FRET is often used in the design of biosensors as it allows for the specific and sensitive detection of biomolecules in a highly specific manner with high sensitivity, without the need to induce a change in the biomolecule. The fluorescence of the acceptor molecule is activated only when both the donor fluorophore and the acceptor molecule are in proximity. This means that any changes in their surrounding environment that affect the distance between them will also impact the fluorescence of the molecule. This mechanism of action enables the detection of changes in the environment, even if they are subtle, without the need to genetically modify the molecule. FRET is a non-radiative process, which means it does not produce any ionizing radiation. This makes this type of biosensor safer to use and handle compared to others. Additionally, they are very sensitive and versatile biosensors, allowing them to detect the presence of a wide variety of biomolecules, as well as changes in the environment. They can detect protein-protein interactions, monitor changes in pH, measure enzyme activity, among others [3] .

Characterization

Gene amplification, Gibson Assembly and Transformation

We performed a Gibson assembly reaction to insert the Rifmo gene (K444703) into the previous iteration of the ECFP_EryK_mVENUS construct (BBa_K4447004). To accomplish this, the Rifmo gene was amplified using PCR with primers designed to recognize its Open Reading Frame (ORF) and add homology arms for recombination with the pET28b(+) vector. These primers also include NcoI and XhoI restriction sites for further validation. The PCR reaction components are detailed in Table 1, which lists the reagents used for amplification, including DreamTaq Polymerase (Thermo Fisher).

The results of the PCR amplification were analyzed by electrophoresis, as shown in Figure 2A. A single clear band around 1.5 kb was observed, corresponding to the predicted molecular weight for the Rifmo gene. The absence of additional bands confirmed the specificity of the primers, with no evidence of primer dimers or nonspecific amplifications, indicating that the PCR was successful.

E. coli BL21 cells were transformed with the Gibson Assembly product containing the RifMo gene and the ECFP_mVenus construct. After heat shock, the transformed cells were plated on LB agar with kanamycin (50 μg/mL) and incubated overnight at 37°C. Colonies appeared Figure 2, indicating successful transformation and assembly of the RifMo construct.

Restriction Enzyme Digestion

Although the appearance of colonies suggested successful transformation, further validation was needed to confirm the assembly of the RifMo gene. A restriction assay was performed using NcoI and XhoI enzymes, which flank the region containing the assembled RifMo gene. An in silico digestion using Benchling predicted two bands: one for the ECFP_mVenus construct and one for RifMo. In the electrophoresis gel Figure 4, a band around 5 kb was observed, corresponding to the ECFP_mVenus construct, and a band around 3 kb, matching the expected size of RifMo (2.9 kb). These results confirmed the assembly of the RifMo construct.

Protein Expression

After successfully transforming E. coli BL21 with the Gibson Assembly product containing RifMo, the transformants were induced with 0.4 mM IPTG to overexpress the full ECFP_RifMo_mVenus construct at 16°C overnight. Supernatants from selected clones were collected following cell disruption. The samples were analyzed by SDS-PAGE, which revealed a well-defined band around 109 kDa, corresponding to the expected molecular weight of the ECFP_RifMo_mVenus construct Figure 5 ). This band was absent in the control (induced pET-28b(+) without insert), confirming that no other protein of similar size was expressed.

Validation of our assembled FRET system ECFP_RIFMO_mVENUS

After validating the overexpression of the RifMo construct, the induced E. coli samples were grown on LB plates containing rifampicin to test the response of the FRET system. The concentrations of rifampicin were based on the reported IC50 for E. coli, using 1.25 mg/mL, 0.625 mg/mL, and 0.312 mg/mL [4] [5]. However, as shown in Figure 6, no fluorescence was observed on the plates. This could be due to the limited time window during which the FRET system fluoresced, which may not have aligned with the observation time on the transilluminator.

References