Impact of exogenous aminoacyl-tRNA synthetase and tRNA on temperature sensitivity inEscherichia coli

Abstract

AbstractGenetic code expansion (GCE) is a powerful strategy that expands the genetic code of an organism for incorporating non-canonical amino acids (ncAAs) into proteins using engineered tRNAs and aminoacyl-tRNA synthetases (aaRSs). While GCE has opened up new possibilities for synthetic biology, little is known about the potential side effects of exogenous aaRS/tRNA pairs. In this study, we investigated the impact of exogenous aaRS and amber suppressor tRNA on gene expression inEscherichia coli. We discovered that in DH10β ΔcyaA, transformed with the F1RP/F2P two-hybrid system, high consumption rate of cellular ATP by exogenous aaRS/tRNA at elevated temperatures induces temperature sensitivity in the expression of genes regulated by the catabolite activator protein. We harnessed this temperature sensitivity to create a novel biological AND gate inE. coli, responsive to bothp-benzoylphenylalanine (BzF) and low temperature, using a BzF-dependent variant ofE. colichorismate mutase and split subunits ofBordetella pertussisadenylate cyclase. Our study provides new insights into the unexpected effects of exogenous aaRS/tRNA pairs and offers a new approach for constructing a biological logic gate.