TY - JOUR T1 - Distinct timescales of RNA regulators enable the construction of a genetic pulse generator JF - bioRxiv DO - 10.1101/377572 SP - 377572 AU - Alexandra Westbrook AU - Xun Tang AU - Ryan Marshall AU - Colin S. Maxwell AU - James Chappell AU - Deepak K. Agrawal AU - Mary J. Dunlop AU - Vincent Noireaux AU - Chase L. Beisel AU - Julius Lucks AU - Elisa Franco Y1 - 2018/01/01 UR - http://biorxiv.org/content/early/2018/07/25/377572.abstract N2 - To build complex genetic networks with predictable behaviours, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely employed to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and CRISPR interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ODE-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity. ER -