Automated prototyping of genetic codes

A robotic, cell-free platform rapidly prototypes redesigned genetic codes, enabling the translation of proteins with reassigned codons and non-standard amino acids without altering living genomes. The platform, called AGENTEX, represents a significant advance in synthetic biology and genetic engineering capabilities.

Scientists have long sought to expand the genetic code beyond its natural 20 amino acids to create proteins with novel properties. While previous research has demonstrated the incorporation of more than 400 non-standard amino acids into proteins using engineered ribosomes, tRNAs, and aminoacyl-tRNA synthetases (aaRSs), reassigning more than a handful of sense codons across an entire genome remains challenging due to cellular toxicity and low efficiency in incorporating multiple non-standard amino acids.

The AGENTEX platform overcomes these limitations by combining custom lysate-based translation systems with synthetic pools of tRNAs carrying engineered anticodons. By engineering the universal Watson-Crick interaction between the 3' CCA sequence of tRNAs and specific bases in the 23S rRNA of E. coli ribosomes, researchers created a system where only specially designed tRNAs are accommodated for translation, preventing crosstalk with natural tRNA pools.

A key innovation is the development of tSCAN (tRNA sequencing of charging by automated NGS), a multiplexed method to measure aminoacylation of synthetic tRNAs in complex pools. This allowed the team to study the full sequence variation of engineered tRNA pools and identify permissive 3' tRNA sequences that function as substrates for all aaRSs.

Using AGENTEX, the researchers built and tested minimal genetic codes for polypeptide transcription and non-standard amino acid incorporation. The system enables rapid generation and testing of genetic code experiments through an automated workflow on an open-source robotic platform (Opentrons OT-2), significantly accelerating the pace of genetic code engineering.

The work provides insights into the evolution of the translation system and enables further evolution and expansion of the genetic code. Potential applications include creating organisms with increased virus resistance, reduced horizontal gene transfer, production of genetically encoded materials, novel therapeutics, and improved biocatalysts. The biocontainment features of the system ensure that the components are not functional if transferred to living organisms in the environment, addressing safety concerns associated with synthetic biology research.

**Sources:** Nature