(259d) Engineering and Optimizing Small RNAs That Activate Transcription

                  Manipulation of gene expression lies at the very heart of synthetic biology, and small RNAs offer an attractive means of rapidly controlling gene expression with minimal use of cellular resources.  Naturally occurring sRNA regulators encompass both translational activators and repressors, as well as transcriptional repressors, but no sRNA-based transcriptional activators have been reported in natural systems. To address this, we designed and engineered a number of highly orthogonal synthetic RNA-based transcriptional activators, thus expanding the synthetic biology toolkit and allowing for the construction of increasingly complex RNA circuitry.  These Small Transcription Activating RNAs (STARs) act in trans by preventing the formation of a bacterial intrinsic terminator hairpin in the 5’ UTR of a nascent mRNA, thus activating gene expression.  We describe a protocol for enhancing their performance based on engineering the stability of the trans acting sRNA, the relative dosage of the sRNA to target, and the strength of the disrupted terminator hairpin.  Additionally, we report the construction of a number of ligand-dependent STARs, made to sense the small molecule theophylline and to function only in its presence.  These ligand-dependent STARs were isolated through a high-throughput screening process that allows for easy testing of hundreds of library members in order to identify functional ligand-sensitive variants.  The development of RNA transcriptional activators, coupled with the ability to easily optimize additional STARs and develop ligand-sensitive activators, significantly expands the building blocks available for synthetic biology and bolsters our capability to construct RNA-based genetic circuits responsive to changes in their environment.