Developing a synthetic signalling system capable of the precise spatial and temporal control of protein function in living cells

合成生物学的目的是以在医疗保健，能源生产，食品安全和环境领域产生社会和经济效益的方式修改细胞或生物系统。真核细胞在合成生物学中具有巨大的潜力，然而，由于它们的复杂性，使用传统的合成生物学方法对其进行修饰，包括构建基因网络，是具有挑战性的。相反，直接调节这些细胞内蛋白质的功能可能提供一种更简单的方法来控制它们的行为。目前有许多选择，包括抗体，可以做到这一点，但这些方法的缺点是它们不能轻易关闭，因此它们会持续影响细胞的行为。为了更精确地控制细胞行为，我们需要开发一种可以快速打开或关闭的新系统。理想的系统将包括细胞内的开关样“效应”蛋白，该蛋白可以被工程化以结合和调节其他蛋白质，以及细胞表面的“受体”蛋白，当特定化学物质被添加到其环境中时，该蛋白可以打开效应蛋白。G蛋白偶联受体（GPCR）信号通路是细胞用于通信的天然系统;因此，它们是开发控制细胞行为的新工具的理想模板。GPCR是细胞表面受体，检测细胞外的化学物质，并激活细胞内的效应蛋白，称为“G蛋白”。G蛋白充当开关，当被GPCR激活时，G蛋白结合并调节其靶蛋白，然后以时间依赖性方式关闭。到目前为止，还不可能修饰G蛋白以结合和调节不同的细胞蛋白，因为这些途径中进化出的高度复杂性。然而，我最近开发了一种简化的G蛋白，这可能是第一次，使这成为可能。这项建议旨在修改GPCR信号通路，以创建一种新的基于细胞的工具，使我们能够控制活的真核细胞内不同蛋白质的活性。这将使我们能够在真实的时间内研究蛋白质的功能，或者直接控制细胞过程和行为。这个系统的关键组成部分将是简化的G蛋白，它将被修改，以便它可以结合和调节不同的细胞蛋白。这种G蛋白可以被天然GPCR激活，在这种情况下，该工具可以使用天然存在的化学物质（如激素）进行控制，或者被修饰的GPCR激活，在这种情况下，它可以由专门设计的化学物质进行控制。这个工具将在几个不同的领域中得到应用。首先，它将支持基础研究，以了解对动物和人类健康和疾病至关重要的天然细胞蛋白质的功能。其次，它将应用于开发一系列基于细胞的生物传感器，这些传感器能够真实的实时检测数百种生理相关的化学物质。第三，它将促进再生医学领域的研究，促进对人类细胞行为的精确控制，例如细胞分裂。奖学金的前四年将用于开发和优化该工具，并与能够将其转化为可行的医疗保健产品的公司建立伙伴关系。最后三年将专注于开发生物传感器应用，目的是生产生物医学诊断设备，并探索控制人类细胞分裂的应用，目的是开发改善退行性疾病（如骨关节炎）的治疗方法。这项研究的长期意义将是一种新的基于细胞的工具，通过简化合成生物学在真核细胞中的实施并加速其承诺的社会和经济效益，使学术和工业研究人员受益。

The aim of synthetic biology is to modify cells or biological systems in ways that produce societal and economic benefits in the areas of healthcare, energy production, food security and the environment. Eukaryotic cells hold huge potential in synthetic biology, however, due to their complexity, modifying them using conventional synthetic biology approaches, which involve constructing gene networks, is challenging. In contrast, directly regulating the function of proteins inside these cells may offer a simpler method to control their behaviour. A number of options, including antibodies, are currently available to do this, but the drawback of these approaches is that they cannot be easily switched off, so they affect the cell's behaviour continuously. In order to control cell behaviour more precisely, we need to develop a new system that can be turned on or off rapidly. The Ideal system would comprise a switch-like 'effector' protein inside the cell that can be engineered to bind and regulate other proteins, and a 'receptor' protein on the cell surface that can switch the effector protein on when a specific chemical is added to its environment. G protein-coupled receptor (GPCR) signalling pathways are natural systems that cells use to communicate; they are therefore ideal templates for the development of a new tool to control cell behaviour. GPCRs are cell-surface receptors that detect chemicals outside the cell, and activate effector proteins, called 'G proteins', inside the cell. The G proteins act as switches, which, when activated by GPCRs, bind and regulate their target protein, before switching themselves off in a time-dependent manner. Until now, it has not been possible to modify G proteins to bind and regulate different cellular proteins because of the high degree of complexity that has evolved within these pathways. However, I recently developed a simplified G protein that may, for the first time, make this possible.This proposal aims to modify the GPCR signalling pathway to create a novel cell-based tool that will allow us to control the activity of different proteins inside live eukaryotic cells. This will enable us to either study the protein's function in real time or directly control cellular processes and behaviours. The key component of this system will be the simplified G protein, which will be modified so that it can bind and regulate different cellular proteins. This G protein can be activated by either native GPCRs, in which case the tool could be controlled using naturally occurring chemicals (such as a hormone), or a modified GPCR, in which case it could be controlled by a specially designed chemical. This tool will have applications in several different areas. First, it will underpin basic research to understand the function of native cellular proteins that are central to the health and disease of animals and humans. Second, it will have applications in the development of a range of cell-based biosensors capable of detecting hundreds of physiologically-relevant chemicals in real time. Third, it will facilitate research in the field of regenerative medicine, by facilitating the precise control of human cell behaviour, for example, cell division. The first four years of the Fellowship will be used to develop and optimise the tool, and to foster partnerships with companies that are capable of translating it into viable healthcare products. The final three years will focus on both developing the biosensor applications, with the aim of producing miniaturised medical diagnostic devices, and exploring applications to control human cell division, with the aim of developing improved treatments for degenerative conditions such as osteoarthritis. The long-term implication of this research will be a novel cell-based tool that will benefit both academic and industrial researchers, by simplifying the implementation of synthetic biology in eukaryotic cells and expediting its promised societal and economic benefits.