Difference between revisions of "Part:BBa K5477048"
 
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===Summary===
 
===Summary===
  
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This system provides the ability to detect and detoxify endocrine-disrupting chemicals (EDCs), with a particular focus on BPA and related compounds, by utilizing a sophisticated combination of ligand detection, signal transduction, and enzymatic detoxification.
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This device allows testing of the SUPERMOM concept, if detection of BPA, can induce both signal output, and expression of detoxification enzyme (CYP3A4-myc). Due to the use of CYP3A4-myc in this device, it allows for testing of presence with western blot.
  
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1.) Ligand Detection: The receptor module (pRET2-LexA-mERα(LBD)) contains a mutant Estrogen Receptor Alpha (mERα), specifically modified to detect and bind to BPA and similar ligands. Once BPA binds, the receptor undergoes a conformational change, enabling transcriptional activation of the downstream modules.
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1.) Ligand Detection: The receptor module (pRET2-LexA-mERα(LBD)) contains a mutant Estrogen Receptor Alpha (mERα), engineered to detect and bind to BPA. Once BPA binds, the receptor undergoes a conformational change, enabling transcriptional activation of the downstream modules (6) (7).
  
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2.) Signal Transduction: The activated LexA-mERα(LBD) fusion protein then binds to the Lex6Op operator sequences in the reporter module (Lex6Op-pLEU2-NanoLuc), triggering the expression of NanoLuc luciferase, which produces a bioluminescent signal. This signal provides a quantitative and real-time indication of ligand binding and detection.
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2.) Signal Transduction: The activated LexA-mERα(LBD) fusion protein then binds to the Lex6Op operator sequences in the reporter module (Lex6Op-pLEU2-NanoLuc), inducing the expression of NanoLuc luciferase, which produces a bioluminescent signal (7).  
  
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3.) Detoxification: Concurrently, the Lex6Op sequences also control the detoxification module (CYP3A4-MYC-pGAL1/10-POR), which is responsible for metabolizing BPA or related EDCs. CYP3A4, in conjunction with POR (NADPH-cytochrome P450 reductase), functions to oxidize and detoxify the harmful compounds, converting them into less toxic or more readily excretable metabolites.
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3.) Detoxification: The Lex6Op sequences also control the detoxification module (CYP3A4-MYC-pGAL1/10-POR).
  
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/devices/supermoms/supermom2.png" width="300"></div></html>
  
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===Usage and Biology===
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Figure 1: SUPERMOM concept testing
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In this biosensor system, the combination of a mutant ERα receptor module, a reporter module, and a detoxification system provides a robust setup for detecting and detoxifying estrogen-like compounds, particularly BPA. The integration of these modules allows for ligand detection, signal transduction, and metabolic detoxification all within a single yeast-based system.
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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. Below is a figure of the whole device consisting of the following composites.
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===Usage===
  
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https://static.igem.wiki/teams/5477/for-registry/correct-ones/supermom-w-cont-resized-800.png
 
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This figure illustrates the SUPERMOM biosensor in the absence of a BPA. 1) In the absence of BPA, the LexA-mERα (LBD) complex remains bound to HSP90 in the cytoplasm, preventing its translocation into the nucleus. 2) No signal is generated as the complex does not reach the nucleus to bind with Lex60p. 3) Additionally, without the presence of BPA, no detoxification process occurs.
 
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https://static.igem.wiki/teams/5477/for-registry/correct-ones/supermom-cyp3a4.png
 
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This figure shows the SUPERMOM dual-function biosensor in the presence of BPA, illustrating its role in both detection and detoxification. 1) The LexA-mERα (LBD) complex is expressed and 2) translocated into the nucleus. 3) Due to the presence of BPA, binding to the Lex60p operator sequence, it triggers the signal output and the expression of POR and CYP3A4-MYC. 4) The products formed from CYP3A4’s oxidation of BPA are intermediates that are more easily metabolized (1). These compounds are further modified and prepared for elimination from the cell. 5) The detoxified and metabolized BPA is now in a form that can be excreted from the cell, often through secretion mechanisms like urine.
 
  
 
Below is a figure of the whole device consisting of these composites.  
 
Below is a figure of the whole device consisting of these composites.  
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<h2>Receptor Module </h2>
 
<h2>Receptor Module </h2>
  
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pRET2-LexA-mERα(LBD) [https://parts.igem.org/Part:BBa_K5477029 | BBa_K5477029]: This receptor module includes the pRET2 promoter, driving the expression of a LexA-mERα(LBD) fusion protein. The LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of a mutant Estrogen Receptor Alpha (mERα), which is engineered to exhibit high specificity towards BPA. Upon ligand binding, the LexA-mERα(LBD) fusion protein undergoes a conformational shift, enabling it to bind to Lex6Op sequences in the reporter and detox modules, thus initiating the transcriptional response.
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pRET2-LexA-mERα(LBD) [https://parts.igem.org/Part:BBa_K5477029 BBa_K5477029]: This receptor module includes the pRET2 promoter, driving the expression of a LexA-mERα(LBD) fusion protein. The LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of a mutant Estrogen Receptor Alpha (mERα), which is engineered to exhibit high specificity towards BPA. Upon ligand binding, the LexA-mERα(LBD) fusion protein undergoes a conformational shift, enabling it to bind to Lex6Op sequences in the reporter and detox modules, thus initiating the transcriptional response.
  
 
<h2>Reporter Module </h2>
 
<h2>Reporter Module </h2>
  
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Lex6Op-pLEU2-NanoLuc [https://parts.igem.org/Part:BBa_K5477031 | BBa_K5477031]: The reporter module contains Lex6Op operator sequences, which serve as binding sites for the activated LexA-mERα(LBD) protein. Binding of LexA to these sequences activates the pLEU2 promoter, leading to the expression of NanoLuc luciferase. NanoLuc, a highly efficient luciferase, emits bioluminescence in the presence of its substrate. The intensity of this signal correlates directly with the concentration of the ligand (BPA or similar compounds) bound to the mERα receptor, enabling real-time detection.
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Lex6Op-pLEU2-NanoLuc [https://parts.igem.org/Part:BBa_K5477031 BBa_K5477031]: The reporter module contains Lex6Op operator sequences, which serve as binding sites for the activated LexA-mERα(LBD) protein. Binding of LexA to these sequences activates the pLEU2 promoter, leading to the expression of NanoLuc luciferase. NanoLuc, a highly efficient luciferase, emits bioluminescence in the presence of its substrate. The intensity of this signal correlates directly with the concentration of the ligand (BPA or similar compounds) bound to the mERα receptor, enabling real-time detection.
  
 
<h2>Detoxification Module</h2>
 
<h2>Detoxification Module</h2>
  
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CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR [https://parts.igem.org/Part:BBa_K5477032 | BBa_K5477032]: The detoxification module incorporates the cytochrome P450 enzyme CYP3A4, which plays a key role in the oxidative metabolism of various EDCs including BPA. CYP3A4 is regulated by the pPDC1-Lex6Op-pENO1 bidirectional synthetic promoter and is fused with a MYC tag for detection. The co-expression of NADPH-cytochrome P450 reductase (POR), driven by the same promoter, ensures efficient electron transfer to CYP3A4, facilitating its catalytic activity. This module enables the oxidation and detoxification of BPA, converting it into more hydrophilic and excretable metabolites, reducing its harmful effects.
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CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR [https://parts.igem.org/Part:BBa_K5477032 BBa_K5477032]: The detoxification module incorporates the cytochrome P450 enzyme CYP3A4, which plays a role in phase 1 oxidative metabolism. CYP3A4 is regulated by the pPDC1-Lex6Op-pENO1 bidirectional synthetic promoter and is fused with a MYC tag for detection of presence. The co-expression of NADPH-cytochrome P450 reductase (POR), driven by the same promoter, ensures efficient electron transfer to CYP3A4, facilitating its catalytic activity. This module enables the oxidation and detoxification of BPA, converting it into more hydrophilic and excretable metabolites, reducing its harmful effects.
  
  
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/sds.png" width="300"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/sds.png" width="300"></div></html>
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Figure 4: SDS page with all of the protein content of the tested yeasts induced with BPA or DMSO.
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<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/westernblot.png" width="300"></div></html>
 
<html><div style="text-align: center;"><img src="https://static.igem.wiki/teams/5477/for-registry/detox/westernblot.png" width="300"></div></html>
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Figure 5: Western Blot for the SUPERMOM system
  
 
<!-- Add more about the biology of this part here
 
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===References===
 
===References===
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1. Nakamura, Shigeo & Tezuka, Yoshito & Ushiyama, Atsuko & Kawashima, Chiaki & Kitagawara, Yumina & Takahashi, Kyoko & Ohta, Shigeru & Mashino, Tadahiko. (2011). Ipso substitution of bisphenol A catalyzed by microsomal cytochrome P450 and enhancement of estrogenic activity. Toxicology letters. 203. 92-5. 10.1016/j.toxlet.2011.03.010.
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1. Bardal SK, Waechter JE, Martin DS. Chapter 6 - Pharmacogenetics. In: Bardal SK, Waechter JE, Martin DS, editors. Applied Pharmacology [Internet]. Philadelphia: W.B. Saunders; 2011. p. 53–8. Available from: https://www.sciencedirect.com/science/article/pii/B9781437703108000063
 +
 
 +
2. Guengerich, F.. (2008). Cytochrome P450 and Chemical Toxicology. Chemical research in toxicology. 21. 70-83. 10.1021/tx700079z.
 +
 
 +
3. Klyushova LS, Perepechaeva ML, Grishanova AY. The Role of CYP3A in Health and Disease. Biomedicines. 2022 Oct 24;10(11):2686. doi: 10.3390/biomedicines10112686. PMID: 36359206; PMCID: PMC9687714.
 +
 
 +
4. Lynch T, Price A. The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am Fam Physician. 2007;76(3):391-396.
 +
 
 +
5. Pandey AV, Flück CE. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther. 2013;138(2):229-254. doi:10.1016/j.pharmthera.2013.01.010  
 +
 
 +
6. 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
 +
 
 +
7. 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 08:06, 2 October 2024


Testing of the SUPERMOM Concept

Summary

This device allows testing of the SUPERMOM concept, if detection of BPA, can induce both signal output, and expression of detoxification enzyme (CYP3A4-myc). Due to the use of CYP3A4-myc in this device, it allows for testing of presence with western blot.

1.) Ligand Detection: The receptor module (pRET2-LexA-mERα(LBD)) contains a mutant Estrogen Receptor Alpha (mERα), engineered to detect and bind to BPA. Once BPA binds, the receptor undergoes a conformational change, enabling transcriptional activation of the downstream modules (6) (7).

2.) Signal Transduction: The activated LexA-mERα(LBD) fusion protein then binds to the Lex6Op operator sequences in the reporter module (Lex6Op-pLEU2-NanoLuc), inducing the expression of NanoLuc luciferase, which produces a bioluminescent signal (7).

3.) Detoxification: The Lex6Op sequences also control the detoxification module (CYP3A4-MYC-pGAL1/10-POR).

Figure 1: SUPERMOM concept testing

Usage

Below is a figure of the whole device consisting of these composites.

supermommy.png


Receptor Module

pRET2-LexA-mERα(LBD) BBa_K5477029: This receptor module includes the pRET2 promoter, driving the expression of a LexA-mERα(LBD) fusion protein. The LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of a mutant Estrogen Receptor Alpha (mERα), which is engineered to exhibit high specificity towards BPA. Upon ligand binding, the LexA-mERα(LBD) fusion protein undergoes a conformational shift, enabling it to bind to Lex6Op sequences in the reporter and detox modules, thus initiating the transcriptional response.

Reporter Module

Lex6Op-pLEU2-NanoLuc BBa_K5477031: The reporter module contains Lex6Op operator sequences, which serve as binding sites for the activated LexA-mERα(LBD) protein. Binding of LexA to these sequences activates the pLEU2 promoter, leading to the expression of NanoLuc luciferase. NanoLuc, a highly efficient luciferase, emits bioluminescence in the presence of its substrate. The intensity of this signal correlates directly with the concentration of the ligand (BPA or similar compounds) bound to the mERα receptor, enabling real-time detection.

Detoxification Module

CYP3A4-MYC-pPDC1-Lex6Op-pENO1-POR BBa_K5477032: The detoxification module incorporates the cytochrome P450 enzyme CYP3A4, which plays a role in phase 1 oxidative metabolism. CYP3A4 is regulated by the pPDC1-Lex6Op-pENO1 bidirectional synthetic promoter and is fused with a MYC tag for detection of presence. The co-expression of NADPH-cytochrome P450 reductase (POR), driven by the same promoter, ensures efficient electron transfer to CYP3A4, facilitating its catalytic activity. This module enables the oxidation and detoxification of BPA, converting it into more hydrophilic and excretable metabolites, reducing its harmful effects.


Results

Objective: To verify that the enzyme is being produced in response to the contaminant, bisphenol A (BPA).

Methodology: An SDS-PAGE and Western blot were performed according to standard protocol. BPA treatment was applied alongside a negative control using DMSO. Two positive controls were also included, which lacked the biosensor but were induced with galactose. However, due to human error—specifically, a selective amino acid was added 3 hours after the incubation began—the positive control results are unreliable.

Results: The results showed non-specific bands. In the SDS-PAGE (Figure 4), no significant difference was observed between the DMSO-treated and BPA-induced samples. Similarly, the Western blot (Figure 5) confirmed the lack of a clear result, displaying multiple bands across the gel. Additional detoxification data should be incorporated here for further analysis.


Presence of the detoxification module CYP3A4-MYC

Figure 4: SDS page with all of the protein content of the tested yeasts induced with BPA or DMSO.


Figure 5: Western Blot for the SUPERMOM system

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1606
    Illegal XbaI site found at 4766
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 182
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1740
    Illegal BglII site found at 2751
    Illegal XhoI site found at 4823
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1606
    Illegal XbaI site found at 4766
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1606
    Illegal XbaI site found at 4766
    Illegal PstI site found at 1790
    Illegal PstI site found at 1961
    Illegal PstI site found at 5712
    Illegal PstI site found at 5909
    Illegal PstI site found at 6293
    Illegal PstI site found at 6733
    Illegal PstI site found at 6793
    Illegal NgoMIV site found at 6115
    Illegal NgoMIV site found at 6234
    Illegal AgeI site found at 37
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Bardal SK, Waechter JE, Martin DS. Chapter 6 - Pharmacogenetics. In: Bardal SK, Waechter JE, Martin DS, editors. Applied Pharmacology [Internet]. Philadelphia: W.B. Saunders; 2011. p. 53–8. Available from: https://www.sciencedirect.com/science/article/pii/B9781437703108000063

2. Guengerich, F.. (2008). Cytochrome P450 and Chemical Toxicology. Chemical research in toxicology. 21. 70-83. 10.1021/tx700079z.

3. Klyushova LS, Perepechaeva ML, Grishanova AY. The Role of CYP3A in Health and Disease. Biomedicines. 2022 Oct 24;10(11):2686. doi: 10.3390/biomedicines10112686. PMID: 36359206; PMCID: PMC9687714.

4. Lynch T, Price A. The effect of cytochrome P450 metabolism on drug response, interactions, and adverse effects. Am Fam Physician. 2007;76(3):391-396.

5. Pandey AV, Flück CE. NADPH P450 oxidoreductase: structure, function, and pathology of diseases. Pharmacol Ther. 2013;138(2):229-254. doi:10.1016/j.pharmthera.2013.01.010

6. 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

7. 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