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Abstract

Cell-free systems have emerged as a versatile platform for constructing complex biological systems from the bottom-up. In particular, they enable the rapid engineering and characterization of gene regulatory networks, a critical cellular subsystem that allows a cell to sense and respond to a myriad of signals with a computational-like logic. Building synthetic gene circuits provides the means to both program new functionalities and augment our understanding of natural networks. Genetic circuits are composed of elements that control transcription, translation and post-translational processes through biomolecular interactions involving DNA, RNA, proteins and small molecules. Even the design space of simple gene circuits can be sizeable, necessitating high-throughput methods capable of characterizing large libraries of regulatory components in a comprehensive manner that can guide the engineering of functional circuits. Therefore, we have developed microfluidic platforms that can be coupled with cell-free systems to facilitate the high-throughput screening of gene regulatory elements. In this work, we begin by presenting a microfluidic device capable of performing hundreds of independent cell-free transcription-translation reactions in parallel, using different combinations of surface immobilised DNA as the reaction templates. We employ this device to study different mechanisms for tuning transcriptional repression using synthetic zinc-fingers, whose affinity, specificity and coopertivity can be rationally engineered. Functional repression assays were combined with quantitative affinity measurements and thermodynamic modeling to generate a library of well-characterized synthetic transcription factors and corresponding promoters, with which we were able to build gene regulatory circuits de novo. As the first platform was limited to carrying out reactions in batch-mode, we then adapted the microfluidic device to enable the implementation of cell-free transcription-translation reactions at steady state, while maintaining high-throughput screening capabilities. The modified device consisted of individual reaction compartments that could be periodically supplied with cell-free reagents mixed on-chip to create programmable concentration gradients. For a proof of concept we measured the steady state repression levels for a subset of the synthetic components we had previously characterized in batch and demonstrated that we could implement a genetic toggle switch. Lastly, we developed a microfluidic device for generating double emulsion droplets to encapsulate cell-free gene expression reactions. The flow focusing device integrates pneumatic valves that enable on-chip mixing with minimal reagent volumes and drop sorting. Utilizing this device we were able to precisely titrate DNA template concentrations and monitor in vitro protein synthesis profiles in the droplets.

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