Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.821661
Title: Identification and evolution of new orthogonal aminoacyl-tRNA synthetase/tRNA pairs for genetic code expansion
Author: Cervettini, Daniele
ISNI:       0000 0004 9360 0186
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
Genetic code expansion is the branch of molecular biology aiming to expand the repertoire of amino acids which can be incorporated into proteins in vivo. A central challenge in expanding the genetic code of cells to incorporate non-canonical amino acids is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)–tRNA pairs (the components of the cellular translational machinery which specify the matching between codons and amino acids) that are orthogonal in their aminoacylation specificity. An orthogonal pair is composed of an aaRS which can interact with its partner tRNA, but not with any other tRNAs in the host, and a tRNA which is substrate to its partner aaRS, but not to any other aaRS in the host. In this research, candidate orthogonal tRNAs were identified from millions of sequences by implementing a computational analysis which scored their likelihood to be recognised by the endogenous aaRSs in E. coli, our model organism. I then developed a rapid, scalable new in vitro approach, named tRNA Extension (tREX), to determine the in vivo aminoacylation status of tRNAs. Using tREX, 243 candidate tRNAs were tested in E. coli and 71 orthogonal tRNAs were identified, covering 16 isoacceptor classes. 23 of those formed functional orthogonal tRNA–cognate aaRS pairs. By performing additional characterisation and molecular evolution of these newly identified functional pairs, we discovered 5 orthogonal pairs, 3 of which displayed high activity in amber suppression, the technique of choice used to implement genetic code expansion in model organisms. I additionally evolved new amino acid substrate specificities for two pairs. Finally, I use tREX to characterize a matrix of 64 orthogonal synthetase-orthogonal tRNA specificities. This work expanded the number of orthogonal pairs available for genetic code expansion, provided a robust pipeline for the discovery of additional orthogonal pairs, and established a foundation for encoding the cellular synthesis of non-canonical biopolymers.
Supervisor: Chin, Jason Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.821661  DOI:
Keywords: genetic code expansion ; directed evolution ; molecular biology ; mutagenesis
Share: