Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although longterm fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dualtimescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.L ong-term cell fates can often be selectively triggered by specific temporal patterns (dynamics) of stimulation (Fig. 1A). Relatively few cellular systems that "decode" time-varying inputs have been characterized in detail, but recurrent network motifs are beginning to emerge (1, 2). One key feature that is often observed in such systems is the interlinking of circuits that operate on distinct timescales (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In perhaps the best example of a biological "dynamic gate," the synaptic remodeling of neurons is mediated by two layers of regulation ( Fig. 1B): first, an upstream circuit of rapid but transient allosteric and posttranslational changes detects incoming stimuli and filters for high-frequency pulses; second, the signal is transmitted to downstream circuits regulated by slower processes (gene expression, trafficking, and morphological changes), which ultimately can yield stable alterations in receptor localization and synaptic function (4, 6). This common motif suggests that a simple solution for achieving tunable dynamic control systems is to link fast and slow subnetworks, whereby the upstream fast system processes how the intrinsically slow downstream switch receives and responds to external dynamic inputs.To test this hypothesis, we engineered synthetic cellular circuits based on linked fast (phosphorylation)-and slow (gene expression)-timescale modules (Fig. 1C). We first developed a versatile method for building fast-timescale signaling circuits in yeast using modular phospho-regulons. We then linked engineered phospho-feedback circuits with an intrinsically slow downstream transcription-based bistable switch, and were thereby able to generate a dynamic cell fate switch in yeast ...

No abstract