflammation. Semin Immunopathol 43, 563–575 (2021). https://doi.org/10.1007/s00281-021-00867-8
2. Goedtke L, Sprenger H, Hofmann U, Schmidt FF, Hammer HS, Zanger UM, Poetz O, Seidel A, Braeuning A, Hessel-Pras S. Polycyclic Aromatic Hydrocarbons Activate the Aryl Hydrocarbon Receptor and the Constitutive Androstane Receptor to Regulate Xenobiotic Metabolism in Human Liver Cells. Int J Mol Sci. 2020 Dec 31;22(1):372. doi: 10.3390/ijms22010372. PMID: 33396476; PMCID: PMC7796163.
3. Kafafi SA, Afeefy HY, Ali AH, Said HK, Kafafi AG. Binding of polychlorinated biphenyls to the aryl hydrocarbon receptor. Environ Health Perspect. 1993 Oct;101(5):422-8. doi: 10.1289/ehp.93101422. PMID: 8119253; PMCID: PMC1519849.
4. Mandal A, Biswas N, Alam MN. Implications of xenobiotic-response element(s) and aryl hydrocarbon receptor in health and diseases. Hum Cell. 2023 Sep;36(5):1638-1655. doi: 10.1007/s13577-023-00931-5. Epub 2023 Jun 17. PMID: 37329424.
5. Onate SA, Boonyaratanakornkit V, Spencer TE, et al. The steroid receptor coactivator-1 contains multiple receptor interacting and activation domains that cooperatively enhance the activation function 1 (AF1) and AF2 domains of steroid receptors. J Biol Chem. 1998;273(20):12101-12108. doi:10.1074/jbc.273.20.12101
6. Oñate SA, Tsai SY, Tsai MJ, O'Malley BW. Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science. 1995;270(5240):1354-1357. doi:10.1126/science.270.5240.1354
7. England CG, Ehlerding EB, Cai W. NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence. Bioconjug Chem. 2016 May 18;27(5):1175-1187. doi: 10.1021/acs.bioconjchem.6b00112. Epub 2016 Apr 19. PMID: 27045664; PMCID: PMC4871753.
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