Coding
ECFP-RIFMO

Part:BBa_K5439003

Designed by: Osvaldo Sanchez   Group: iGEM24_TecMonterreyGDL   (2024-09-30)
Revision as of 23:20, 1 October 2024 by Osvaldosan21 (Talk | contribs)


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

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 2. DNA ligation conditions.
Reactive Quantity
T4 DNA Ligase Buffer (10X) 2 µL
Vector DNA 100 ng
Insert DNA 773.5 ng
Nuclease-free water To 20 µL
T4 DNA Ligase 1.5 µL
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Categories
Parameters
None