Part:BBa_K4207067
Toehold sensor plasmid A70-mScarlet-I
Sensor plasmid for detecting BYDV
1. Usage and biology
This sensor plasmid was designed to detect a conserved RNA sequence in the barley yellow dwarf virus (BYDV) genome. It should express the reporter protein mScarlet-I only in the presence of its specific trigger sequence. The translation is controlled by a toehold switch, which sequesters the RBS and the start codon in a stable RNA secondary structure. This structure is unfolded in the presence of the specific trigger, which is a conserved sequence in the BYDV genome.
The translational activity can be measured by fluorescence intensity at 569/593 nm. The construct can be used in various reaction set ups. If used in a PURE (protein expression using recombinant elements) system or any other environment with reduced nuclease activity, it can be used as a linear plasmid. However, if used in a reaction with active nucleases, for instance a lysate-based cell-free expression system, the construct should be used as a circular plasmid to reduce degradation.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 171
Illegal EcoRI site found at 219 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 171
Illegal EcoRI site found at 219 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 171
Illegal EcoRI site found at 219 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 171
Illegal EcoRI site found at 219 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 171
Illegal EcoRI site found at 219 - 1000COMPATIBLE WITH RFC[1000]
2. Characterization
This part was not evaluated experimentally. However, two sensor constructs ( BBa_K4207061, BBa_K4207065, BBa_K4207073, BBa_K4207074) that only differ from this construct in context of their protein coding sequence were evaluated. They exhibited 0.46-fold activity of the reporter protein β-galactosidase in the reactions. This sensor construct is likely to exhibit similar properties.
3. Conclusion
This part lacks experimental data. However, based on the experimental data about similar constructs as well as the modeling data of the toehold switch contained in this construct suggests that this part is not likely to detect BYDV genome.
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