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 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 co-operativity 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.


Published in:
Proceedings Of The National Academy Of Sciences Of The United States Of America, 116, 13, 5892-5901
Year:
Mar 26 2019
ISSN:
0027-8424
Keywords:
Laboratories:




 Record created 2019-04-10, last modified 2020-04-20


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