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Biosensor device III for detection of BPA

Summary

In this system, the pRET2-LexA-mERα(LBD) receptor module has been engineered according to the study of Rajasärkkä et al. 2011. The chimeric activator binds to BPA with higher affinity than estrogen (3). Upon ligand binding, the mutant ERα activates the LexA DBD, allowing it to bind to the Lex6Op sequences in the reporter module. This interaction activates the pLEU2 promoter, leading to the expression of NanoLuc and resulting in a bioluminescent signal (4).

Usage and Biology

This biosensor system consists of a receptor and reporter module to detect the presence of compounds like bisphenol A (BPA), which can activate the mutant Estrogen Receptor Alpha (mERα). This system is designed to provide a quantifiable luminescent signal using NanoLuc as a reporter (4).

Figure 1: pRET2-mERα biosensor in the absence of BPA

Figure 1 illustrates the LexA-mERα (LBD) biosensor's behavior in the absence of BPA. 1) The LexA-mERα (LBD) complex is expressed, but without BPA present, it remains bound to HSP90 in the cytoplasm. 2) As a result, the LexA-mERα (LBD) complex is not translocated into the nucleus and stays inactive in the cytoplasm. 3) Because the complex does not enter the nucleus, it cannot bind to the Lex60p operator sequence, and thus no signal output is generated.

Figure 2: pRET2-mERα biosensor in the presence of BPA

Figure 2 shows the LexA-mERα (LBD) biosensor's response when Bisphenol A (BPA) is present. 1) The LexA-mERα (LBD) complex is expressed in the cytoplasm. 2) Upon binding to BPA, the LexA-mERα (LBD) complex undergoes a conformational change and is translocated into the nucleus. 3) Inside the nucleus, the complex binds to the Lex6Op operator sequence, triggering transcription and resulting in a signal output of NanoLuc, indicating the detection of BPA. Below is a figure of the whole device consisting of our composites.

Figure 3: Parts and composite parts in this device

Receptor Module

1.pRET2-LexA-mERα(LBD)| BBa_K5477029

This receptor module expresses a mutant version of Estrogen Receptor Alpha (mERα) under the control of the pRET2 promoter. The LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of the mutant ERα (mERα). The mutation in the ERα LBD alters its ligand-binding specificity, enabling the receptor to respond to ligands such as BPA more effectively than the wild-type receptor. Upon binding a ligand like BPA, the mutant ERα undergoes a conformational change that activates the LexA DBD. This allows the LexA-mERα (LBD) fusion protein to bind to Lex6Op operator sequences in the reporter module, initiating the transcription of downstream genes.

Reporter Module

1. pLex6Op-pLEU2-NanoLuc| BBa_K5477031

The reporter module contains six LexA operator sequences (Lex6Op), which serve as binding sites for the activated LexA-mERα (LBD) fusion protein. Upon binding to the Lex6Op sequences, the LexA-mERα protein activates the pLEU2 promoter, driving the expression of the NanoLuc reporter gene.

Results

Response of pRET2-LexA-mERα biosensor to BPA

Aim: To establish a dose-response curve for BPA using engineered estrogen receptor biosensors.

Methodology: A 3-hour luminescent bioassay was performed with yeast cells at an OD of 5. The assay focused on a single column of measurements.

Results: An incomplete dose-response curve was observed (Figure 4). The estimated minimum plateau appears to be in the range of 4000-4500 luminescence units. Based on visual estimation, the EC50 is approximately 6500 luminescence units, though further refinement and validation through additional experiments are required. If these results hold true, a more detailed focus on concentrations between 0-10 nM would be necessary, as the luminescence signal plateaus beyond this range. Given that this data is derived from a single column, it should be interpreted with caution; while compelling, it may not be fully representative, and further experimentation is needed to corroborate these findings.

Figure 4 pRET2- mERα biosensor response to BPA, shown on a Line plot.

Response of pRET2-LexA-mERα biosensor to BPA in milk

Aim: To evaluate the effect of milk on the mERα biosensor’s ability to detect Bisphenol A (BPA).

Methodology: The experimental conditions followed the previously established 3-hour incubation protocol as described on the experiments page, with a few modifications. Full-fat milk (3.5%) was used as the matrix for BPA detection. A constant BPA concentration of 10 µM was employed to assess biosensor response in the presence of milk, selected based on prior experiments that demonstrated a measurable signal at this concentration. A control row containing 50 µL of water was included to compare with the milk samples. Each well received 50 µL of yeast cells at an OD of 10 for the assay.

Results: The experiment showed that cells incubated with water and highly diluted milk produced higher luminescence. As the proportion of milk increased, a corresponding decrease in signal was observed. Notably, a significant drop in luminescence occurred at the 1/64 milk dilution, followed by a gradual decline as the milk concentration increased.

Although luminescence remained detectable, it demonstrated an inverse relationship with the percentage of milk in the reaction. This decline in signal could potentially be attributed to either a detrimental effect of milk on yeast cell viability or interference caused by the milk matrix, obstructing accurate luminescence readings by the plate reader.

Figure 5 Line plot showing the variations in luminescence signal of mERα for 10 µM concentration of BPA, when incubated with various milk dilutions.

Figure 6 Line plot showing the variations in luminescence signal of mERα in BPA and milk.

pRET2-LexA-ERα biosensor vs. pRET2-LexA-mERα biosensor

Aim: The objective of this study was to compare the performance of mERα and ERα biosensors in detecting Bisphenol A (BPA).

Methodology: A 3-hour incubation assay was conducted using yeast cells at an optical density (OD) of 5. BPA was serially diluted seven times, starting from a concentration of 1M to 1μM, and distilled water was used as the negative control. Each well received 1 μL of the BPA dilutions, followed by 100 μL of the yeast cell suspension, resulting in a final BPA concentration in the reaction mixture ranging from 1 mM to 1 nM, with the negative control wells containing no BPA.

Previous concerns arose regarding the biosensor’s potential to generate background signals in milk due to the presence of estrogen, a known milk component, albeit in low concentrations.

Following the work by Rajasärkkä et al., 2011, the mutant mERα was hypothesized to exhibit both higher specificity to BPA and reduced sensitivity to its natural ligands. To investigate this, we performed a similar experiment using estradiol as the ligand to compare mERα and ERα responses. Estradiol is the most potent and biologically active form of estrogen. Thus, a lower response from mERα to estradiol would indicate a reduced likelihood of activation by estrogenic compounds present in milk.

Results: The experimental results demonstrated that mERα exhibited greater specificity towards BPA compared to ERα. Consistent with the findings of Rajasärkkä et al., 2011, mERα produced stronger signals in the presence of BPA. Additionally, mERα showed reduced activity in response to estradiol, suggesting its potential for more selective detection of BPA, even in the presence of estrogenic compounds in complex matrices like milk.

Figure 7 Line plots comparing the ability of mERα and ERα to detect BPA.

After comparing the mERα activation with BPA and Estradiol based on figure 8, we found that mERα shows lower signal for Estradiol as compared to BPA. Thus, we believe the system has potential to work in milk conditions without giving an excess background signal due to estrogen present in breast milk.

Figure 8 Line plots comparing the mERα activation with BPA and Estradiol.

On the other hand, Era showed higher signal in presence of estradiol than with BPA. Thus, supporting our decision of engineering mERα.

Figure 9 Line plots comparing the ERα activation with BPA and Estradiol.

When comparing the signals generated by ERα and mERα due to estradiol, we see mERα gives a higher signal in comparison. However, this should not be a problem. This effect nullifies because mERα gives a much higher signal in presence of BPA. Thus, we expect little or nearly no background luminescence due to Estrogen when mERα is tested in milk.

Figure 10 Line plots comparing the ERα and mERα activation with Estradiol.

pPOP6-LexA-ERα biosensor vs. pRET2-LexA-mERα biosensor - Promoter Optimization by proxy

Aim: Given that pRET2-mERα biosensor and pPOP6-ERα biosensor both exhibit significantly higher activity compared to pRET2-mERα, we sought to determine whether pRET2-mERα is more effective than pPOP6-ERα. Although time constraints prevented us from recloning our mutant construct, we hypothesized that if the pPOP6-ERα construct demonstrates superior strength, the mutant receptor could potentially perform even better under the POP6 promoter.

Results: The pPOP6-ERα biosensor demonstrated significantly higher potency against BPA compared to the mutant ERα construct with the RET2 promoter, as shown in Figure 11. These results indicate that the pPOP6-ERα biosensor outperforms the pRET2-mERα construct. In previous experiments, we concluded that mERα produces stronger luminescence than ERα, suggesting that the difference in performance observed here is likely due to the distinct promoters used.

Based on these findings, we hypothesize that the mutant ERα could exhibit even greater performance under the POP6 promoter. Therefore, we recommend future researchers prioritize the POP6 promoter over RET2 in similar experiments. Given more time, we would have recloned the mutant gene with the POP6 promoter to further test and validate this hypothesis.

Figure 11 Line plot showing the variations in luminescence signal of mERα for 1 µM concentration of Aroclor, when incubated with various milk dilutions.

Response of pRET2-LexA-mERα biosensor to PCB3

Aim: To determine whether mERα produces a detectable response to PCB3.

Methodology: The experiment was conducted using duplicate samples with an overnight incubation protocol.

Results: A quasi-linear response to PCB3 was observed within the luminescence range of 2000-5000 units, with minimal variation between the two replicates at lower concentrations (0-50 nM). This consistency suggests that the observed response in this range may accurately reflect the system's behavior. However, at higher concentrations, particularly at 5 µM, the response exhibited greater variability and, in some instances, dropped below the baseline. The cause of this anomaly is unclear. Based on the trendline, we recommend expanding the concentration range examined in future experiments to establish a more definitive dose-response curve.

Figure 12 Line plot showing the response to PCB3 of mERα.

Response of pRET2-LexA-mERα biosensor to PAH

Aim: To evaluate the response of the engineered ERα biosensor to benzanthracene.

Methodology: A 3-hour incubation assay was conducted using duplicate samples.

Results: the PRET2-mERα biosensor produced a strong signal in response to benzanthracene, though the resulting dose-response curve lacked consistency (Figure 13). While this trend is promising, the high variability observed between replicates prevents us from drawing definitive conclusions at this stage. Further investigation with additional replicates would be necessary to confirm these findings.

Figure 13 Line plot showing the response of mERα to different concentrations of PAH.

Response of pRET2-LexA-mERα biosensor to PAH in Milk

Aim: To test the effect of milk on mERα’s ability to detect PAH in milk

Results: From this experiment, we see that higher luminescence is given by cells incubated with water and highly diluted milk. As the milk ratio increased the signals dropped, figure 14. However, we see that unlike the results obtained with BPA, here the signals drop gradually. Based on the graph we see that there is almost 1/2 decrease in signal intensity between cells incubated with water and half fraction of milk.

Figure 14 Line plot showing the variations in luminescence signal of mERα for 2.5 µM concentration of PAH, when incubated with various milk dilutions.

Response of pRET2-LexA-mERα biosensor to Aroclor 1260

Aim: To establish a dose-response curve for Aroclor 1260 detection using the engineered ERα biosensor.

Methodology: A 3-hour incubation assay was performed in duplicate.

Results: The dynamic range of the biosensor response was observed between 7000 and 11,500 luminescence units. However, significant variability was noted across replicates. The biosensor does not appear to be highly sensitive to Aroclor 1260 within the tested concentration range, as indicated by the relatively consistent mean values between 1 µM and 100 µM. Due to the large error bars, definitive conclusions regarding sensitivity cannot be drawn, and further investigation is required to clarify these results.

Figure 15 Line plot exhibiting the signal given by mERα in response to different concentrations of Aroclor 1260.

Response of pRET2-LexA-mERα biosensor to Aroclor 1260 in Milk

Aim: To test the effect of milk on mERα’s ability to detect Aroclor 1260 in milk Result: From this experiment we see that the luminescence given by mERα decreases in a step-like trend with increasing milk fraction. The signal remains at the same level for water and 1/128 dilution and then decreases rapidly at 1/64 dilution. The same is seen to be repeated for dilution 1/64 and 1/32 after which there is again a sharp decrease.

From all the milk fraction experiments we conclude that the mERα biosensor behaves differently with each contaminant in milk conditions and the level of impact on the system due to the milk environment also varies.

Figure 16 Line plot showing the variations in luminescence signal of mERα for 1 µM concentration of Aroclor, when incubated with various milk dilutions.

Sequence and Features

References

1. Çiftçi S, Yalçın SS, Samur G. Bisphenol A Exposure in Exclusively Breastfed Infants and Lactating Women: An Observational Cross-sectional Study. J Clin Res Pediatr Endocrinol. 2021 Nov 25;13(4):375-383. doi: 10.4274/jcrpe.galenos.2020.2021.0305. Epub 2021 Mar 22. PMID: 33749218; PMCID: PMC8638632.

2. Park, Choa & Song, Heewon & Choi, Junyeong & Sim, Seunghye & Kojima, Hiroyuki & Park, Joonwoo & Iida, Mitsuru & Lee, Youngjoo. (2020). The mixture effects of bisphenol derivatives on estrogen receptor and androgen receptor. Environmental Pollution. 260. 114036. 10.1016/j.envpol.2020.114036.

3. Rajasärkkä, J., Hakkila, K. and Virta, M. (2011), Developing a compound-specific receptor for bisphenol a by directed evolution of human estrogen receptor ᆇ. Biotechnol. Bioeng., 108: 2526-2534. https://doi.org/10.1002/bit.23214 Zhou, T., Liang, Z. & Marchisio, M.A. Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol. Sci Rep 12, 21791 (2022). https://doi.org/10.1038/s41598-022-26195-x

4. Zhou, T., Liang, Z. & Marchisio, M.A. Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with β-estradiol. Sci Rep 12, 21791 (2022). https://doi.org/10.1038/s41598-022-26195-x