Abstract：
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 DH10β 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.
摘要：
遗传密码扩展（GCE）是一种强大的策略，可使用工程改造的tRNA和氨酰tRNA合成酶（aaRSs）扩展生物体的遗传密码，以将非规范氨基酸掺入蛋白质中。虽然GCE为合成生物学开辟了新的可能性，关于外源性aaRS/tRNA对的潜在副作用知之甚少。在这项研究中,我们研究了外源aaRS和琥珀抑制子tRNA对大肠杆菌基因表达的影响。我们发现在DH10βΔcyaA中，用F1RP/F2P双混合系统改造，在升高的温度下，外源aaRS/tRNA对细胞三磷酸腺苷的高消耗率诱导了由环状AMP受体蛋白（CRP）调节的基因表达的温度敏感性。我们利用这种温度敏感性在大肠杆菌中创造了一种新型的生物与门，对对苯甲酰苯丙氨酸（BzF）和低温，使用大肠杆菌分支杆菌酸变位酶的BzF依赖性变体和百日咳博德特氏菌腺苷酸环化酶的分裂亚基。我们的研究为外源aaRS/tRNA对的意外影响提供了新的见解，并为构建生物逻辑门提供了新的方法。
