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The realization of artificial biochemical reaction networks with unique functionality is one of the main challenges for the development of synthetic biology. Due to the reduced number of components, biochemical circuits constructed in vitro promise to be more amenable to systematic design and quantitative assessment than circuits embedded within living organisms. To make good on that promise, effective methods for composing subsystems into larger systems are needed. Here we used an artificial biochemical oscillator based on in vitro transcription and RNA degradation reactions to drive a variety of "load" processes such as the operation of a DNA-based nanomechanical device ("DNA tweezers") or the production of a functional RNA molecule (an aptamer for malachite green). We implemented several mechanisms for coupling the load processes to the oscillator circuit and compared them based on how much the load affected the frequency and amplitude of the core oscillator, and how much of the load was effectively driven. Based on heuristic insights and computational modeling, an "insulator circuit" was developed, which strongly reduced the detrimental influence of the load on the oscillator circuit. Understanding how to design effective insulation between biochemical subsystems will be critical for the synthesis of larger and more complex systems.cell-free circuits | modularity | genelets | DNA nanotechnology I n biology, chemical oscillators control the timing of cellular processes and provide day-night rhythms, as in circadian clocks (1). In the past decade, synthetic clock systems with a reduced number of components have been constructed in vivo in order to study the design principles underlying oscillatory behavior (2-7). Most of these artificial gene regulatory systems are still relatively complex and difficult to understand quantitatively, as they make use of the full transcriptional and translational machinery of their host organisms. The cellular environment also puts significant limits on the types of chemistries that these oscillators can orchestrate. At the other extreme, inorganic oscillators can be quite robust, but difficult to systematically couple to a wide range of downstream processes (8, 9).Synthetic cell-free biochemical circuits offer interesting possibilities for the design of complex molecular processes, both because of their relative simplicity and their potential applicability for controlling a wide range of in vitro chemistries (10-16). Systems whose behavior is dependent on DNA templates are particularly promising because they can be systematically rewired to obtain new functionalities. Recently, a simple oscillator based on only transcription and degradation in vitro has been demonstrated (17), raising the prospect of orchestrating the temporal expression of other synthetic chemical processes.In the present work we demonstrate how this synthetic transcriptional circuit can be used as a molecular clock for timing biochemical processes in vitro. Specifically, we address the question of how these d...

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