scholarly journals Two-Tier Screening Platform for Directed Evolution of Aminoacyl-tRNA Synthetases with Enhanced Stop Codon Suppression Efficiency

ChemBioChem ◽  
2017 ◽  
Vol 18 (12) ◽  
pp. 1109-1116 ◽  
Author(s):  
Andrew E. Owens ◽  
Katherine T. Grasso ◽  
Christine A. Ziegler ◽  
Rudi Fasan
Keyword(s):  
Directed Evolution ◽  
Stop Codon ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Suppression Efficiency ◽  
Screening Platform

scholarly journals Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases

2019 ◽  
Vol 20 (9) ◽  
pp. 2294 ◽  
Author(s):  
Ana Crnković ◽  
Oscar Vargas-Rodriguez ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Directed Evolution ◽  
Active Site ◽  
Nonsense Suppression ◽  
Natural Amino Acid ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genetic Incorporation

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.

scholarly journals Continuous directed evolution of aminoacyl-tRNA synthetases

2017 ◽  
Vol 13 (12) ◽  
pp. 1253-1260 ◽  
Author(s):  
David I Bryson ◽  
Chenguang Fan ◽  
Li-Tao Guo ◽  
Corwin Miller ◽  
Dieter Söll ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

scholarly journals Erratum: Errata: Continuous directed evolution of aminoacyl-tRNA synthetases

2018 ◽  
Vol 14 (2) ◽  
pp. 186-186
Author(s):  
David I Bryson ◽  
Chenguang Fan ◽  
Li-Tao Guo ◽  
Corwin Miller ◽  
Dieter Söll ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases

scholarly journals A high-throughput platform for feedback-controlled directed evolution

2020 ◽  
Author(s):  
Erika A. DeBenedictis ◽  
Emma J. Chory ◽  
Dana Gretton ◽  
Brian Wang ◽  
Kevin Esvelt
Keyword(s):  
Directed Evolution ◽  
Real Time ◽  
Parallel Evolution ◽  
T7 Rna Polymerase ◽  
Massively Parallel ◽  
Feedback Control System ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Evolutionary Trajectories ◽  
And Robotics

Continuous directed evolution rapidly implements cycles of mutagenesis, selection, and replication to accelerate protein engineering. However, individual experiments are typically cumbersome, reagent-intensive, and require manual readjustment, limiting the number of evolutionary trajectories that can be explored. We report the design and validation of Phage-and-Robotics-Assisted Near-Continuous Evolution (PRANCE), an automation platform for the continuous directed evolution of biomolecules that enables real-time activitydependent reporter and absorbance monitoring of up to 96 parallel evolution experiments. We use this platform to characterize the evolutionary stochasticity of T7 RNA polymerase evolution, conserve precious reagents with miniaturized evolution volumes during evolution of aminoacyl-tRNA synthetases, and perform a massively parallel evolution of diverse candidate quadruplet tRNAs. Finally, we implement a feedback control system that autonomously modifies the selection strength in response to real-time fitness measurements. By addressing many of the limitations of previous methods within a single platform, PRANCE simultaneously enables multiplexed, miniaturized, and feedback-controlled continuous directed evolution.

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