Difference between revisions of "Part:BBa K5477043"
 
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Methodology: An overnight incubation was conducted with cells at an OD of 0.5.
 
Methodology: An overnight incubation was conducted with cells at an OD of 0.5.
  
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Results: The pRET2-ERα construct demonstrated peak sensitivity at approximately 1 µM of BPA, with no observable plateau, preventing an estimation of the EC50 (Figure X). The dynamic range for this construct was found to be between 1700 and 7400 luminescence units.
+
Results: The pRET2-ERα construct demonstrated peak sensitivity at approximately 1 µM of BPA, with no observable plateau, preventing an estimation of the EC50 (Figure 4). The dynamic range for this construct was found to be between 1700 and 7400 luminescence units.
  
 
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/ret2-era-response-bpa.png" width="400"></div></html>
  
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Figure X Line plot showing the response to BPA by the pRET2-ERα device.
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Figure 4 Line plot showing the response to BPA by the pRET2-ERα device.
  
 
<h2>Response of pRET2-LexA-ERα biosensor to BPA in Milk</h2>
 
<h2>Response of pRET2-LexA-ERα biosensor to BPA in Milk</h2>
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/era-bpa-milk.png" width="400"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/era-bpa-milk.png" width="400"></div></html>
  
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Figure X Line plot showing the variations in luminescence signal of ERα when incubated with different BPA concentrations.
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Figure 5 Line plot showing the variations in luminescence signal of ERα when incubated with different BPA concentrations.
  
  
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Figure 7 Line plots comparing the ability of mERα and ERα to detect BPA.
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Figure 6 Line plots comparing the ability of mERα and ERα to detect BPA.
  
  
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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.
+
After comparing the mERα activation with BPA and Estradiol based on figure 7, 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.
  
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/bpa-mera-estradiol-bpa.png" width="400"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/bpa-mera-estradiol-bpa.png" width="400"></div></html>
  
  
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Figure 8 Line plots comparing the mERα activation with BPA and Estradiol.
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Figure 7 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.
 
On the other hand, Era showed higher signal in presence of estradiol than with BPA. Thus, supporting our decision of engineering mERα.
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/bpa-era-estradiol-bpa.png" width="400"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/bpa-era-estradiol-bpa.png" width="400"></div></html>
  
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Figure 9 Line plots comparing the ERα activation with BPA and Estradiol.
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Figure 8 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.
 
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.
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Figure 10 Line plots comparing the ERα and mERα activation with Estradiol.
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Figure 9 Line plots comparing the ERα and mERα activation with Estradiol.
  
  
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Results: Our findings indicate that the POP6 promoter produces higher luminescence signals compared to RET2 when paired with ERα for the detection of BPA (Figure X). However, the signal intensity did not show a strong dependence on the concentration of BPA added. Based on these results, we suggest that ERα may not be the most optimal system for this application.
+
Results: Our findings indicate that the POP6 promoter produces higher luminescence signals compared to RET2 when paired with ERα for the detection of BPA (Figure 9). However, the signal intensity did not show a strong dependence on the concentration of BPA added. Based on these results, we suggest that ERα may not be the most optimal system for this application.
  
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/ret2-pop6-era-bpa.png" width="400"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/ret2-pop6-era-bpa.png" width="400"></div></html>
  
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Figure X Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different BPA concentrations.
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Figure 10 Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different BPA concentrations.
  
  
−
We also observed that the POP6 promoter generates higher luminescence signals compared to pRET2 when paired with ERα for the detection of Aroclor 1260 (Figure X). This effect of the POP6 promoter is consistent across both compounds, BPA and Aroclor 1260.
+
We also observed that the POP6 promoter generates higher luminescence signals compared to pRET2 when paired with ERα for the detection of Aroclor 1260 (Figure 11). This effect of the POP6 promoter is consistent across both compounds, BPA and Aroclor 1260.
  
 
In the case of Aroclor 1260, the POP6-ERα system displayed a gradual increase in signal intensity corresponding to increasing concentrations of the compound.
 
In the case of Aroclor 1260, the POP6-ERα system displayed a gradual increase in signal intensity corresponding to increasing concentrations of the compound.
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/ret2-pop6-era-aroclor.png" width="400"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/ret2-pop6-era-aroclor.png" width="400"></div></html>
  
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Figure X Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different Aroclor 1260 concentrations.
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Figure 11 Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different Aroclor 1260 concentrations.
  
  
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/ret2-era-response-pah.png" width="400"></div></html>
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Figure 12 pRET2-LexA-ERα biosensor incubation in PAH
  
  

Latest revision as of 07:17, 2 October 2024


Biosensor device I for detection of BPA

Summary

In this system, the pRET2-LexA-ERα(LBD) receptor module responds to the presence of estrogen or estrogen-like compounds, in this case BPA. When these ligands bind to the ERα LBD, it induces a transcriptional response by allowing the LexA-ERα chimeric activator to bind to the Lex6Op sequences in the pLex6Op-pLEU2-NanoLuc reporter module. This activates the pLEU2 promoter, leading to the expression of NanoLuc, which produces a luminescent signal (4).

Usage and Biology

In this biosensor device, the combination of receptor and reporter modules is designed to monitor the activity of Estrogen Receptor Alpha (ERα) in response to estrogenic compounds, such as estrogen or bisphenol A (BPA), and generate a luminescent signal through the expression of NanoLuc.

era-w-cont-resized-800.png

This figure illustrates the LexA-ERα (LBD) biosensor's behavior in the absence of BPA. 1) The LexA-ERα (LBD) complex is expressed, but without BPA present, it remains bound to HSP90 in the cytoplasm. 2) As a result, the LexA-ERα (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.

era-cont-resized-800.png

This figure shows the LexA-ERα (LBD) biosensor's response when Bisphenol A (BPA) or estrogen, the native ligand, is present. 1) The LexA-ERα (LBD) complex is expressed in the cytoplasm. 2) Upon binding to BPA, the LexA-ERα (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.


Receptor Module

1.pRET2-LexA-ERα(LBD) BBa_K5477027: This receptor module utilizes the pRET2 promoter to drive the expression of a fusion protein composed of the LexA DNA-binding domain (DBD) and the ligand-binding domain (LBD) of Estrogen Receptor Alpha (ERα). The LexA-ERα(LBD) fusion allows the system to detect estrogen or estrogen-like molecules such as BPA. When BPA binds to the ERα LBD, it induces a conformational change in the receptor, activating the LexA DBD to bind to specific LexA operator sequences (Lex6Op).

Reporter Module

1. pLex6Op-pLEU2-NanoLuc BBa_K5477031: The reporter module contains Lex6Op, a cassette of six LexA operator sequences that serve as the binding sites for the LexA DBD in the receptor module. When LexA-ERα(LBD) is activated by estrogen binding, the LexA DBD binds to these operator sequences, activating the pLEU2 promoter. The pLEU2 promoter then drives the expression of the NanoLuc reporter gene. NanoLuc is a highly sensitive luciferase enzyme that produces bioluminescence when its substrate is added, allowing the system to output a quantifiable luminescent signal that directly correlates with the presence of estrogen or BPA.


Results

Response of pRET2-LexA-ERα biosensor to BPA

Aim: To evaluate the response of the RET2+ERα construct to BPA.

Methodology: An overnight incubation was conducted with cells at an OD of 0.5.

Results: The pRET2-ERα construct demonstrated peak sensitivity at approximately 1 µM of BPA, with no observable plateau, preventing an estimation of the EC50 (Figure 4). The dynamic range for this construct was found to be between 1700 and 7400 luminescence units.

Figure 4 Line plot showing the response to BPA by the pRET2-ERα device.

Response of pRET2-LexA-ERα biosensor to BPA in Milk

Aim: To evaluate the performance of the RET2-ERα-based biosensor in detecting BPA in milk.

Methodology: A 3-hour incubation assay was conducted using yeast cells with an optical density (OD) of 5. Bisphenol A (BPA) was serially diluted seven times, from 1M to 1μM, while water was used in control wells without BPA to serve as a negative control. A volume of 1μL from each BPA dilution was added to respective wells containing 100μL of yeast cells, resulting in a final BPA concentration range of 1mM to 1nM, with the control having no BPA. Additionally, 50μL of 3.5% fat milk was added to each well to test the sensor's performance in the presence of milk.

Results: The presence of milk led to significantly reduced ERα luminescence signals, with readings in the range of 200-300 RLU. However, an upward trend in signal intensity was observed with increasing BPA concentrations, indicating that while luminescence is diminished in the presence of milk, the system still demonstrates a detectable response to varying BPA levels.


Figure 5 Line plot showing the variations in luminescence signal of ERα when incubated with different BPA concentrations.


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


After comparing the mERα activation with BPA and Estradiol based on figure 7, 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 7 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 8 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 9 Line plots comparing the ERα and mERα activation with Estradiol.


pRET2-LexA-ERα biosensor vs. pPOP6-LexA-ERα biosensor in response to BPA - Promoter optimization

Aim: To evaluate the variations in luminescence signal of ERα under the RET2 and POP6 promoters when exposed to different concentrations of BPA and Aroclor 1260.

Methodology: A total of 12 columns were tested: 6 for the RET2 promoter and 6 for the POP6 promoter, with 6 columns dedicated to BPA and 6 to Aroclor 1260. The assay was conducted using an overnight incubation protocol.


Results: Our findings indicate that the POP6 promoter produces higher luminescence signals compared to RET2 when paired with ERα for the detection of BPA (Figure 9). However, the signal intensity did not show a strong dependence on the concentration of BPA added. Based on these results, we suggest that ERα may not be the most optimal system for this application.

Figure 10 Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different BPA concentrations.


We also observed that the POP6 promoter generates higher luminescence signals compared to pRET2 when paired with ERα for the detection of Aroclor 1260 (Figure 11). This effect of the POP6 promoter is consistent across both compounds, BPA and Aroclor 1260.

In the case of Aroclor 1260, the POP6-ERα system displayed a gradual increase in signal intensity corresponding to increasing concentrations of the compound.


Figure 11 Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different Aroclor 1260 concentrations.


pRET2-LexA-ERα biosensor and its response to PAH

Aim: To determine whether ERα exhibits a detectable response to PAH.

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

Results: The data suggest that extending the concentration range below 2.5 nM may be necessary, as the response appears to plateau at higher concentrations. Further testing at lower concentrations could provide a more comprehensive understanding of the biosensor's response to PAH.

Figure 12 pRET2-LexA-ERα biosensor incubation in PAH


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1606
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 182
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1740
    Illegal BglII site found at 2751
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1606
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1606
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal AgeI site found at 37
  • 1000
    COMPATIBLE WITH RFC[1000]


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