In vivo application of an inhibitory RNA aptamer against T7 RNA polymerase

Juan Quijano, Jongmin Kim, Enoch Yeung and Richard M. Murray
Submitted, 2014 Winter q-bio Conference (5 Nov 2013)

Synthetic biology involves the design of complex artificial biological systems with exploitable features for human benefit through an ecological and sustainable way. Recent advances in nucleic acids engineering introduced several RNA-based regulatory components for synthetic gene circuits, expanding the toolsets to engineer organisms. In this work, we designed genetic circuits implementing an RNA aptamer previously described to have the capability of binding to the T7 RNA polymerase and inhibiting its activity in vitro. Using in vitro transcription assays, we first demonstrated the utility of the RNA aptamer in combination with programmable synthetic transcription networks. As a step to quickly assess the feasibility of aptamer functions in vivo, a cell-free expression system was used as a breadboard to emulate the in vivo conditions of E. coli. We tested the aptamer and its three sequence variants in the cell-free expression system, verifying the aptamer functionality in the cell-free testbed. In vivo expression of aptamer and its variants demonstrated control over GFP expression driven by T7 RNA polymerase with different response curves, indicating its ability to serve as building blocks for both logic circuits and transcriptional cascades. This work elucidates the potential of RNA-based regulators for implementing different “biocodes” for cell programming and for improved controllability thanks to the fast production and degradation time scales of RNA molecules.

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