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

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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.