Orthogonality of Redesigned tRNA Molecules with Three Stop Codons

Comprehensive Summary The ability to expand genetic code in living cells has emerged as a powerful method with diverse applications. Here, we design replacement of the anticodons of E. coli tRNAs that recognize codons for 20 natural amino acids, with three anti‐stop codons, resulting in a total of 60 engineered tRNA constructs. We test these constructs one by one in E. coli, and found that six tRNAsCUA (tyrV, serX, hisR, trpT, glnV and leuX), two tRNAsUCA (trpT and leuX) and one tRNAUUA (tyrV) allowed efficient expression of Red Fluorescence Protein (RFP) with the presence of a corresponding stop codon in frame. Furthermore, we exploit the mutual orthogonality of tRNASerCUA, tRNATrpUCA and tRNATyrUUA with corresponding stop codons and demonstrated that the tRNASerCUA and the tRNATrpUCA can provide dynamic range and low crosstalk. Finally, we show the TAG and TGA can not only be used as an “AND gate” circuit to control the translation of target gene, but also be used to control the translation of a prodeoxyviolacein (PDV) pathway and a reporter in parallel. Overall, this work provides flexible tools for translational control and holds great potential to promote synthetic biology studies. This study revealed the ability of stop codons to participate in encoding natural amino acids, and proved that the tRNASerCUA and the tRNATrpUCA can provide dynamic range and low crosstalk by orthogonal test. We demonstrated that mutually orthogonal nonsense suppressor tRNA‐stop codon pairs can be used to not only control single gene expression in series, but also control two genetic functional units in parallel. Overall, this work provides useful tools with minimal size for translational control and holds great potential to promote synthetic biology studies.