Part:BBa_K3258017
Self-amplifying fluorescent RNA (MDV-Q-beta-Spinach)
This is a part composite composed of self-encoded Qbeta replicase with a Spinach aptamer attached, both sequences flanked by midivariant regions recognized by Qbeta, referred to as MDV regions upstream and downstream. When expressed in E. coli cell free extract supplemented with DFHBI, Spinach fluorescence increases due to the replication of the RNA by the Qbeta replicase enzyme. This creates a self replicative cycle, and the amplification of the Qbeta and Spinach aptamer.
Testing in In Vitro
The functionality and operation of the MDV-Qbeta-Spinach construct was tested in Sigma 70 myTXTL Cell Free Cell Lysate. Initial tests were conducted comparing fluorescent output from another construct, mRFP-Spinach, with the fluorescent output of MDV-Qbeta-Spinach. Trials were conducted running reactions with each construct at the same time at 29 degrees Celsius for 18 hours and measuring fluorescent output. Results showed significant differences in fluorescent output between MDV-Qbeta-Spinach and mRFP-Spinach, suggesting that MDV-Qbeta-Spinach was operational in vitro in the used cell free lysate.
Testing MDV Region Impact on Replicability of Qbeta System
After confirming the functionality of the MDV-Qbeta-Spinach construct with in vitro experimentation, the impact of the MDV regions was tested. Each MDV region (BBa_K3258015 and BBa_K3258016, or midivariant region, is a RNA sequence experimentally classified in previous literature to be a good substrate for Qbeta binding and replication. Flanking a given gene of interest, especially Qbeta, should make the whole sequence more conducive to replication and amplification by Qbeta. This was tested by comparing the fluorescent output of the MDV-Qbeta-Spinach construct in vitro with trials of a Qbeta-Spinach only construct, which lacked the MDV flanking regions. Run in triplicate, the results suggested a clear improvement of the MDV flanked Qbeta-Spinach over the control Qbeta-Spinach only, with no trial of the MDV-Qbeta-Spinach dipping below the highest performing trial of the Qbeta-Spinach. The differences in amplification of each construct suggested a ~30% improvement in the amplification rate with the MDV flanked construct versus the non-MDV flanked construct.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 180
Illegal XbaI site found at 2231
Illegal SpeI site found at 2107 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 146
Illegal SpeI site found at 2107
Illegal NotI site found at 2213 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 172
Illegal BglII site found at 2228
Illegal BamHI site found at 1538
Illegal BamHI site found at 1681
Illegal XhoI site found at 168
Illegal XhoI site found at 1212
Illegal XhoI site found at 2222 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 180
Illegal XbaI site found at 2231
Illegal SpeI site found at 2107 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 180
Illegal XbaI site found at 2231
Illegal SpeI site found at 2107
Illegal NgoMIV site found at 2120
Illegal NgoMIV site found at 2149
Illegal AgeI site found at 1694 - 1000COMPATIBLE WITH RFC[1000]
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