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

一些被称为“病毒”的病毒在数百万年的时间里已经发展出了入侵细菌的策略，它们在细菌中繁殖并从内部杀死细菌。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。这一点很重要，因为抗菌素耐药性的迅速出现和传播已经演变成一个主要的医疗保健威胁。然而，新的噬菌体启发疗法的开发取决于我们对细菌如何与细菌相互作用的理解。例如，有些细菌会感染细菌，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。我们需要的是新的技术，可以在溶解剂存在时单独跟踪细菌。在这个项目中，我们将把细菌和益生菌封装在微小的油包水微滴中，并记录图像，这样我们就可以计算出益生菌杀死的每一个细菌。这将有助于我们更好地了解细菌是如何以及在什么时候被溶解的。拟议的研究将提供一种工具，用于了解多达一百万个同时实验的这些相互作用，使我们能够完整地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饰，使它们在杀死细菌时更有效，这一过程称为定向进化。然而，这一过程是缓慢的，繁琐的和昂贵的，往往限于测试少数的变化，在基因序列编码的酶，而且，集中在一个相对较小的池先前表征的酶。显然需要一种技术，使我们能够对酶进行大量（数百万）改变并快速筛选它们。这些技术还应该分离出最好的酶，这样我们就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同时将数千个DNA片段（编码酶的分子）表达为溶素。然后我们将使用尖端的微管道技术将所有这些蛋白质与细菌联合收割机结合起来。使用最先进的光学和电子仪器，我们将建立一个新的平台，用于检测和计数裂解细菌的数量。这种创新和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目前使用的技术快1000倍左右。我们提出的项目将打开一系列新方法的大门，这些方法既可以研究细菌和细菌之间的相互作用，也可以了解溶素的进化与其溶解功能的关系，还可以开发新的抗微生物药物。

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.