Difference between revisions of "Part:BBa K5439003"
Osvaldosan21 (Talk | contribs) (→Usage and Biology) |
Osvaldosan21 (Talk | contribs) (→Characterization) |
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We performed a Gibson assembly reaction, in order to insert Rifmo gene (K444703) into the previos iteration of the ECFP_EryK_mVENUS (BBa_K4447004). | We performed a Gibson assembly reaction, in order to insert Rifmo gene (K444703) into the previos iteration of the ECFP_EryK_mVENUS (BBa_K4447004). | ||
− | To carry out the Gibson assembly reaction, | + | To carry out the Gibson assembly reaction, the Rifmo gene was amplified via PCR using primers that recognize its Open Reading Frame (ORF) and include homology arms for recombination with the pET28b(+) vector. These primers also contain NcoI and XhoI restriction sites for validation. The PCR results, observed in an electrophoresis gel, showed a single band around 1.5 kb (figure 1A), matching the predicted molecular weight for Rifmo. This confirmed that the primers were effective and there were no primer dimers or nonspecific amplifications. |
− | |||
{| class="wikitable" style="margin:auto; text-align:center; length: 80%" | {| class="wikitable" style="margin:auto; text-align:center; length: 80%" | ||
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<figure> | <figure> | ||
<img src="https://static.igem.wiki/teams/5439/amplificacion-pcr.png" width="600"> | <img src="https://static.igem.wiki/teams/5439/amplificacion-pcr.png" width="600"> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 2.</b> (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. .</figcaption> |
</figure> | </figure> | ||
</body> | </body> | ||
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{| class="wikitable" style="margin:auto; text-align:center; length: 80%" | {| class="wikitable" style="margin:auto; text-align:center; length: 80%" | ||
− | |+ Table | + | |+ Table 2. Restriction digest conditions |
|- | |- | ||
!Componets !! 2-3 fragment assembly !! Positive control | !Componets !! 2-3 fragment assembly !! Positive control | ||
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<figure> | <figure> | ||
<img src="https://static.igem.wiki/teams/5439/bacteria-transformtion-rifmo.png" width="400"> | <img src="https://static.igem.wiki/teams/5439/bacteria-transformtion-rifmo.png" width="400"> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 3.</b>Bacterial transformation of ECFP_mVENUS with Rifmo in E.coli BL21 in LB agar with kanamycin (50 µg/mL) .</figcaption> |
</figure> | </figure> | ||
</body> | </body> | ||
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{| class="wikitable" style="margin:auto; text-align:center; length: 80%" | {| class="wikitable" style="margin:auto; text-align:center; length: 80%" | ||
− | |+ Table | + | |+ Table 3. Restriction digest conditions |
|- | |- | ||
!Reactive !! Quantity | !Reactive !! Quantity | ||
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<figure> | <figure> | ||
<img src="https://static.igem.wiki/teams/5439/fotos-11.png" width="600"> | <img src="https://static.igem.wiki/teams/5439/fotos-11.png" width="600"> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 4.</b> 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. |
.</figcaption> | .</figcaption> | ||
</figure> | </figure> | ||
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<figure> | <figure> | ||
<img src="https://static.igem.wiki/teams/5439/sds-page-rifmo.png" width="600"> | <img src="https://static.igem.wiki/teams/5439/sds-page-rifmo.png" width="600"> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 5.</b> 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. .</figcaption> |
</figure> | </figure> | ||
</body> | </body> | ||
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<figure> | <figure> | ||
<img src="https://static.igem.wiki/teams/5439/placas-de-rifmo.png" width="600"> | <img src="https://static.igem.wiki/teams/5439/placas-de-rifmo.png" width="600"> | ||
− | <figcaption><b>Figure | + | <figcaption><b>Figure 6.</b> Validation of the construct with the substrates of the interest (rifampicin) at a different concentrations .</figcaption> |
</figure> | </figure> | ||
</body> | </body> |
Revision as of 05:49, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1913
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2562
Contents
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
Gene amplification, Gibson Assembly and Transformation in vivo: iGEM24_TecMonterreyGDL
We performed a Gibson assembly reaction, in order to insert Rifmo gene (K444703) into the previos iteration of the ECFP_EryK_mVENUS (BBa_K4447004). To carry out the Gibson assembly reaction, the Rifmo gene was amplified via PCR using primers that recognize its Open Reading Frame (ORF) and include homology arms for recombination with the pET28b(+) vector. These primers also contain NcoI and XhoI restriction sites for validation. The PCR results, observed in an electrophoresis gel, showed a single band around 1.5 kb (figure 1A), matching the predicted molecular weight for Rifmo. This confirmed that the primers were effective and there were no primer dimers or nonspecific amplifications.
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
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
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 |
Protein Expression
Validation
References
1.
2.