Coding

Part:BBa_K5439006

Designed by: Diego Cota Barocio, Ruben Valenzuela Sanchez   Group: iGEM24_TecMonterreyGDL   (2024-09-30)
Revision as of 01:42, 1 October 2024 by Ranchvzla (Talk | contribs)


FRET-based system for the detection of ibuprofen

FRET-based sensor system for the detection of ibuprofen that consists of long-chain fatty acid CoA ligase from Sphingomonas spp.(BBa_K5439005),an enzyme that catalyzes the conversion of ibuprofen into isobutylcatechol, flanked by two fluorescent proteins: ECFP(BBa_K1159302)as energy donor and mVenus(BBa_K1907000)as an energy acceptor

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2306
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1177
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2174
    Illegal BsaI.rc site found at 2959

Usage and Biology

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

[1]. Jan-Roblero, J., & Cruz-Maya, J. A. (2023). Ibuprofen: Toxicology and Biodegradation of an Emerging Contaminant. Molecules (Basel, Switzerland), 28(5), 2097. https://doi.org/10.3390/molecules28052097

[2]. Kumar-Verma, A., Noumani, A., Yadav, A. K., & Solanki , P. R. (2023). FRET Based Biosensor: Principle Applications Recent Advances and Challenges. MDPI, 13(8), 1375–1375. https://doi.org/10.3390/diagnostics13081375
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