Cell-free gene-regulatory network engineering with synthetic transcription factors.
Proc Natl Acad Sci U S A
; 116(13): 5892-5901, 2019 03 26.
Article
em En
| MEDLINE
| ID: mdl-30850530
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Fatores de Transcrição
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Engenharia Genética
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Sistema Livre de Células
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Redes Reguladoras de Genes
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article