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bioRxiv · 10.1101/2024.05.02.592135

Impact of exogenous aminoacyl-tRNA synthetase and tRNA on temperature sensitivity in Escherichia coli: towards a genetic code-expanded biological logic gate

Choi, J.·Ahn, J.·Bae, J.·Yoon, M.·Yun, H.·Koh, M.
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Abstract

Genetic 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 in Escherichia coli. We discovered that in DH10{beta} {Delta}cyaA, 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 in E. coli, responsive to both p-benzoylphenylalanine (BzF) and low temperature, using a BzF-dependent variant of E. coli chorismate mutase and split subunits of Bordetella pertussis adenylate 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.

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Choi, J., Ahn, J., Bae, J., Yoon, M., Yun, H., Koh, M.. 2024-05-02. Impact of exogenous aminoacyl-tRNA synthetase and tRNA on temperature sensitivity in Escherichia coli: towards a genetic code-expanded biological logic gate. https://doi.org/10.1101/2024.05.02.592135

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