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
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 pRAD27-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 pRAD27-AhR biosensor to Aroclor in milk
Aim: To find out whether the AhR biosensor system works in milk when sensing Aroclor 1260.
Method: This experiment followed the 3 hour protocol, using the RAD27+AhR strain. There were a couple of changes made however: we used a full-fat (3.5%) milk dilution and a constant, unchanging concentration of Aroclor 1260 to measure the response in milk. Each well got added 50 µl of cells which were concentrated to be OD=10.
Result: The mean activity shows a response curve of what we would expect, however, there are massive deviations from the mean in both sides, especially in the 3.1%-12.5% milk range. The plot (Figure 5) indicates that the biosensor remains functional in milk.
Figure 5 Line plot showing the RAD27+AhR promoter biosensor function in milk against Aroclor 1260 .
Response of pRAD27-AhR biosensor to 2.5 µM PAH in milk
Objective: To assess the functionality of the AhR biosensor system in detecting benzanthracene in milk.
Methodology: The standard 3-hour incubation protocol was followed as detailed in the experimental section. Milk dilutions (Table X) were used along with a constant concentration of PAH to measure the response in milk. A control row with 50 µL of water was included. Each well received 50 µL of cells concentrated to an OD of 10.
Results: A basal signal was observed, which dropped by approximately 3000 RLU when using a 50% milk and 50% media solution (Figure X). The signal remained stable, between 10,000 and 10,500 RLU, across milk dilutions from ¼ to 1/128, but decreased to an average of 9000 RLU in the absence of milk. This suggests the possible presence of a cofactor in milk that enhances the biosensor's response. However, further replicates and experimentation are needed to confirm this hypothesis and better define the biosensor's dynamic range. Nonetheless, we conclude that the biosensor is functional in milk.
Figure 6 Line plot showing the RAD27+AhR promoter biosensor function in milk against PAH.
Response of pRAD27-AhR Biosensor compared to pSTE12-AhR Biosensor (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 7). 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 7 ), 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.
Figure 7 Line plot showing the performance of two different AhR biosensor promoters as a function of media pH change, aiming to find the optimal pH for the biosensor systems. On the x-axis: pH levels, ranging from 4.5 to 8 in increments of 0.5, on the y-axis: luminescence in RLU (biosensor response).
Response of pRAD27-AhR Biosensor compared to pSTE12-AhR Biosensor (2.5 µM PAH) in milk
Objective: To evaluate whether the AhR biosensor system can detect benzanthracene in a milk matrix.
Methodology: The standard 3-hour incubation protocol was followed as outlined in the experimental procedure. A series of milk dilutions were used with a constant concentration of PAH to assess the biosensor's response in milk. A control row with 50 µL of water was included. Each well received 50 µL of cells concentrated to an OD of 10.
Results: A basal luminescent signal was observed, which decreased by approximately 3000 RLU in a half milk, half media solution (Figure 8). The signal stabilized around 10,000-10,500 RLU across milk dilutions ranging from ¼ to 1/128, but dropped to an average of 9000 RLU in the absence of milk. While these results suggest that a potential cofactor in milk might enhance the system's response, further replicates and experiments are needed to confirm this hypothesis and better understand the dynamic range of the biosensor. Despite these uncertainties, we conclude that the biosensor is functional in milk.
Figure 8 Line plot showing the pRAD27-AhR promoter biosensor function in milk against PAH.
pRAD27-AhR biosensor incubation Overnight OD = 0.5 vs. 3-hours OD = 0.5
Objective: To optimize between the overnight and 3-hour incubation protocols.
Methodology: Two 96-well plates were prepared simultaneously under identical conditions, with the only difference being the optical density (OD) of the cultures. Aroclor 1260 and PCB#3 were used as test compounds, with at least triplicates per condition on each plate. The overnight protocol was conducted at an OD of 0.5, while the 3-hour protocol was performed at an OD of 5.
Results: The experiments revealed two key findings: the 3-hour protocol produced a higher overall signal, while the overnight protocol demonstrated a significantly better signal-to-noise ratio seen from figure 9.
Figure 9 Line plot comparing the effect of overnight versus 3 hour protocol on RAD27+AhR’s ability to report Aroclor 1260 and PCB3 presence.
Response of pRAD27-AhR Biosensor to PCB3
Objective: To establish a dose-response curve for the RAD27+AhR biosensor in response to PCB3.
Methodology: A luminescent bioassay was performed following an overnight incubation, analyzing four columns with varying concentrations of PCB3.
Results: The RAD27+AhR biosensor exhibited a highly variable response to different PCB3 concentrations. The detection range was limited, and the response did not follow a consistent or predictable pattern (Figure 10). Based on these findings, we hypothesize that this construct may not be suitable for further experiments.
Figure 10 Line plot delineating PCB effect on the light signal given by the RAD27+AhR system.
Response of pRAD27-AhR Biosensor to PAH
Objective: To investigate the response of the RAD27+AhR biosensor to the tested PAH.
Methodology: A 3-hour incubation was conducted with an optical density (OD) of 5, using duplicate samples.
Results: The experiment produced a signal range between 12,000 and 20,000, with considerable variability and large error margins. Despite the fluctuations, this represents the highest signal range observed in contaminant-specific studies, supporting the conclusion that the AhR biosensor is indeed sensitive to PAHs.
Figure 11 Line plot showing the response of RAD27+AhR to PAH in our experiment.
Response of pRAD27-AhR Biosensor to Aroclor 1260
Objective: To determine the dose-response curve of the RAD27+AhR biosensor in response to Aroclor 1260.
Methodology: A 3-hour incubation protocol was followed, using duplicate samples.
Results: While the data showed wide error bars, the response range of 12,000 to 16,000 (Figure 12) was relatively consistent compared to other experiments. However, we were unable to successfully establish a clear dose-response curve.
Figure 12 Line plot exhibiting RAD27 AhR response to Aroclor 1260. Duplicate data.
Sequence and Features
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References
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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.
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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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