Part:BBa_K5439006

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). 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.

Figure 2.(A) Agarose gel (0.8%) showing the PCR amplification for the Gibson assembly primer validation of IpfF and its respective control. The marked bands correspond to the expected molecular weight for the gene of 1.5 kb. (B) Agarose gel (0.8%) showing the amplification of the pET28b(+) backbone along with the two fluorescent proteins, ECFP and mVenus, each amplified using specific primers targeting homologous regions for their respective genes. 3000 bp bands correspond to not-amplified sequences in the supercoil form. .

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).

Figure 3.Bacterial transformation of ECFP_mVenus with IpfF in E.coli BL21 in LB agar with kanamycin (50 μg/mL). .