Part:BBa_K5477043

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 triggers 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. The intensity of the luminescence is proportional to the concentration of the estrogenic compound in the environment, providing a measurable output of receptor activation.

Usage and Biology

In this biosensor system, 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.

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.

This figure shows the LexA-ERα (LBD) biosensor's response when Bisphenol A (BPA) 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.

Why do we want to detect BPA in breast milk?

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 our composites.

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

Sequence and Features

Assembly Compatibility: