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In vitro ribosome synthesis and evolution through ribosome display

Author

Listed:
  • Michael J. Hammerling

    (Northwestern University)

  • Brian R. Fritz

    (Northwestern University)

  • Danielle J. Yoesep

    (Northwestern University)

  • Do Soon Kim

    (Northwestern University)

  • Erik D. Carlson

    (Northwestern University
    Stanford University)

  • Michael C. Jewett

    (Northwestern University
    Northwestern University
    Northwestern University
    Northwestern University)

Abstract

Directed evolution of the ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent ribosomes with ribosome display to develop a fully in vitro methodology for ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of ribosomes with altered properties.

Suggested Citation

  • Michael J. Hammerling & Brian R. Fritz & Danielle J. Yoesep & Do Soon Kim & Erik D. Carlson & Michael C. Jewett, 2020. "In vitro ribosome synthesis and evolution through ribosome display," Nature Communications, Nature, vol. 11(1), pages 1-10, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-020-14705-2
    DOI: 10.1038/s41467-020-14705-2
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    Cited by:

    1. Joongoo Lee & Jaime N. Coronado & Namjin Cho & Jongdoo Lim & Brandon M. Hosford & Sangwon Seo & Do Soon Kim & Camila Kofman & Jeffrey S. Moore & Andrew D. Ellington & Eric V. Anslyn & Michael C. Jewet, 2022. "Ribosome-mediated biosynthesis of pyridazinone oligomers in vitro," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
    2. Antje Krüger & Andrew M. Watkins & Roger Wellington-Oguri & Jonathan Romano & Camila Kofman & Alysse DeFoe & Yejun Kim & Jeff Anderson-Lee & Eli Fisker & Jill Townley & Anne E. d’Aquino & Rhiju Das & , 2023. "Community science designed ribosomes with beneficial phenotypes," Nature Communications, Nature, vol. 14(1), pages 1-11, December.

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