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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space.

Conor J McClune1,2, Aurora Alvarez-Buylla1, Christopher A Voigt2

  • 1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.

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|October 25, 2019
PubMed
Summary

Researchers created new signaling pathways in E. coli by engineering functional kinase-substrate pairs. This demonstrates that sequence space is not crowded, allowing for easy evolution or de novo design of novel, insulated cellular pathways.

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Systems Biology

Background:

  • Gene duplication creates new signaling pathways, but requires insulation to prevent crosstalk.
  • Engineering new pathways or transferring them between genomes faces challenges due to existing paralogs.
  • Pathway introduction ease depends on paralog density in sequence space, defined by specificity-determining residues.

Purpose of the Study:

  • To investigate the density and distribution of paralogous pathways in sequence space.
  • To generate novel, insulated two-component signaling pathways in Escherichia coli.
  • To assess the potential for de novo design of orthogonal signaling pathways.

Main Methods:

  • Utilized cell sorting coupled with deep sequencing to analyze large libraries of engineered proteins.
  • Designed libraries based on coevolutionary patterns to generate novel kinase-substrate pairs.
  • Tested generated pathways for orthogonality against existing pathways in E. coli.

Main Results:

  • Successfully produced 58 insulated pathways with novel kinase-substrate specificities.
  • Demonstrated that several new pathways are orthogonal to all 27 existing E. coli pathways.
  • Identified sets of six mutually orthogonal kinase-substrate pairs, increasing signaling capacity.

Conclusions:

  • Sequence space is not densely occupied by paralogous pathways.
  • The sparsity suggests new insulated pathways can evolve easily or be designed de novo.
  • Successfully engineered a novel pathway responding to plant cytokinin without crosstalk, validating de novo design.