Cell-free gene regulatory network engineering with synthetic transcription factors

Gene regulatory networks are ubiquitous in nature and critical for bottom-up engineering of synthetic networks. Transcriptional repression is a fundamental function in gene regulatory networks and 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, specificity, 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. By integrating these methods we generated a library of well-characterized synthetic transcription factors and corresponding promoters, and used these standardized parts to build gene regulatory networks de novo in a cell-free environment. 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.