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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 Selection and Optimization for Enhanced Sensitivity

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.

Figure 4: Line plot comparing the performance of RAD27+AhR (pink) and pSTE12-AhR (STE12) biosensors in detecting benzanthracene using a luminescent bioassay. In the X-axis: PAH (benzanthracene) concentration, ranging from 2.5 mM to 2.5 nM, in the Y-axis: luminescence signal in Relative Light Units (RLU) indicating the output of the strains. N=3.

Result: While RAD27 exhibits a higher luminescence overall, the STE12 promoter appears to be more sensitive at lower PAH concentrations, as it responds to increases in PAH from the very beginning (0 to 25 nM), see figure 4. In contrast, RAD27 seems to have a higher baseline but requires higher concentrations to show a significant change in luminescence. Thus, pSTE12-AhR is likely more sensitive to low concentrations of PAH, whereas RAD27 shows a stronger response at higher concentrations.

Response of pSTE12-AhR Biosensor to 2.5 µM PAH under different pH conditions

Objective: To determine the optimal pH levels for the AhR biosensor.

Methodology: Two 96-well plates were prepared, one for the STE12 promoter and the other for the RAD27 promoter. Glucose yeast media with pH values ranging from 4.5 to 8.0, in 0.5 increments, were aliquoted into columns on each plate (as illustrated in Figure 5). Each well received 2.5 μM PAH, a concentration selected based on the promoter optimization assay, as it produced the highest signal. The original cell cultures were diluted to an optical density (OD) of 0.5, and the plates were incubated at 30°C. The luminescent bioassay was conducted the following morning to measure the biosensor response across the pH range.

Results: Both biosensor strains demonstrated functionality at pH 5, with a significantly reduced signal at pH 4.5, suggesting that the biosensor system is less effective under highly acidic conditions. This finding was consistent across replicates, indicating that it is likely an inherent characteristic of the AhR biosensor system rather than a result of technical error.

The pSTE12-AhR exhibited greater robustness across a range of pH levels, consistently generating a higher luminescent signal than RAD27, particularly in more acidic conditions. In contrast, RAD27 performed better in neutral to slightly alkaline conditions, specifically within the pH 7.0–7.5 range. This difference explains the variation between these results and those from the promoter optimization assay, where the media pH was between 6.5 and 6.7.

As milk’s pH with yeast is consistently around 6.7 (Milk pH Testing, Figure 5), we recommend prioritizing the use of the RAD27 promoter for assays conducted in milk-based environments. However, given that pSTE12-AhR shows a strong response in more acidic environments, it may be better suited for applications in lower pH conditions. Both promoters performed poorly at extreme pH values (4.5 and 8.0), indicating that the AhR biosensor system operates optimally within a moderate pH range.

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

Objective: To assess the response of the pSTE12-AhR biosensor to higher concentrations of BPA.

Methodology: A 3-hour incubation protocol was followed, examining four columns for varying BPA concentrations.

Result: It appears that at 10 mM the sensor loses sensitivity to the compound, and previously it is not increasing in a manner proportional to the increase in concentration see figure below.

Detection of Aroclor 1260 by pSTE12-AhR Biosensor

Objective: To evaluate the AhR response at the higher range of BPA concentrations.

Methodology: A 3-hour incubation protocol was conducted, testing four columns with varying concentrations of BPA.

Results: The luminescent response fluctuated across different BPA concentrations. Luminescence increased between 0.5 µM and 1 µM, followed by a decrease at 50 µM. The signal then rose again at 100 µM before decreasing at 0.5 mM and 1 mM. A significant increase in luminescence was observed at the highest BPA concentrations (5 mM and 10 mM), indicating a strong activation or interaction with the pSTE12-AhR biosensor at these levels see figure below.

Figure 7 Line plot showing the response of pSTE12-AhR to the higher end of Aroclor concentration.

Sequence and Features

References

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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.