21ENGBIO Engineered orthogonal ribosomes for programmable protein modification

蛋白质是微小的纳米级分子机器，充当所有活细胞的主力。它们支撑着重要的任务，包括感知和信号传导，新陈代谢的协调，甚至细胞结构元件的自组装。这些功能中的许多都可以通过修改相关蛋白质来定制，为细胞提供了一种使其行为多样化的方法。蛋白质在生物系统中的广泛应用使其成为工程新生物形式或利用材料科学等其他领域的生物成分和功能的重要目标。能够按需合成和修饰蛋白质可以释放这一巨大的潜力。在这个项目中，我们的目标是通过创造一种所谓的“正交核糖体”来直接应对这一挑战，这种核糖体可以与细胞的天然过程平行合成蛋白质。重要的是，我们的正交核糖体将被工程化，以包括能够修饰正在合成的蛋白质的次级组分的附着点。通过用正交机制合成我们的蛋白质，我们避免以有害的方式修饰天然细胞蛋白质，从而可以自由地以不同的方式修饰我们自己的蛋白质。此外，通过切换存在的修饰附件，我们可以轻松地改变所做的修饰类型，为可编程蛋白质合成和修饰创建一个平台。为了实现这一雄心勃勃的目标，我们将使用新开发的实验方法，可以创建大量具有不同附着点的正交核糖体设计，并评估这些设计对核糖体有效合成核糖体的能力的影响。蛋白将选择那些工作良好的设计，然后修改使用计算机模型和模拟精确设计的附件，以具有确保参与修改的区域完美定位在核糖体上的形状。最后，我们将联合收割机结合工程正交核糖体和活细胞内的修饰附件，并测试它们修饰靶蛋白的能力，使其在改变时定位于细胞边缘--我们将能够使用单细胞显微镜轻松监测这种变化。一种天然的生物分子机器核糖体。我们使用“即插即用”组件的灵活和模块化方法提供了快速改变对目标蛋白质进行的修饰的能力，而无需从头开始构建新系统，并为生物学家，生物工程师和材料科学家提供了机会，以更好地了解蛋白质在其天然环境中的功能，精确地设计其在活细胞中的特性，并利用高度修饰的蛋白质作为纳米级构建模块，用于新形式的可持续高性能材料。更广泛地说，我们的方法还提供了一条利用其他核心细胞过程并将其功能重新用于工程生物学新兴领域的新应用的途径。

Proteins are tiny nano-scale molecular machines that act as the workhorses of all living cells. They underpin crucial tasks spanning sensing and signalling, the coordination of metabolism and even the self-assembly of structural elements of the cell. Many of these functions can be tailored by the modification of the proteins involved, offering a way for a cell to diversity its behaviour. The broad applications of proteins in biological systems makes them an important target for engineering new forms of biology or harnessing biological components and functions in other areas like Material Science. Being able to synthesise and modify proteins on demand could unlock this huge potential.In this project we aim to directly tackle this challenge by creating what is termed an "orthogonal ribosome" that can synthesise proteins in parallel to a cell's native process. Importantly, our orthogonal ribosomes will be engineered to include attachment points for secondary components that are able to modify the protein being synthesised. By synthesising our proteins with orthogonal machinery, we avoid modifying native cellular proteins in a detrimental way and thus have the freedom to modify our own in diverse ways. Furthermore, by switching the modifying attachment that is present, we can easily change the type of modification made, creating a platform for programmable protein synthesis and modification.To achieve this ambitious goal, we will use newly developed experimental methods that can create vast numbers of orthogonal ribosome designs with different attachment points and assess the impact these have on the ability for the ribosome to effectively synthesise a protein. Those designs that work well will be selected and then modifying attachments precisely designed using computer models and simulation to have shapes that ensure the region involved in modification is perfectly positioned on the ribosome. Finally, we will combine the engineered orthogonal ribosomes and modifying attachments within living cells and test their ability to modifying a target protein such that it becomes localised to the edge of a cell when altered - a change we will be able to easily monitor using single-cell microscopy.This project is an attempt to develop the new methods needed to engineer the complex biological process of protein synthesis through the "augmentation" of a native biomolecular machine - the ribosome. Our flexible and modular approach using "plug-n-play" components offers the ability to rapidly alter the modifications made to a target protein without the need to build a new system from scratch, and opens opportunities for Biologists, Biological Engineers, and Material Scientists to better understand the function of proteins in their native context, precisely engineer their properties in living cells, and make use of highly modified proteins as nanoscale building blocks for new forms of sustainable, high-performance material. More broadly, our methodology also offers a path to harnessing other core cellular processes and repurposing their functionalities for novel applications in the emerging area of Engineering Biology.