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Area of Science:

  • Synthetic Biology
  • Metabolic Engineering
  • Molecular Biology

Background:

  • Heterologous gene expression imposes a metabolic burden on cellular resources.
  • This burden can lead to reduced growth rates and instability in engineered microbial systems.
  • Existing methods lack dynamic control to mitigate these negative effects.

Purpose of the Study:

  • To engineer a novel feedforward controller for dynamic gene activation in bacteria.
  • To compensate for the non-physiological burden of gene expression on cellular resources.
  • To enable sustained and stable heterologous gene expression without compromising cell growth.

Main Methods:

  • Introduction of a feedforward controller activating a modified SpoT enzyme (SpoTH) upon gene of interest (GOI) activation.
  • Utilizing an inducible RelA+ expression cassette to precisely control basal ppGpp levels and nominal growth rate.
  • Co-culture experiments to assess population-level gene activation stability.

Main Results:

  • The controller successfully compensated for the burden of GOI activation, preventing growth rate defects.
  • Without the controller, GOI activation decreased growth rate by over 50%.
  • The controller enabled persistent population-level activation of the GOI in co-culture, unlike control strains.

Conclusions:

  • The developed feedforward controller is a tunable, modular, and portable tool for dynamic gene activation.
  • This system allows for precise control over gene expression without impacting bacterial growth.
  • It offers a significant advancement for synthetic biology applications requiring stable and inducible gene expression.