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

The basis for the assembly of the construct was the previous iteration of the ECFP_EryK_mVenus biosensor(BBa_K4447004), which was made up of the gene(BBa_K5439005)in a pET-28b backbone. In order to successfully assemble the intended construct through Gibson Assembly, we amplified and purified the vector using primers that bind to the ends of both fluorescent proteins and exclude the center EryK gene, obtaining an empty FRET backbone with the homology regions corresponding to the gene of interest. This PCR step proved to be particularly difficult and in need of optimization, as it was performed numerous times varying the number of cycles, elongation step duration, and especially primer annealing temperatures. Once we obtained a slightly more visible band and a satisfactory concentration after purification, we proceeded to the amplification of the insert. Along with the amplified vector, we amplified and purified the gene of interest(BBa_K5439005)with its corresponding primers in order to obtain the gene with the corresponding regions homologous to the backbone (Figure X). Having the required fragments amplified and ready, we proceeded to assemble the fragments together using New England Biolabs’ Gibson Assembly Master Mix. The next step was to transform the assembled ECFP_IpfF_mVenus product into E. coli BL21, an expression strain. This step also required some optimization, particularly regarding heat shock timings and the efficiency of our competent cells. Once those problems were sorted out, we obtained successfully transformed colonies for our constructs (Figure X).

Restriction Enzyme Digestion

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). As seen in the Figure X, the digested bands corresponding to the pair bases of the construct and the gene of interest are seen.

Protein Expression

Validation of our assembled FRET systems ECFP_IpfF_mVENUs

References