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Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells
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Noise-reducing optogenetic negative-feedback gene circuits in human cells.

Michael Tyler Guinn1,2,3, Gábor Balázsi1,2

  • 1Biomedical Engineering Department, Stony Brook University, Stony Brook, NY 11794, USA.

Nucleic Acids Research
|July 4, 2019
PubMed
Summary

Researchers engineered novel optogenetic gene circuits, called Light-Inducible Tuners (LITer), for precise control of gene expression in mammalian cells. These circuits significantly reduce gene expression noise, enabling new applications in biology and medicine.

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Area of Science:

  • Synthetic biology
  • Optogenetics
  • Gene circuit engineering

Background:

  • Gene autorepression is crucial for controlling gene expression and reducing noise.
  • Optogenetic systems for mammalian cells often rely on activators, with limited use of negative feedback for noise reduction.
  • In silico control is insufficient for real-time, in vitro gene expression modulation.

Purpose of the Study:

  • To engineer optogenetic gene circuits for precise gene expression control in mammalian cells.
  • To achieve noise reduction through genetic, in vitro negative feedback using optogenetics.
  • To develop a toolset of Light-Inducible Tuner (LITer) gene circuits for versatile applications.

Main Methods:

  • Fusion of TetR repressor with Tet-inhibiting peptide (TIP) or degradation tag via the light-sensitive LOV2 protein domain.
  • Construction of noise-reducing Light-Inducible Tuner (LITer) gene circuits.
  • Application of LITer circuits to control KRAS(G12V) oncogene expression and study downstream effects.

Main Results:

  • LITer circuits provide a 4-fold range of gene expression control.
  • Achieved up to 5-fold noise reduction compared to existing optogenetic systems.
  • Demonstrated successful control of KRAS(G12V) expression and its impact on phospho-ERK levels and cell proliferation.

Conclusions:

  • Novel LITer optogenetic platforms enable precise spatiotemporal control of gene expression with reduced noise.
  • These systems offer significant improvements over existing optogenetic tools for gene expression modulation.
  • LITer circuits are valuable for studying multicellular phenotypes in developmental biology, oncology, and biomedical research.