To create an biosensor capable of detecting the anti-inflammatory drug ibuprofen, the FRET-based sensor system (BBa_K4447004) that we propose by changing the gene(BBa_K4447001), for the gene(BBa_K5439005). By changing the genes, our composite part can detect ibuprofen from water bodies. Ibuprofen is an anti-inflammatory treatment drug widely used in the world that can be bought without any necessary prescription. This makes ibuprofen a drug that everyone can consume easily, bringing problems because its disposal makes it an emerging contaminant in water bodies (Jan-Roblero, J., & Cruz-Maya, J. A., 2023). FRET (fluorescence resonance energy transfer) is a biosensor technique that detects biomolecules without modifying them. It relies on the proximity of fluorophore molecules to trigger fluorescence. This non-radiative process allows for sensitive and specific detection of environmental changes and biomolecule interactions. FRET biosensors are safe and versatile, able to detect protein-protein interactions, pH changes, enzyme activity, and more. They provide a reliable means of monitoring various biomolecular activities without the need for genetic modifications, making them valuable tools in research and diagnostics (Kumar-Verma, A., et al., 2023).

Characterization

Gene Aplification, Gibson Assembly and Transformation

Restriction Enzyme Digestion and Ligation

Finally, as a confirmation step, we performed minipreps on colonies from all 3 constructs and digested the plasmid using NcoI and XhoI enzymes, in order to ensure the transformed colonies contained the interest plasmid with the full construct (Figure X).

Protein Expression

Validation of our assembled FRET systems ECFP_IpfF_mVENUs

References