Part:BBa_K2904142
asRNA helping modular Btub riboswitch turn on gene expression
Design
Background of 2019 OUC-China's project——RiboLego
Thermodynamic switches are found being energetic equilibrium between their on- and off-state. If switching is triggered, the equilibrium distribution shifts towards the new energetically best conformation. This implies that thermodynamic switches can reversibly and repeatedly toggle between on- and off-states. In contrast, kinetic switches are trapped in one state, depending on whether the ligand was present at the time of folding. Because ligands are hard to be degraded, the state of the dynamic riboswitch is difficult to be changed without ligand washing. However, the ligand washing or medium replacement is almost impossible for many actual industrial application scenarios. So a new approach is expected to utilize to regulate the state of riboswitch.
The construction of this part
Antisense RNA is endogenous in E.coli that do not require heterologous proteins to function. Owing to its simple design principles, small size, and highly orthogonal behavior, the engineered genetic parts of asRNA has been incorporated into genetic circuits. So we used asRNA to regulate the state of riboswitch. Antisense RNA can be thought to consist of two regions: a target binding region (TBR) containing a sequence that is complementary to the target gene, and an Hfq binding site which allows for binding of the Hfq protein. Hfq is a native chaperone protein that mediates RNA interactions by binding to a particular RNA binding site on the asRNA molecule. In our work, the engineered MicF binding site (MicF M7.4) was used as Hfq binding site because it performed well with low off-target effect in previous studies. By using model to design artificial TBR, we hope to utilize asRNA to turn on Btub riboswitch. when targeting the complementary sequences of RBS of Tuner E, asRNA is able to open modular Btub riboswitch even in presence of VB12.
Result
After testing the translation-activating riboswitch, the translation-repressing riboswitch was also utilized to be combined with asRNA to turn on its output in the presence of the ligand. We employed modular Btub riboswitch containing Tuner E and designed asRNA to target the RBS of Tuner E. By adding IPTG and aTc, an increase of fluorescence intensity can be observed compared with the riboswitch system only induced by aTc.
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]
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