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Spatial engineering of E. coli with addressable phase-separated RNAs.

Haotian Guo1, Joseph C Ryan1, Xiaohu Song1

  • 1Université de Paris, INSERM U1284, Center for Research and Interdisciplinarity (CRI), 75006 Paris, France.

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|September 30, 2022
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Summary

Researchers created synthetic membraneless organelles in bacteria called transcriptionally engineered addressable RNA solvent droplets (TEARS). These TEARS enable spatial control of biochemical processes, advancing bacterial bioengineering.

Keywords:
RNA condensatesRNA triplet repeatsbioengineeringexpression noise bufferingliquid-liquid phase separationmembraneless organellesmetabolic engineeringsynthetic biologysynthetic organelles

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Area of Science:

  • Synthetic biology
  • Bacterial cell biology
  • Biochemistry

Background:

  • Biochemical processes require spatial regulation and specific microenvironments.
  • Bacteria lack organelles, limiting complex intracellular reaction bioengineering.
  • Need for tools to control spatial biochemistry in bacteria.

Purpose of the Study:

  • To develop synthetic membraneless organelles in Escherichia coli.
  • To demonstrate the utility of these organelles for controlling intracellular biochemistry.

Main Methods:

  • Assembly of synthetic membraneless organelles (TEARS) using RNA-binding protein recruiting domains and poly-CAG repeats.
  • Induction of liquid-liquid phase separation in the cytoplasm.
  • Targeting TEARS with fluorescent proteins to visualize structure.
  • Assessing TEARS for bioprocess isolation and control.

Main Results:

  • TEARS spontaneously assembled and formed multilayered structures.
  • TEARS provided organelle-like isolation for sequestering biochemical pathways.
  • Demonstrated control over metabolic branch points and mRNA translation rates.
  • Scaffolding of protein-protein interactions within TEARS.

Conclusions:

  • TEARS are a simple and versatile tool for spatial control of E. coli biochemistry.
  • The modular design simplifies bioengineering design-build-test cycles.
  • TEARS offer organelle-like functionality in bacteria without expression fine-tuning.