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Transfer RNA (tRNA) is a dynamic molecule used by all forms of life as a key component of the translation apparatus. Each tRNA is highly processed, structured, and modified, to accurately deliver amino acids to the ribosome for protein synthesis. The tRNA molecule is a critical component in synthetic biology methods for the synthesis of proteins designed to contain non-canonical amino acids (ncAAs). The multiple interactions and maturation requirements of a tRNA pose engineering challenges, but also offer tunable features. Major advances in the field of genetic code expansion have repeatedly demonstrated the central importance of suppressor tRNAs for efficient incorporation of ncAAs. Here we review the current status of two fundamentally different translation systems (TSs), selenocysteine (Sec)- and pyrrolysine (Pyl)-TSs. Idiosyncratic requirements of each of these TSs mandate how their tRNAs are adapted and dictate the techniques used to select or identify the best synthetic variants.
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Engineering tRNA abundances for synthetic cellular systems
Abstract Routinizing the engineering of synthetic cells requires specifying beforehand how many of each molecule are needed. Physics-based tools for estimating desired molecular abundances in whole-cell synthetic biology are missing. Here, we use a colloidal dynamics simulator to make predictions for how tRNA abundances impact protein synthesis rates. We use rational design and direct RNA synthesis to make 21 synthetic tRNA surrogates from scratch. We use evolutionary algorithms within a computer aided design framework to engineer translation systems predicted to work faster or slower depending on tRNA abundance differences. We build and test the so-specified synthetic systems and find qualitative agreement between expected and observed systems. First principles modeling combined with bottom-up experiments can help molecular-to-cellular scale synthetic biology realize design-build-work frameworks that transcend tinker-and-test.
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- Award ID(s):
- 1929752
- PAR ID:
- 10436919
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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