RESUMEN
Genetic code expansion (GCE) is a powerful strategy that expands the genetic code of an organism for incorporating noncanonical amino acids 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 DH10ß ΔcyaA, transformed with the F1RP/F2P two-hybrid system, the high consumption rate of cellular adenosine triphosphate by exogenous aaRS/tRNA at elevated temperatures induces temperature sensitivity in the expression of genes regulated by the cyclic AMP receptor protein (CRP). 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.
Asunto(s)
Aminoácidos , Aminoacil-ARNt Sintetasas , Escherichia coli , ARN de Transferencia , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Aminoácidos/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Código Genético , Proteína Receptora de AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Biología Sintética/métodos , Corismato Mutasa/genética , Corismato Mutasa/metabolismo , Fenilalanina/metabolismo , Fenilalanina/análogos & derivados , Adenosina Trifosfato/metabolismo , Regulación Bacteriana de la Expresión Génica , BenzofenonasRESUMEN
The expression of genes of interest (GOI) can be initiated by providing external stimuli such as temperature shifts and light irradiation. The application of thermal or light stimuli triggers structural changes in stimuli-sensitive biomolecules within the cell, thereby inducing or repressing gene expression. Over the past two decades, several groups have reported genetic circuits that use natural or engineered stimuli-sensitive modules to manipulate gene expression. Here, we summarize versatile strategies of thermosensors and light-driven systems for the conditional expression of GOI in bacterial hosts.