Difference between revisions of "Part:BBa K5439003"

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==Dosis-response assessment to characterize the effect of erythromycin on ECFP–EryK–mVENUS <i>in vivo</i>: iGEM24_TecMonterreyGDL==
  
 
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 <b>Figure 3</b>. The total vector concentration was 100 nanograms, whereas the reaction volume was 20 µL. Next, <b>Table 2</b> displays the protocol followed for the reaction.
 
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 <b>Figure 3</b>. The total vector concentration was 100 nanograms, whereas the reaction volume was 20 µL. Next, <b>Table 2</b> displays the protocol followed for the reaction.
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Revision as of 05:17, 2 October 2024


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 7. 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

Dosis-response assessment to characterize the effect of erythromycin on ECFP–EryK–mVENUS in vivo: iGEM24_TecMonterreyGDL

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


Once we obtained


Figure 7. (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 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


The next step


Figure 7.Bacterial transformation of ECFP_mVENUS with Rifmo in E.coli BL21 in LB agar with kanamycin (50 µg/mL) .


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


Figure 7. 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 7. 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 7. Validation of the construct with the substrates of the interest (rifampicin) at a different concentrations .