RT Journal Article SR Electronic T1 Cell-free gene regulatory network engineering with synthetic transcription factors JF bioRxiv FD Cold Spring Harbor Laboratory SP 407999 DO 10.1101/407999 A1 Zoe Swank A1 Nadanai Laohakunakorn A1 Sebastian J. Maerkl YR 2019 UL http://biorxiv.org/content/early/2019/01/23/407999.abstract AB 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.