Difference between revisions of "Part:BBa K5477048"

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.

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

Sequence and Features

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