Istituto Italiano di Tecnologia, Largo Barsanti e Matteucci, Naples, Italy.
University of Genoa, Genoa, Italy.
Methods Mol Biol. 2021;2229:331-346. doi: 10.1007/978-1-0716-1032-9_16.
Synthetic biology has been advancing cellular and molecular biology studies through the design of synthetic circuits capable to examine diverse endogenously or exogenously driven regulatory pathways. While early genetic devices were engineered to be insulated from intracellular crosstalk, more recently the need of achieving dynamic control of cellular behavior has led to the development of smart interfaces that connect signal information (sensor) to desired output activation (actuator). Sensor-actuator circuits can respond to diverse inputs, including small molecules, exogenous and endogenous mRNA, noncoding RNA (i.e., miRNA), and proteins to regulate downstream events, transcriptionally, posttranscriptionally, and translationally. These devices require attentive engineering to either create complex chimeric proteins or modify protein structures to be amenable to the specific circuits' architecture and/or purpose.In this chapter, we describe how to implement two different protein-based devices in mammalian cells: (1) a modular platform that sense and respond to disease-associated proteins and (2) a protein-based system that allows simultaneous regulation of RNA translation and protein activity, via RNA-protein and newly engineered protein-protein interactions.
合成生物学通过设计能够检测多种内源性或外源性驱动的调控途径的合成回路，推动了细胞和分子生物学研究的进展。虽然早期的遗传装置被设计为与细胞内串扰隔离，但最近实现对细胞行为的动态控制的需求促使开发了智能接口，将信号信息（传感器）连接到所需的输出激活（执行器）。传感器-执行器电路可以响应多种输入，包括小分子、外源性和内源性 mRNA、非编码 RNA（即 miRNA）和蛋白质，以在转录、转录后和翻译水平上调节下游事件。这些装置需要精心设计，要么创建复杂的嵌合蛋白，要么修饰蛋白质结构，以适应特定电路的结构和/或目的。在本章中，我们描述了如何在哺乳动物细胞中实现两种不同的基于蛋白质的设备：（1）一种模块化平台，用于感知和响应与疾病相关的蛋白质；（2）一种基于蛋白质的系统，通过 RNA-蛋白质和新设计的蛋白质-蛋白质相互作用，允许同时调节 RNA 翻译和蛋白质活性。
