The Ser/Leu-swapped genetic code can act as a genetic firewall, mitigating 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. In this study, to advance this system for protein engineering, we introduce a natural/in vitro transcribed-hybrid tRNA set. This set combines natural tRNAs from Escherichia coli (excluding Ser, Leu, and Tyr) and in vitro transcribed tRNAs, encompassing anticodon-swapped tRNASerGAG and tRNALeuGGA. This approach reduces the number of in vitro transcribed tRNAs required from 21 to only 4. In this optimized system, the production of a model protein, superfolder green fluorescent protein, increases 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 with reduced biohazard concerns in future biological endeavors.
Cell-Free System , Escherichia coli , Protein Biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , RNA, Transfer, Leu/genetics , RNA, Transfer, Leu/metabolism , RNA, Transfer, Ser/metabolism , RNA, Transfer, Ser/genetics , Genetic Code , RNA, Transfer/genetics , RNA, Transfer/metabolism , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Protein Engineering/methods , Transcription, Genetic , Anticodon/genetics , Anticodon/metabolism
