21ENGBIO Engineered orthogonal ribosomes for programmable protein modification

蛋白质是微小的纳米级分子机器，是所有活细胞的主力。它们支撑着重要的任务，包括感知和信号，新陈代谢的协调，甚至是细胞结构元素的自我组装。这些功能中的许多都可以通过修饰相关的蛋白质来定制，从而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它们成为工程生物新形式或在材料科学等其他领域利用生物成分和功能的重要目标。能够按需合成和修改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”来直接解决这一挑战，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设计成包括二级组分的附着点，这些附着点能够修饰正在合成的蛋白质。通过用正交机制合成我们的蛋白质，我们避免了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，通过切换现有的修饰附件，我们可以很容易地改变所做修饰的类型，为可编程蛋白质合成和修饰创造了一个平台。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蛋白质的能力的影响。将选择那些工作良好的设计，然后使用计算机模型和模拟精确地设计修改附件，以确保涉及修改的区域完美地定位在核糖体上。最后，我们将结合工程正交核糖体和修饰活细胞内的附着物，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——这种变化我们将能够使用单细胞显微镜轻松监测。该项目试图通过“增强”一种天然生物分子机器——核糖体，来开发设计复杂的蛋白质合成生物过程所需的新方法。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的修饰而无需从头开始构建新系统的能力，并为生物学家，生物工程师和材料科学家提供了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的机会。并利用高度修饰的蛋白质作为纳米级的构建块，用于新型的可持续的高性能材料。更广泛地说，我们的方法也为利用其他核心细胞过程和将其功能重新用于工程生物学新兴领域的新应用提供了一条途径。

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.