A portable regulatory RNA array design enables tunable and complex regulation across diverse bacteria

ABSTRACT A lack of composable and tunable gene regulators has hindered efforts to engineer non-model bacteria and consortia. To address this, we explore the broad-host potential of small transcription activating RNA (STAR) and propose a novel design strategy to achieve tunable gene control. First, we demonstrate that STARs optimized for E. coli function across different Gram-negative species and can actuate using phage RNA polymerase, suggesting that RNA systems acting at the level of transcription are portable. Second, we explore a novel RNA design strategy that uses arrays of tandem and transcriptionally fused RNA regulators to precisely alter regulator concentration from 1 to 8 copies. This provides a simple means to predictably tune output gain across species and does not require access to large regulatory part libraries. Finally, we show RNA arrays can be used to achieve tunable cascading and multiplexing circuits across species, analogous to the motifs used in artificial neural networks.