Part:BBa_K5477042
Biosensor device II for detection of PAHs, dioxin or dioxin-like PCBs
Summary
This system is designed to detect contaminants utilizing the pRAD27-AhR receptor module. The AhR pathway detects toxic compounds (1) (2) (3). 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 (5) (6). This complex binds to the XRE sequence in the reporter module, activating the pMEL1 promoter and inducing the expression of NanoLuc (4) (7). The resulting bioluminescent signal serves as a direct indicator of the presence 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.
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.
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
Response of pRAD27-AhR Biosensor compared to pSTE12-AhR Biosensor (2.5 µM PAH) under different pH conditions
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
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1064
Illegal EcoRI site found at 2718
Illegal EcoRI site found at 3824
Illegal EcoRI site found at 5856
Illegal EcoRI site found at 6478
Illegal EcoRI site found at 9170
Illegal SpeI site found at 289
Illegal SpeI site found at 426
Illegal SpeI site found at 1896
Illegal SpeI site found at 3668
Illegal SpeI site found at 4059
Illegal SpeI site found at 5305
Illegal SpeI site found at 5606
Illegal PstI site found at 5365 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1064
Illegal EcoRI site found at 2718
Illegal EcoRI site found at 3824
Illegal EcoRI site found at 5856
Illegal EcoRI site found at 6478
Illegal EcoRI site found at 9170
Illegal NheI site found at 869
Illegal NheI site found at 2090
Illegal NheI site found at 4253
Illegal NheI site found at 7618
Illegal SpeI site found at 289
Illegal SpeI site found at 426
Illegal SpeI site found at 1896
Illegal SpeI site found at 3668
Illegal SpeI site found at 4059
Illegal SpeI site found at 5305
Illegal SpeI site found at 5606
Illegal PstI site found at 5365 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1064
Illegal EcoRI site found at 2718
Illegal EcoRI site found at 3824
Illegal EcoRI site found at 5856
Illegal EcoRI site found at 6478
Illegal EcoRI site found at 9170
Illegal BglII site found at 102
Illegal BglII site found at 1879
Illegal BamHI site found at 7120
Illegal BamHI site found at 9114 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1064
Illegal EcoRI site found at 2718
Illegal EcoRI site found at 3824
Illegal EcoRI site found at 5856
Illegal EcoRI site found at 6478
Illegal EcoRI site found at 9170
Illegal SpeI site found at 289
Illegal SpeI site found at 426
Illegal SpeI site found at 1896
Illegal SpeI site found at 3668
Illegal SpeI site found at 4059
Illegal SpeI site found at 5305
Illegal SpeI site found at 5606
Illegal PstI site found at 5365 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1064
Illegal EcoRI site found at 2718
Illegal EcoRI site found at 3824
Illegal EcoRI site found at 5856
Illegal EcoRI site found at 6478
Illegal EcoRI site found at 9170
Illegal SpeI site found at 289
Illegal SpeI site found at 426
Illegal SpeI site found at 1896
Illegal SpeI site found at 3668
Illegal SpeI site found at 4059
Illegal SpeI site found at 5305
Illegal SpeI site found at 5606
Illegal PstI site found at 5365
Illegal AgeI site found at 1945
Illegal AgeI site found at 4108 - 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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