Part:BBa_K4207063
Toehold sensor plasmid A95-LacZ
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 β-galactosidase 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 activity of the translated β-galactosidase can be measured spectrophotometrically if the reaction contains a colorimetric substrate for β-galactosidase. 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
2. Characterization
This part was not evaluated experimentally. However, four sensor constructs ( BBa_K4207061, BBa_K4207065, BBa_K4207073, BBa_K4207074) that only differ from this construct in context of their toehold switches were evaluated. Two of them contain toehold switches with similar predicted performance as this one (BBa_K4207061, BBa_K4207065) and 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 these constructs suggests that this part is not likely to detect BYDV genome.
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