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Francesca Ceroni1,2, Alice Boo2,3, Simone Furini4

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Researchers developed a novel feedback system for Escherichia coli to manage the burden of foreign protein expression. This system enhances protein yield and robust cell growth by automatically regulating synthetic construct expression.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Microbial engineering

Background:

  • Cells possess inherent feedback mechanisms for growth regulation under varying conditions.
  • Expressing foreign proteins imposes an unnatural burden on cellular resources, impacting growth.
  • Understanding cellular transcriptional responses to synthetic expression is crucial for metabolic engineering.

Purpose of the Study:

  • To identify transcriptional changes in Escherichia coli during synthetic construct expression.
  • To engineer a feedback-regulation system to mitigate the burden of foreign protein production.
  • To enhance cellular robustness and protein yield in engineered microbial systems.

Main Methods:

  • RNA sequencing (RNA-seq) to analyze global transcriptional changes.
  • In vivo assays to assess cellular responses to synthetic expression.
  • Development of a dCas9-based feedback controller utilizing native heat-shock promoters.

Main Results:

  • Synthetic expression rapidly activated native heat-shock response promoters in Escherichia coli.
  • A dCas9-based system was constructed to dynamically regulate synthetic gene expression based on cellular burden.
  • Engineered cells maintained native gene expression, demonstrating robust growth and superior protein yield compared to unregulated controls.

Conclusions:

  • The developed dCas9-based system represents a universal, burden-sensitive biomolecular control mechanism.
  • This modular, tunable, and portable feedback system improves protein production in engineered cells.
  • The system ensures cellular robustness by balancing synthetic demands with native cellular functions.