Universal Genetic Code Expansion Platform for Bacteria and Eukaryotes
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Solution Overview
Problem
Current genetic code expansion methods require separate platforms for bacteria and eukaryotes, limiting the ability to incorporate unnatural amino acids into proteins expressed in both cell types.
Innovation Solution
Development of a universal platform using the endogenous tryptophanyl-tRNA/tryptophanyl-tRNA synthetase pair in E. coli, which is functionally replaced with a yeast-derived counterpart, allowing for the directed evolution of orthogonal tRNA/aaRS pairs that can incorporate unnatural amino acids into both E. coli and eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If separate tRNA/aaRS platforms are used for bacteria and eukaryotes, then genetic code expansion can be achieved in each system, but the complexity of maintaining multiple platforms increases and the ability to use a single system for both cell types is limited
Solution Approach 1:
The pyrrolysyl tRNA/PylRS pair from archaebacteria serves as a universal platform that functions in both bacterial and eukaryotic cells. This single orthogonal system can be used to incorporate unnatural amino acids into proteins expressed in either cell type, eliminating the need to maintain separate bacterial and eukaryotic platforms and enabling researchers to work with a unified system across different biological contexts.
2Ease of manufacture
If the E. coli based selection system is used for directed evolution, then the process is facile and successful for encoding new UAAs, but access to new active site topologies for structurally distinct UAAs is limited
Solution Approach 1:
The pyrrolysyl tRNA/PylRS pair acts as an intermediary system that bridges the facile E. coli-based selection process with the ability to access diverse active site topologies. By using this universal orthogonal pair as the foundation, researchers can perform directed evolution in the easy-to-use E. coli system while still achieving access to a broader range of unnatural amino acid structures through the PylRS platform's versatility.
3Device complexity
If endogenous tRNA/aaRS pairs are used, then the system is simple, but orthogonality with host counterparts is compromised leading to cross-reactivity
Solution Approach 1:
The system segments the translation machinery into orthogonal components: the endogenous E. coli tRNA/aaRS system handles natural amino acids, while the imported pyrrolysyl tRNA/PylRS pair handles unnatural amino acids. This segmentation ensures that the two systems operate independently without cross-reactivity, maintaining both simplicity and reliability by keeping the pathways separate.
Solution Approach 2:
The pyrrolysyl tRNA/PylRS pair is extracted from archaebacteria and introduced into E. coli as a separate, orthogonal system. This extraction allows the E. coli host to maintain its native simple translation system while the imported pair provides dedicated functionality for unnatural amino acid incorporation without interfering with endogenous processes.
Data Source
AI summary
Methods and compositions are described for selecting and identifying orthogonal aminoacyl synthetase-tRNA pairs and their use to incorporate unnatural amino acids in a site-specific manner in proteins. Specifically described is a novel E. coli tyrptophanyl synthetase-tRNA pair that functions as both an opal and amber suppressor and that incorporates tryptophan analogs into proteins.