Difference between revisions of "Part:BBa K5477044"
 
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In this biosensor system, the pPOP6-LexA-ERα(LBD) receptor module responds to estrogenic compounds, such as estrogen or BPA. When these ligands bind to the ERα LBD, it activates the LexA DBD, allowing the fusion protein to bind to the Lex6Op operator sequences in the reporter module. This interaction activates the pLEU2 promoter, leading to the expression of NanoLuc. The resulting bioluminescent signal provides a quantitative measure of ligand binding to the receptor.
 
In this biosensor system, the pPOP6-LexA-ERα(LBD) receptor module responds to estrogenic compounds, such as estrogen or BPA. When these ligands bind to the ERα LBD, it activates the LexA DBD, allowing the fusion protein to bind to the Lex6Op operator sequences in the reporter module. This interaction activates the pLEU2 promoter, leading to the expression of NanoLuc. The resulting bioluminescent signal provides a quantitative measure of ligand binding to the receptor.
  
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<i>Why do we want to detect BPA in breast milk?</i>
 
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Detecting BPA (bisphenol A) in breast milk is important due to the potential health risks it poses to infants. BPA is an endocrine-disrupting chemical commonly found in plastics and can leach into food and liquids, including breast milk. Even at low levels, BPA can interfere with the hormonal systems of infants, potentially affecting their development, reproductive health, and neurological function. Infants are particularly vulnerable to BPA exposure because their bodies are still developing and they may not be able to effectively metabolize and eliminate the chemical. Monitoring BPA levels in breast milk helps ensure the safety and well-being of infants during this critical developmental period.
 
  
 
===Usage and Biology===
 
===Usage and Biology===
 +
 
In this biosensor system, a combination of receptor and reporter modules is used to detect the presence of estrogenic compounds like estrogen or bisphenol A (BPA) and provide a luminescent output using the NanoLuc luciferase reporter.
 
In this biosensor system, a combination of receptor and reporter modules is used to detect the presence of estrogenic compounds like estrogen or bisphenol A (BPA) and provide a luminescent output using the NanoLuc luciferase reporter.
 +
 +
https://static.igem.wiki/teams/5477/for-registry/correct-ones/era-w-cont-resized-800.png
 +
 +
This figure illustrates the LexA-ERα (LBD) biosensor's behavior in the absence of BPA or estrogen. 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.
 +
 +
https://static.igem.wiki/teams/5477/for-registry/correct-ones/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. Below is a figure of the whole device consisting of our composites.
 +
 +
 +
https://static.igem.wiki/teams/5477/for-registry/correct-ones/pop6-era-biosensor-resized-800-resized-500.png
  
 
<h2>Receptor Module</h2>
 
<h2>Receptor Module</h2>
−
1.pPOP6-LexA-ERα(LBD)[https://parts.igem.org/Part:BBa_K5477028 | BBa_K5477028]: The pPOP6 promoter drives the expression of a LexA-ERα(LBD) fusion protein, where the LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of Estrogen Receptor Alpha (ERα). The LexA-ERα(LBD) protein allows for the detection of estrogen-like ligands such as BPA. Upon binding to estrogen or BPA, the ERα LBD undergoes a conformational change that activates the LexA DBD. This enables the fusion protein to bind to the Lex6Op operator sequences present in the reporter module, thereby regulating gene expression downstream of these operators.
+
1.pPOP6-LexA-ERα(LBD)[https://parts.igem.org/Part:BBa_K5477028 BBa_K5477028]: The pPOP6 promoter drives the expression of a LexA-ERα(LBD) fusion protein, where the LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of Estrogen Receptor Alpha (ERα). The LexA-ERα(LBD) protein allows for the detection of estrogen-like ligands such as BPA. Upon binding to estrogen or BPA, the ERα LBD undergoes a conformational change that activates the LexA DBD. This enables the fusion protein to bind to the Lex6Op operator sequences present in the reporter module, thereby regulating gene expression downstream of these operators.
  
 
<h2>Reporter Module</h2>
 
<h2>Reporter Module</h2>
−
1. pLex6Op-pLEU2-NanoLuc[https://parts.igem.org/Part:BBa_K5477031 | BBa_K5477031]: The reporter module contains Lex6Op (six LexA operator sequences) that function as the binding sites for the LexA DBD in the receptor module. When the LexA-ERα(LBD) fusion protein is activated by ligand binding, it binds to these operator sequences, triggering the activity of the pLEU2 promoter. The pLEU2 promoter then drives the expression of the NanoLuc reporter gene.
+
1. pLex6Op-pLEU2-NanoLuc[https://parts.igem.org/Part:BBa_K5477031 BBa_K5477031]: The reporter module contains Lex6Op (six LexA operator sequences) that function as the binding sites for the LexA DBD in the receptor module. When the LexA-ERα(LBD) fusion protein is activated by ligand binding, it binds to these operator sequences, triggering the activity of the pLEU2 promoter. The pLEU2 promoter then drives the expression of the NanoLuc reporter gene.
  
  
 
===Results===
 
===Results===
 +
 +
 +
<h2>pRET2-LexA-ERα biosensor vs. pPOP6-LexA-ERα biosensor in response to BPA - Promoter optimization</h2>
 +
 +
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 4). 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>
 +
 +
Figure 4 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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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 5). 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.
 +
 +
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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>
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Figure 5 Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different Aroclor 1260 concentrations.
 +
 +
<h2> pPOP6-LexA-ERα biosensor vs. pRET2-LexA-mERα biosensor - Promoter Optimization by proxy </h2>
 +
 +
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 6. 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.
 +
 +
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/era-and-mera/pop6-vs-mera.jpg" width="400"></div></html>
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Figure 6 Line plot showing the variations in luminescence signal of mERα for 1 µM concentration of Aroclor, when incubated with various milk dilutions.
 +
  
 
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===References===
 
===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

Latest revision as of 07:13, 2 October 2024


Biosensor device II for detection of BPA

Summary

In this biosensor system, the pPOP6-LexA-ERα(LBD) receptor module responds to estrogenic compounds, such as estrogen or BPA. When these ligands bind to the ERα LBD, it activates the LexA DBD, allowing the fusion protein to bind to the Lex6Op operator sequences in the reporter module. This interaction activates the pLEU2 promoter, leading to the expression of NanoLuc. The resulting bioluminescent signal provides a quantitative measure of ligand binding to the receptor.


Usage and Biology

In this biosensor system, a combination of receptor and reporter modules is used to detect the presence of estrogenic compounds like estrogen or bisphenol A (BPA) and provide a luminescent output using the NanoLuc luciferase reporter.

era-w-cont-resized-800.png

This figure illustrates the LexA-ERα (LBD) biosensor's behavior in the absence of BPA or estrogen. 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. Below is a figure of the whole device consisting of our composites.


pop6-era-biosensor-resized-800-resized-500.png

Receptor Module

1.pPOP6-LexA-ERα(LBD)BBa_K5477028: The pPOP6 promoter drives the expression of a LexA-ERα(LBD) fusion protein, where the LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of Estrogen Receptor Alpha (ERα). The LexA-ERα(LBD) protein allows for the detection of estrogen-like ligands such as BPA. Upon binding to estrogen or BPA, the ERα LBD undergoes a conformational change that activates the LexA DBD. This enables the fusion protein to bind to the Lex6Op operator sequences present in the reporter module, thereby regulating gene expression downstream of these operators.

Reporter Module

1. pLex6Op-pLEU2-NanoLucBBa_K5477031: The reporter module contains Lex6Op (six LexA operator sequences) that function as the binding sites for the LexA DBD in the receptor module. When the LexA-ERα(LBD) fusion protein is activated by ligand binding, it binds to these operator sequences, triggering the activity of the pLEU2 promoter. The pLEU2 promoter then drives the expression of the NanoLuc reporter gene.


Results

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 4). 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 4 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 5). 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 5 Line plot showing the variations in luminescence signal of ERα under pRET2 and pPOP6 promoters when incubated with different Aroclor 1260 concentrations.

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 6. 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 6 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


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 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
    Illegal BamHI site found at 73
    Illegal BamHI site found at 390
  • 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
  • 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