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Sebastian R Schmidl1,2, Felix Ekness3, Katri Sofjan4

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Engineers can swap DNA-binding domains in two-component systems (TCSs), enabling rewiring to new promoters. This advances synthetic biology by creating novel sensors and overcoming challenges like cross-regulation and host silencing.

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Area of Science:

  • Microbiology
  • Synthetic Biology
  • Molecular Biology

Background:

  • Two-component systems (TCSs) are crucial signal transduction pathways, widely used as sensors in synthetic biology.
  • Many TCSs are uncharacterized, difficult to apply, or face challenges like unknown output promoters, cross-regulation, and silencing in new hosts.

Purpose of the Study:

  • To demonstrate the interchangeability of response regulator DNA-binding domains in TCSs.
  • To enable TCS rewiring to synthetic output promoters and overcome application challenges.
  • To discover new TCSs and engineer novel biosensors.

Main Methods:

  • Exploiting the flexibility of DNA-binding domain swapping between TCS families.
  • Rewiring TCSs to synthetic output promoters.
  • Screening uncharacterized TCSs from Shewanella oneidensis in Escherichia coli.

Main Results:

  • Demonstrated remarkable flexibility in interchanging response regulator DNA-binding domains.
  • Successfully eliminated cross-regulation and un-silenced a Gram-negative TCS in a Gram-positive host.
  • Engineered a TCS with over 1,300-fold activation and discovered a novel pH sensor.

Conclusions:

  • DNA-binding domain swapping is a powerful strategy for TCS engineering and functional studies.
  • This approach accelerates the development of genetically encoded sensors for diverse synthetic biology applications.
  • Facilitates the discovery and characterization of novel TCSs from various organisms.