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 .

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

With the DNA fragments purified from an agarose gel, we performed ligation at a molar ratio of 1:5 for vector and insert, as shown in Figure 3. The total vector concentration was 100 nanograms, whereas the reaction volume was 20 µL. Next, Table 2 displays the protocol followed for the reaction.

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

[Figure 1. Three-dimensional structure of the RIFMO-FRET system.]

Table 1. 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

Table 1. 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