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Proc Natl Acad Sci U S A. 2019 Mar 26;116(13):5892-5901. doi: 10.1073/pnas.1816591116. Epub 2019 Mar 8.

Cell-free gene-regulatory network engineering with synthetic transcription factors.

Author information

1
Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.
2
Institute of Bioengineering, School of Engineering, École Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland sebastian.maerkl@epfl.ch.

Abstract

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.

KEYWORDS:

biophysics; cell-free synthetic biology; gene regulatory network; synthetic transcription factors; transcriptional regulation

PMID:
30850530
PMCID:
PMC6442555
[Available on 2019-09-26]
DOI:
10.1073/pnas.1816591116

Conflict of interest statement

The authors declare no conflict of interest.

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