Difference between revisions of "Part:BBa K5439003"
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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.  
 
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.  
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===Usage and Biology===
 
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<b>Table 1</b> displays the protocol followed for a 50 µL reaction.
 
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{| class="wikitable" style="margin:auto; text-align:center; length: 80%"
 
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|+ Table 1. Restriction digest conditions.
 
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!Reactive !! Quantity
 
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| style="text-align:center;" style="width: 80%;" | Nuclease-free water || To 50 µL
 
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| style="text-align:center;" style="width: 80%;" | rCutSmart Buffer || 5 µL
 
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| style="text-align:center;" style="width: 80%;" | Template DNA (up to 4000 ng) || X µL
 
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| style="text-align:center;" style="width: 80%;" | <i>NcoI</i> restriction enzyme|| 1 µL
 
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| style="text-align:center;" style="width: 80%;" | <i>XhoI</i> restriction enzyme || 1 µL
 
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| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase || 1.5 µL
 
| style="text-align:center;" style="width: 80%;" | T4 DNA Ligase || 1.5 µL
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K5439003 SequenceAndFeatures</partinfo>
 
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===Functional Parameters===
 
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<partinfo>BBa_K5439003 parameters</partinfo>
 
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Revision as of 23:19, 1 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.


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