Biosensors that transduce target chemical and biochemical inputs into genetic outputs are essential for bioengineering and synthetic biology. Current biosensor design strategies are often limited by a low signal-to-noise ratio, the extensive optimization required for each new input, and poor performance in mammalian cells. Here we report the development of a proximity-dependent split RNA polymerase (RNAP) as a general platform for biosensor engineering. After discovering that interactions between fused proteins modulate the assembly of a split T7 RNAP, we optimized the split RNAP components for protein–protein interaction detection by phage-assisted continuous evolution (PACE). We then applied the resulting activity-responsive RNAP (AR) system to create biosensors that can be activated by light and small molecules, demonstrating the ‘plug-and-play’ nature of the platform. Finally, we validated that ARs can interrogate multidimensional protein–protein interactions and trigger RNA nanostructure production, protein synthesis, and gene knockdown in mammalian systems, illustrating the versatility of ARs in synthetic biology applications. We anticipate that ARs will provided a simplified and more robust strategy for engineering gene circuits for applications in screening, directed evolution, and synthetic biology.

Evolution and applications of split RNA polymerase biosensors

254th National Meeting (2017)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.