TY - JOUR T1 - Cell-free gene regulatory network engineering with synthetic transcription factors JF - bioRxiv DO - 10.1101/407999 SP - 407999 AU - Zoe Swank AU - Nadanai Laohakunakorn AU - Sebastian J. Maerkl Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/01/23/407999.abstract N2 - Gene regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function that can be tuned at the level of DNA, protein, and cooperative protein – protein interactions, necessitating high-throughput experimental approaches for in-depth characterization. Here we used a cell-free system in combination with a high-throughput microfluidic device to comprehensively study the different tuning mechanisms of a synthetic zinc-finger repressor library, whose affinity and cooperativity can be rationally engineered. The device is integrated into a comprehensive workflow that includes determination of transcription factor binding energy landscapes and mechanistic modeling, enabling us to generate a library of well-characterized synthetic transcription factors and corresponding promoters, which we then used to build gene regulatory networks de novo. The well-characterized synthetic parts and insights gained should be useful for rationally engineering gene regulatory networks and for studying the biophysics of transcriptional regulation. ER -