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Refactoring and optimization of light-switchable Escherichia coli two-component systems.

Sebastian R Schmidl1, Ravi U Sheth, Andrew Wu

  • 1Department of Bioengineering and ‡Department of Biochemistry and Cell Biology, Rice University , 6100 Main Street, Houston, Texas 77005, United States.

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|September 25, 2014
PubMed
Summary

Researchers engineered improved light-switchable protein systems (two-component signal transduction systems) for precise control of biological processes. These enhanced sensors offer greater dynamic range and reduced leakage for advanced synthetic biology applications.

Keywords:
Escherichia colicyanobacteriochromeoptogeneticsphytochromerefactoringtwo component system

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biotechnology

Background:

  • Light-switchable proteins offer precise control over molecular biological processes in living organisms.
  • Previous two-component signal transduction systems (TCSs) had limitations including bulky DNA, promoter incompatibility, leaky output, and limited dynamic range.

Purpose of the Study:

  • To engineer second-generation light sensors with improved performance and broader utility.
  • To overcome limitations of existing light-switchable TCSs for enhanced synthetic biology applications.

Main Methods:

  • Streamlined four-gene TCSs onto two plasmids.
  • Replaced chemically inducible promoters with engineered constitutive versions.
  • Optimized expression of sensor histidine kinase and response regulator.
  • Redesigned output promoters to minimize leakiness and maximize dynamic range.

Main Results:

  • Achieved significantly reduced leakiness in the engineered TCSs.
  • Demonstrated substantial improvements in dynamic range, reaching 72- and 117-fold.
  • Developed second-generation light sensors with enhanced performance characteristics.

Conclusions:

  • Optimized bacterial TCSs function as high-performance sensors for scientific and engineering applications.
  • Enhanced light sensors enable programming of more genes over a wider range.
  • Improved TCSs facilitate easier combination with additional plasmids and transfer to different host strains.