权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

A Label-free, Ultra-High-Throughput Bacteriolysis Droplet Screening Platform (KillerDrop)

无标记、超高通量溶菌液滴筛选平台 (KillerDrop)

基本信息

批准号：
BB/T011777/1
负责人：
Fabrice GIELEN
金额：
$ 19.09万
依托单位：
UNIVERSITY OF EXETER
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2020
资助国家：
英国
起止时间：
2020 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FT011777%2F1
关键词：
Label free Ultra Throughput Bacteriolysis

项目摘要

Some viruses called 'phages' have developed, over millions of years, strategies to invade bacteria in which they multiply and kill them from inside. We can use and develop similar strategies in the laboratory to find new ways to kill bacteria. This is important because the rapid emergence and spread of antimicrobial resistance has evolved into a major healthcare threat. However, the development of new phage-inspired therapies depends on our understanding of how phages interact with bacteria. For instance, some phages will infect bacteria but not kill them. Current methods to observe these interactions are very slow and hard to interpret because they do not track the fate of every bacteria. What we need are novel techniques that can track bacteria individually when in presence of lytic agents. In this project, we will encapsulate bacteria and phages in tiny water-in-oil microdroplets and record images so that we count every single bacterium that is killed by phages. This will help us better understand how and in what time bacteria get lysed. The proposed research will provide a tool to understand these interactions for up to a million simultaneous experiments, enabling us to have a complete picture of environmental conditions required for cell lysis.The key proteins that help phages kill bacteria are called lysins. They are enzymes that can degrade bacterial membranes until bacteria burst open. Importantly, these enzymes can be modified in the laboratory to make them more efficient at killing bacteria, in a process called directed evolution. However, this process is slow, tedious and costly, often limited to testing a handful of changes in the gene sequence encoding the enzymes and, furthermore, is focused on a relatively small pool of previously characterised enzymes. There is a clear need for technologies that allow us to make huge numbers (millions) of changes to the enzymes and screen them rapidly. Such technologies should also isolate the very best enzymes so that we can test their properties as therapeutic agent in isolation. The new technology we propose will allow us to express thousands of fragments of DNA (molecules encoding enzymes) into lysins all at once. We will then use cutting edge micro-plumbing to combine all these proteins with bacteria. Using state-of-the-art optical and electronics instrumentation, we will build a new platform for detecting and counting the number of lysed bacteria. This innovative and challenging combination of technology will allow us to screen huge numbers of variant lysins and search for DNA sequence encoding the most active ones.The power of the technology proposed will allow us to take high-throughput measurements surpassing current approaches. Specifically, the new technology that will be utilised for detecting bacteria lysis will be around 1000 times faster than current technologies used. Our proposed project will unlock the door to a range of new approaches for both investigating how bacteria and phages interact, understand how lysin evolution relate to their lytic function and the development of new antimicrobial drugs.

一些被称为“噬菌体”的病毒经过数百万年的发展，已经形成了入侵细菌并在其中繁殖并从内部杀死细菌的策略。我们可以在实验室中使用和开发类似的策略来寻找杀死细菌的新方法。这很重要，因为抗菌素耐药性的迅速出现和传播已演变成主要的医疗威胁。然而，新的噬菌体疗法的开发取决于我们对噬菌体如何与细菌相互作用的理解。例如，一些噬菌体会感染细菌但不会杀死它们。目前观察这些相互作用的方法非常缓慢且难以解释，因为它们无法追踪每种细菌的命运。我们需要的是能够在存在裂解剂的情况下单独追踪细菌的新技术。在这个项目中，我们将把细菌和噬菌体封装在微小的油包水微滴中并记录图像，以便我们计算出被噬菌体杀死的每一个细菌。这将帮助我们更好地了解细菌如何以及何时裂解。拟议的研究将提供一种工具来了解这些相互作用，进行多达一百万个同时进行的实验，使我们能够全面了解细胞裂解所需的环境条件。帮助噬菌体杀死细菌的关键蛋白质称为溶素。它们是可以降解细菌膜直到细菌破裂的酶。重要的是，这些酶可以在实验室中进行修改，使其更有效地杀死细菌，这一过程称为定向进化。然而，这个过程缓慢、繁琐且成本高昂，通常仅限于测试编码酶的基因序列中的少数变化，而且集中于相对较小的先前表征的酶库。显然，我们需要能够对酶进行大量（数百万）次改变并快速筛选的技术。此类技术还应该分离出最好的酶，以便我们可以单独测试它们作为治疗剂的特性。我们提出的新技术将使我们能够一次性将数千个 DNA 片段（编码酶的分子）表达到溶素中。然后，我们将使用尖端的微型管道将所有这些蛋白质与细菌结合起来。使用最先进的光学和电子仪器，我们将建立一个新的平台来检测和计数裂解细菌的数量。这种创新且具有挑战性的技术组合将使我们能够筛选大量的变异溶素并搜索编码最活跃的溶素的 DNA 序列。所提出的技术的力量将使我们能够进行超越当前方法的高通量测量。具体来说，用于检测细菌裂解的新技术将比当前使用的技术快约 1000 倍。我们提出的项目将为一系列新方法打开大门，这些新方法可用于研究细菌和噬菌体如何相互作用、了解溶素进化与其裂解功能的关系以及新抗菌药物的开发。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Handbook of Single-Cell Technologies

单细胞技术手册

DOI：
10.1007/978-981-10-8953-4_47
发表时间：
2022
期刊：
影响因子：
0
作者：
Gielen F
通讯作者：
Gielen F

Phenotyping single-cell motility in microfluidic confinement.

DOI：
10.7554/elife.76519
发表时间：
2022-11-23
期刊：
eLife
影响因子：
7.7
作者：
Bentley SA;Laeverenz-Schlogelhofer H;Anagnostidis V;Cammann J;Mazza MG;Gielen F;Wan KY
通讯作者：
Wan KY

Multi-Object Detector YOLOv4-Tiny Enables High-Throughput Combinatorial and Spatially-Resolved Sorting of Cells in Microdroplets