The behavior of gene modules in complex synthetic circuits is often unpredictable1–4. Upon joining modules to create a circuit, downstream elements (such as binding sites for a regulatory protein) apply a load to upstream modules that can negatively affect circuit function1,5. Here we devise a genetic device named a load driver that mitigates the impact of load on circuit function, and we demonstrate its behavior in Saccharomyces cerevisiae. The load driver implements the design principle of time scale separation: inclusion of the load driver’s fast phosphotransfer processes restores the capability of a slower transcriptional circuit to respond to time-varying input signals even in the presence of substantial load. Without the load driver, we observe circuit behavior that suffers from 76% delay in response time and a 25% decrease in system bandwidth due to load. With the addition of a load driver, circuit performance is almost completely restored. Load drivers will serve as fundamental building blocks in the creation of complex, higher level genetic circuits.

While predictable design of a genetic circuit's output is a major goal of synthetic biology, it remains a significant challenge because DNA binding sites in the cell affect the concentration of available transcription factors (TF). To mitigate this problem, we propose to use a TF that results from the (reversible) phosphorylation of protein substrate as a circuit's output. We demonstrate that by comparatively increasing the amounts of substrate and phosphatase, the TF concentration becomes robust to the presence of DNA binding sites and can be kept at a desired value. The circuit's input/output gain can, in turn, be tuned by changing the relative amounts of the substrate and phosphatase, realizing an amplifying buffer circuit with tunable gain. In our experiments in E. coli, we employ phospho-NRI as the output TF, phosphorylated by the NRII kinase, and dephosphorylated by the NRII phosphatase. Amplifying buffer circuits such as ours could be used to insulate a circuit's output from the context, bringing synthetic biology one step closer to modular design.

Abstract-We seek to minimize both the retroactivity to the output and the retroactivity to the input of a phosphorylationbased insulation device by finding an optimal substrate concentration. Characterizing and improving the performance of insulation devices brings us a step closer to their successful implementation in biological circuits, and thus to modularity. Previous works have mainly focused on attenuating retroactivity effects to the output using high substrate concentrations. This, however, worsens the retroactivity to the input, creating an error that propagates back to the output. Employing singular perturbation and contraction theory tools, this work provides a framework to determine an optimal substrate concentration to reach a tradeoff between the retroactivity to the input and the retroactivity to the output.

Abstract-This paper considers the problem of attenuating retroactivity, that is, the effect of loads in biological networks and demonstrates that signal transduction cascades incorporating phosphotransfer modules have remarkable retroactivity attenuation ability. Uncovering the biological mechanisms for retroactivity attenuation is relevant in synthetic biology to enable bottom-up modular composition of complex circuits. It is also important in systems biology for deepening our current understanding of natural principles of modular organization. In this paper, we perform a combined theoretical and computational study of a cascade system comprising two phosphotransfer modules, ubiquitous in eukaryotic signal transduction, when subject to load from downstream targets. Employing singular perturbation on the finite time interval, we demonstrate that this system implements retroactivity attenuation when the input signal is sufficiently slow. Employing trajectory sensitivity analysis about nominal parameters that we have identified from in vivo data, we further demonstrate that the key parameters for retroactivity attenuation are those controlling the timescale of the system.