(146c) Improving RNA Regulator Dynamic Range with a Dual Transcription-Translation Control Mechanism

RNA repressors are emerging as a highly versatile component in genetic circuit construction. However, the dynamic range of RNA transcriptional repressors still doesnâ??t match that of their protein counterparts. This incomplete repression can cause circuit leak, which impedes the construction of large predictable synthetic regulatory networks. Here we demonstrate how naturally-derived antisense RNA-mediated transcriptional regulators can regulate both transcription and translation, increasing repression from 85% to 98% and activation from 10 fold to over 900 fold, using transcriptional termination and RBS sequestration in a single compact RNA molecule that functions in Escherichia coli. We also show that orthogonal versions of this mechanism can be created through engineering minimal versions of the antisense RNAs. To demonstrate the utility of this dual control mechanism, we use it to show that they greatly reduce circuit leak when used in RNA-only transcriptional cascades that activate gene expression as a function of a small molecule input. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to larger and more sophisticated circuits.