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Orthogonal translation enables heterologous ribosome engineering in E. coli

Author

Listed:
  • Natalie S. Kolber

    (Broad Institute of MIT and Harvard
    Stanford University)

  • Ranan Fattal

    (Broad Institute of MIT and Harvard)

  • Sinisa Bratulic

    (Broad Institute of MIT and Harvard
    Chalmers University of Technology)

  • Gavriela D. Carver

    (Broad Institute of MIT and Harvard)

  • Ahmed H. Badran

    (Broad Institute of MIT and Harvard)

Abstract

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells.

Suggested Citation

  • Natalie S. Kolber & Ranan Fattal & Sinisa Bratulic & Gavriela D. Carver & Ahmed H. Badran, 2021. "Orthogonal translation enables heterologous ribosome engineering in E. coli," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-020-20759-z
    DOI: 10.1038/s41467-020-20759-z
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