Part:BBa_K5477048
Testing of the SUPERMOM Concept
Summary
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.
1.) Ligand Detection: The receptor module (pRET2-LexA-mERα(LBD)) contains a mutant Estrogen Receptor Alpha (mERα), specifically modified to detect and bind to pharmaceutical and similar ligands. Once pharmaceutical binds, the receptor undergoes a conformational change, enabling transcriptional activation of the downstream modules.
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.
3.) Detoxification: Concurrently, the Lex6Op sequences also control the detoxification module (CYP3A4-MYC-pGAL1/10-POR), which is responsible for metabolizing pharmaceutical 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.
Usage and Biology
Usage and Biology
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 pharmaceuticals. The integration of these modules allows for ligand detection, signal transduction, and metabolic detoxification all within a single yeast-based system.
Why Target Pharmaceuticals in Breast Milk?
Detecting pharmaceuticals in breast milk is crucial due to the potential health risks they pose to infants. Many medications, such as painkillers, antibiotics, antidepressants, and hormonal drugs, can pass from a mother's bloodstream into her breast milk. Infants are particularly vulnerable to these substances because their bodies are still developing, and they may have limited ability to metabolize or eliminate the drugs efficiently. Prolonged exposure to even low levels of pharmaceuticals can impact an infant’s growth, immune function, neurological development, and overall health. Monitoring pharmaceutical levels in breast milk is essential to safeguard infant well-being during this critical developmental period and to ensure that breastfeeding remains a safe and healthy practice.
This figure illustrates the SUPERMOM biosensor in the absence of a pharmaceutical. 1) In the absence of pharmaceutical, 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 pharmaceutical, no detoxification process occurs.
Below is a figure of the whole device consisting of our composites.
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 pharmaceuticals and related compounds and in this case 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-pGAL1/10-POR | BBa_K5477039: The detoxification module incorporates the cytochrome P450 enzyme CYP3A4, which plays a key role in the oxidative metabolism of various EDCs, pharmaceuticals and including BPA. CYP3A4 is regulated by the pGAL1/10 bidirectional 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 pharmaceuticals, converting it into more hydrophilic and excretable metabolites, reducing its harmful effects.
Sequence and Features
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1740
Illegal BglII site found at 2751
Illegal XhoI site found at 4823 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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