Part:BBa_K2942701

Riboswitch

Riboswitches is an element comprising a ribozyme actuator and RNA aptamer sensor, which could bind to a series of small molecular metabolites and iron ions to regulates transcription, translation, splicing and the stability of RNA [1]. In our project, we use a kind of riboswitch developed from the hammerhead ribozyme from the satellite RNA of tobacco ringspot virus (sTRSV) conjugated to a theophylline aptamer. It’s an OFF-type riboswitch, which inhibits ribozyme cleavage in the absence of theophylline, but addition of theophylline allows the ribozyme to form its active conformation and initiate cleavage, turning gene expression off. By adding theophylline or not, we can control the expression of the pro-drug gene attached to M1 virus, which greatly improves the safety of M1 virus and efficiency of gene expression. This part was mainly used for the construction of composite part. We artificially synthesized the sequence in the article that has mention above, and then we design the primers (see details in the composite part BBa_2942705, BBa_2942706, BBa_2942707) to gain and recycle the PCR product (Fig.1) for the multiple fragment homologous recombination next. Its characterization can be seen in the composite part BBa_2942705, BBa_2942706, BBa_2942707 in details.

Fig.1 PCR result. We followed the protocol of NEB Q5® High-Fidelity 2X Master Mix to perform the PCR, and the PCR product was identified by 1% agarose gel electrophoresis.

[1] Bell, C.L., et al., Control of alphavirus-based gene expression using engineered riboswitches. Virology, 2015. 483: p. 302-311.