Part:BBa_K5477046
SUPERMOM: Dual-Function Biosensor for BPA Detection and Detoxification
Summary
This system provides the ability to detect and detoxify :
1. Ligand Detection: The mutant ERα receptor (mERα) in the pRET2-LexA-mERα(LBD) receptor module is specifically tailored to detect BPA, binding the ligand and triggering transcriptional activation of the downstream modules.
2. Signal Transduction: Upon BPA binding, the LexA-mERα(LBD) protein activates the pLex6Op-pLEU2-NanoLuc reporter module, producing a quantifiable bioluminescent signal. This allows for the real-time detection and measurement of BPA or similar compounds.
3. Detoxification: Simultaneously, the Lex6Op sequences in the detoxification module activate the expression of UDPD and UGT2B15, which work together to detoxify BPA through glucuronidation. UDPD supplies UDP-glucuronic acid, and UGT2B15 attaches it to BPA, making it water-soluble and ready for excretion from the cell.
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 bisphenol A (BPA). The integration of these modules allows for ligand detection, signal transduction, and metabolic detoxification all within a single yeast-based system.
Why BPA?
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.
This figure illustrates the SUPERMOM biosensor in the absence of 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 (glucuronidation) occurs, and thus no BPA Glucuronide is produced.
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 UDPD and UGT2B15. 4) BPA undergoes glucuronidation, a detoxification process facilitated by UGT2B15 and UDPD enzymes, which convert BPA into BPA Glucuronide. 5) BPA Glucuronide is produced as the detoxified form of BPA, which is then secreted via urine, ensuring removal from the system.
Below is a figure of the whole device consisting of the composites.
Receptor Module
pRET2-LexA-mERα(LBD) | BBa_K5477029: The receptor module utilizes the pRET2 promoter to drive the expression of a LexA-mERα(LBD) fusion protein, where the LexA DNA-binding domain (DBD) is fused to the ligand-binding domain (LBD) of a mutant Estrogen Receptor Alpha (mERα). The mutant ERα LBD is engineered to have an altered binding specificity, allowing it to better detect BPA or other endocrine-disrupting chemicals (EDCs). Upon binding BPA or related compounds, the LexA-mERα(LBD) protein undergoes a conformational change that enables the LexA DBD to bind to Lex6Op operator sequences in the downstream reporter and detoxification modules, thereby regulating their expression.
Reporter Module
pLex6Op-pLEU2-NanoLuc | BBa_K5477031: This reporter module contains Lex6Op operator sequences, which act as binding sites for the activated LexA-mERα(LBD) fusion protein. When the LexA DBD binds to the Lex6Op sequences, it activates the pLEU2 promoter, driving the expression of the NanoLuc luciferase gene. NanoLuc produces a bioluminescent signal in the presence of its substrate, providing a real-time and quantifiable measure of BPA or ligand detection. The intensity of the bioluminescent output is directly proportional to the concentration of the ligand binding to the mERα receptor.
Detox Module
UDPD-pPDC1-Lex6Op-pENO1-UGT2B15 | BBa_K5477040: The detoxification system uses two enzymes, UDPD and UGT2B15, controlled by two promoters (pPDC1 and pENO1) arranged in opposite orientations, with regulation provided by the Lex6Op operator sequences. Upon activation by the LexA-mERα(LBD), UDPD (UDP-glucose dehydrogenase) produces UDP-glucuronic acid, the critical substrate for glucuronidation reactions and UGT2B15 (UDP-glucuronosyltransferase 2B15) utilizes this substrate to conjugate glucuronic acid to BPA or other estrogenic compounds. This glucuronidation process makes these compounds more water-soluble, facilitating their excretion from the yeast cell and detoxifying the harmful effects of BPA or related EDCs.
Results
Measuring the luminescence of SUPERMOM
Objective: To validate the activity of the luciferase reporter module from the pRET2-mERα with UGT2B15-UDPD comprising the SUPERMOM system, which serves as a proxy for signal induction in the biosensor system.
Methodology: A luminescent bioassay was conducted with two incubation times: one overnight (ON)and one 3-hour incubation, see Figure 4. Two tests were performed to verify that the unexpected peak at the highest concentrations was not caused by human error or incubation time variability. DMSO was used as a control at a consistent concentration of 1 µL per well.
Results: A basal luminescent signal was observed across all conditions, including when only DMSO (1 µL) or 0 BPA was added. Although a higher signal was detected at the highest concentration of BPA, this effect was mirrored in the DMSO control (Figure x). We hypothesize that the persistent low signal may be due to leaky expression of the detoxification enzyme, which reduces BPA levels, resulting in a feedback loop where the system maintains a constant signal. The spike observed at the 10 mM BPA concentration is yet to be fully explained. It may represent the beginning of the dynamic range, but further tests at higher concentrations are required to support this hypothesis. This spike was not corroborated by the DMSO control.
At this stage, we cannot conclusively determine whether the SUPERMOM system specifically detects BPA. Further experimentation is needed to confirm its sensitivity and response dynamics.
Figure 4: A and B 3 hour and overnight incubation of pRET2-mERα SUPERMOM concept.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6656
Illegal XbaI site found at 1606
Illegal PstI site found at 1790
Illegal PstI site found at 1961
Illegal PstI site found at 3825 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6656
Illegal NheI site found at 182
Illegal PstI site found at 1790
Illegal PstI site found at 1961
Illegal PstI site found at 3825 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6656
Illegal BglII site found at 1740
Illegal BglII site found at 2751
Illegal BglII site found at 4405
Illegal BamHI site found at 6476
Illegal BamHI site found at 6770 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6656
Illegal XbaI site found at 1606
Illegal PstI site found at 1790
Illegal PstI site found at 1961
Illegal PstI site found at 3825 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6656
Illegal XbaI site found at 1606
Illegal PstI site found at 1790
Illegal PstI site found at 1961
Illegal PstI site found at 3825
Illegal AgeI site found at 37 - 1000COMPATIBLE WITH RFC[1000]
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