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). Figure 1 shows the three-dimensional structure of the protein system.

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 which correspond to the ones in the Table 1.

Table 1. PCR Conditions.

Component	Volume
10X DreamTaq buffer	5 µL
dNTP Mix (10 mM each)	1 µL
Upstream primer 10 μM	1 µL
Downstream primer 10 μM	1 µL
DNA template	10 pg - 1 μg
DreamTaq Polymerase	0.25 μL
Nuclease-free water	To 50 μL
Total volume	50 μL

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 2). Having the required fragments amplified and ready, we proceeded to assemble the fragments together using New England Biolabs’ Gibson Assembly Master Mix with the conditions as established in the Table 2.

Table 2. Restriction digest conditions.

Component	2-3 fragment assembly	Positive Control
Total amount of fragments	0.02-0.5 pmol	10 μL
Gibson Assembly 2X Master Mix	10 μL	10 μL
Nuclease-Free Water	To 20 μL	0 μL
Total Volume	20 μL	20 μL

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 3).

==Restriction Enzyme Digestion== Finally, as a confirmation step, we performed minipreps on colonies from the construct 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 4). As seen in the Figure 4, the digested bands corresponding to the pair bases of the construct and the gene of interest are seen. The conditions for the restriction digest are seen in the Table 3. {| class="wikitable" style="margin:auto; text-align:center; length: 80%" |+ Table 3. Restriction digest conditions. |- !Reactive !! Quantity |- |- | style="text-align:center;" style="width: 80%;" | Restriction Enzyme 10X Buffer || 5 µL |-- | style="text-align:center;" style="width: 80%;" | DNA (1 μg/μL) || 1 µL |- | style="text-align:center;" style="width: 80%;" | NcoI restriction enzyme|| 1 µL |- | style="text-align:center;" style="width: 80%;" | XhoI restriction enzyme || 1 µL |- | style="text-align:center;" style="width: 80%;" | BSA (10 μg/μL) || 0.2 µL |- | style="text-align:center;" style="width: 80%;" | Nuclease-free water || To 20 µL |- | style="text-align:center;" style="width: 80%;" | Total Volume || 20 µL |} [[Image:figure-4-registro-de-partes.png|250px|right|thumb|Figure 4. A) Comparison in silico, where 1 corresponds to pET-28b(+) ECFP_RifMo_mVenus Assembly NcoI Xhol, 2, corresponds to pET-28b(+) ECFP_TjPCs_mVenus Assembly NcoI Xhol, and 3, corresponds to pET-28b(+) ECFP_IpfF_mVenus Assembly NcoI Xhol. B) Agarose gel (0.8%) of restriction assay with Ncol and XhoI of the construct ECFP_mVenus cloned with the gene IpfF.]] ==Protein Expression== Once we had confirmation of a successful transformation and a successful assembly, selected colonies were induced with 0.4 mM IPTG to stimulate protein overexpression (Gomes, L., Monteiro, G., & Mergulhão, F., 2020). Various trials were attempted at different temperatures and incubation conditions, and the last trial at 16 °C for 16 hours yielded visible, clear bands corresponding to the approximate molecular weight of the construct of interest (~113 kDa for IpfF). As shown in Figure 5, the bands were not visible in the negative controls, which corresponded to E. coli BL21 transformed with an empty pET-28b backbone containing no insert. This shows evidence for the expression of our fusion construct, and we proceeded to tests with each construct’s substrate. [[Image:figure-5-registro-de-partes.png|250px|right|thumb|Figure 5. SDS-PAGE gel showing the protein overexpression results of the ECFP_mVenus and IpfF. The marked band of 113 kDa correspond to the expected molecular weight of the full protein construct. No band of the same molecular weight as the desired protein was observed in the control sample.]] ==Validation of our assembled FRET systems ECFP_IpfF_mVENUs== We assessed the intended fluorescence generated by our FRET system by plating the same induced samples onto LB plates containing each construct’s substrate at different concentrations: 1.25 mg/mL, 0.625 mg/mL, and 0.312 mg/mL for ibuprofen. This test was done both to verify the viability of the cells in the presence of the substrate and to determine whether the fluorescence signal produced by the FRET system was visible and dependent on substrate concentration (Figure 6). Unfortunately, there was no visible fluorescence in any of the concentrations when compared to the control (pET-28b with no insert). However, these results could be explained by the duration and timing of the fluorescence produced by FRET, as it is possible there was fluorescence for a period of time that didn’t coincide with when the plates were analyzed. Additionally, to absolutely determine and quantify the correlation between substrate concentrations and fluorescence produced, protein purification after overexpression needs to be performed, with the aim of eliminating unwanted variables and removing the risk of mutation in the cells. This will also enable the adequate calibration of our proposed device, adding the possibility of not only detecting the contaminant, but also to quantify its presence in the tested sample. [[Image:figure-6-registro-de-partes.png|250px|right|thumb|Figure 6. Validation of the construct with the substrates of interest at different concentrations.]] =References=