21ENGBIO Engineered orthogonal ribosomes for programmable protein modification

21ENGBIO 用于可编程蛋白质修饰的工程正交核糖体

基本信息

批准号：
BB/W012448/1
负责人：
Thomas Gorochowski
金额：
$ 12.84万
依托单位：
University of Bristol
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2022
资助国家：
英国
起止时间：
2022 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FW012448%2F1
关键词：
21ENGBIO Engineered orthogonal ribosomes programmable

项目摘要

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.

蛋白质是微小的纳米级分子机器，是所有活细胞的主力。它们支撑着传感和信号传导、新陈代谢协调甚至细胞结构元件自组装等关键任务。其中许多功能可以通过修改所涉及的蛋白质来定制，从而为细胞提供多样化其行为的方法。蛋白质在生物系统中的广泛应用使其成为工程新形式生物学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。能够按需合成和修饰蛋白质可以释放这一巨大潜力。在这个项目中，我们的目标是通过创建所谓的“正交核糖体”来直接应对这一挑战，它可以与细胞的天然过程并行合成蛋白质。重要的是，我们的正交核糖体将被设计为包括能够修饰正在合成的蛋白质的次要成分的附着点。通过用正交机制合成我们的蛋白质，我们可以避免以有害的方式修改天然细胞蛋白质，从而可以自由地以多种方式修改我们自己的蛋白质。此外，通过切换现有的修饰附件，我们可以轻松更改所做的修饰类型，从而创建可编程蛋白质合成和修饰的平台。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，这些方法可以创建大量具有不同附着点的正交核糖体设计，并评估它们对核糖体有效合成蛋白质的能力的影响。将选择那些效果良好的设计，然后使用计算机模型和模拟来修改精确设计的附件，以确保所涉及的修饰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活细胞内的修饰附着物，并测试它们修饰目标蛋白的能力，使其在改变时定位于细胞边缘——我们将能够使用单一的方法轻松监测这种变化。细胞显微镜。该项目试图开发通过“增强”天然生物分子机器（核糖体）来设计蛋白质合成的复杂生物过程所需的新方法。我们使用“即插即用”组件的灵活模块化方法能够快速改变对目标蛋白质所做的修饰，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为了更好地了解蛋白质在其天然环境中的功能，精确设计它们在活细胞中的特性，并利用高度修饰的蛋白质作为新型可持续高性能材料的纳米级构建模块。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重新利用其功能以用于工程生物学新兴领域的新应用的途径。