In Vitro Ribosome Synthesis and Evolution Through Ribosome Display
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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 × 10 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 10 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.
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Cell-Free Gene Expression: Methods and Applications.
Hunt A, Rasor B, Seki K, Ekas H, Warfel K, Karim A Chem Rev. 2024; 125(1):91-149.
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Lino B, Williams S, Castor M, Van Deventer J Chem Rev. 2024; 124(21):12145-12175.
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An Automated Cell-Free Workflow for Transcription Factor Engineering.
Ekas H, Wang B, Silverman A, Lucks J, Karim A, Jewett M ACS Synth Biol. 2024; 13(10):3389-3399.
PMID: 39373325 PMC: 11494693. DOI: 10.1021/acssynbio.4c00471.
Fan Y, Feng R, Zhang X, Wang Z, Xiong F, Zhang S Acta Pharm Sin B. 2024; 14(8):3362-3384.
PMID: 39220863 PMC: 11365444. DOI: 10.1016/j.apsb.2024.04.006.