Part:BBa_K5439003

FRET-based system for the detection of rifampicin

FRET-based sensor system for the detection of rifampicin that consists of rifampicin monooxygenase (K4447003), an enzyme that catalyzes the hydroxylation of rifampicin, 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 1913
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2562
  

Usage and Biology

Rifampicin (RAMP) is an antibiotic widely used in the treatment of severe bacterial infections such as tuberculosis, meningitis, leprosy, and HIV-associated infections. RAMP residues contaminate water sources, primarily through human excretions (urine and feces) and waste generated by the pharmaceutical industry and animal husbandry. Due to its high solubility and environmental stability, RAMP is not fully removed by wastewater treatment plants, contributing to the development of antibiotic-resistant bacteria (ARB) 3 .

Figure 1. Predicted structure with the best PAE obtained from ColabFold showing ECFP (green), Rifmo (gray) , and mVENUS (yellow).

Selecting Fluorescent Proteins

FRET (Fluorescence Resonance Energy Transfer) 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 2 .

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).

Table 1. Components and volumes for the PCR with DreamTaq polymerase protocol.

Reactive	Quantity
10X DreamTaq buffer	5 µL
dNTP Mix (10 mM each)	1 µL
IUpstream primer	1 µL
Downstream primer	1 µL
DNA temple	10 pg - 1 µL
DreamTaq Polymerase	0.25 µL
Nuclease-free water	To 50 µL
Total volume	50 µL

The results of the PCR amplification were analyzed by electrophoresis, as shown in Figure 1A. 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.

Figure 2. (A) Agarose gel (0.8%) showing the PCR amplification for the Gibson assembly primer validation of IpfF, TjPCs, RifMo, and their respective controls. The marked bands correspond to the expected molecular weight for each 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

Componets	2-3 fragment assembly	Positive control
Total amount of fragments	0.02 - 0.5 pool	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

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 4), indicating successful transformation and assembly of the RifMo construct.

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

Table 3. Restriction digest conditions

Reactive	Quantity
Restriction Enzyme 10X Buffer	5 µL
DNA (1 µg/ µL)	1 µL
Ncol restriction enzyme	1 µL
Xhol restriction enzyme	1 µL
BSA (1 µg/ µL)	0.2 µL
Nuclease-free water	To 20 µL
Total volume	20 µL

Figure 4. A) In silico prediction of restriction assay, where the patterns of digestions are shown using the enzymes NcoI and XhoI, and the plasmids (1) pET-28b(+) ECFP_RifMo_mVenus, (2) pET-28b(+) ECFP_TjPCs_mVenus, and (3) pET-28b(+) ECFP_IpfF_mVenus. B) Electrophoresis of agarose gel (0.8%) of the restriction assay with Ncol and XhoI of the construct ECFP_mVenus cloned with the genes IpfF, RifMo or TjPCs. .

Protein Expression

Figure 5. SDS-PAGE gel showing the protein overexpression results of the ECFP_RIFMO_mVenus construct, 109 kDa correspond to the expected molecular weight of the full protein construct. No band of the same molecular weight as the desired proteins was observed in the control sample. .

Validation

Figure 6. Validation of the construct with the substrates of the interest (rifampicin) at a different concentrations .

References

1.

2.