Ser/Leu-swapped cell-free translation system constructed with natural/in vitro transcribed-hybrid tRNA set

Abstract

Abstract The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating the biohazard risks arising from horizontal gene transfer in genetically modified organisms. Our prior work demonstrated the orthogonality of this swapped code to the standard genetic code using a cell-free translation system comprised of 21 in vitro transcribed tRNAs. To advance this system for protein engineering, we introduced a natural/in vitro transcribed hybrid tRNA set. This set combined natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduced the requisite in vitro transcribed tRNAs from 21 to a mere 4. In this optimized system, the production of a model protein, super-folder green fluorescent protein, increased to 3.5-fold. With this hybrid tRNA set, the Ser/Leu-swapped cell-free translation system will stand as a potent tool for protein production for reduced biohazard concerns in future biological endeavors.