A multi-user access laser tweezers, fluorescence and interference microscopy facility for understanding force at the molecular level

多用户访问激光镊子、荧光和干涉显微镜设备，用于了解分子水平的力

• 批准号: BB/T017767/1
• 负责人: Neil Kad
• 金额: $ 96.76万
• 依托单位: University of Kent
• 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%2FT017767%2F1
• 关键词: multi; user; access; laser; tweezers

Biological systems are affected by numerous external factors such as temperature, osmotic stress, and force. The latter occurs on multiple size scales, DNA repair proteins are subject to collisions with other proteins bound to DNA, immune cells migrating to a site of infection must push past obstacles and muscles contract against load. These are all examples of the impact of force on biological systems. At present, we lack a clear understanding of these processes because of insufficient access to instruments capable of studying the effects of force. In this proposal, we aim to fill this gap by installing an optical trapping system at the University of Kent known as the Lumicks C-trap. Optical trapping (also known as laser tweezers), the discoverers of which were awarded the Nobel prize last year, is a technique that allows beads or other objects, including vesicles, cells or organelles to be captured by a focused beam of light. This 'tractor beam'-like technology then enables investigators to physically manipulate the biological system of interest. The architecture of such assays could include suspending a single molecule of DNA between two beads and then assembling protein complexes on the DNA by dipping the DNA into different solutions made possible by the Lumicks C-trap microfluidic chamber. Using fluorescence to check for assembly the force-dependence of the system can then be investigated. By applying these forces in vitro we learn the properties of the system and how it would respond in its native environment in the cell, where it is not presently possible to perform such measurements.Alternatively, we can capture pathogenic yeast cells and measure their adhesion to materials on the flow chamber surface, and their response to drugs affecting adhesion can be directly measured. The uses of this system are vast and in this proposal we present seven projects with a diverse spectrum of applications.The system we are proposing to install possesses multiple combined functionalities, microfluidics, optical trapping, TIRF, widefield fluorescence and interference reflection microscopy (IRM). This latter technology uses the interference of light to detect objects without any label. This means that in some experiments where labelling affects activity, using IRM overcomes this limitation. Indeed, one project in this proposal seeks to use IRM to measure the formation of protein complexes in a membrane coated surface. By combining these capabilities the resulting system is very powerful, and also unique. In the UK there are no systems with this capability, and across the world there are only two. We want to ensure wide access to this technology and therefore we are reserving 25% of instrument time for external use. We will bring investigators to Kent to train on the system and to try out force experiments on their biological systems. The environment at Kent is ideal for the C-trap, the PI is an expert in the use and development of optical trapping technologies and this project also includes a second expert in optical trapping from the University of Nottingham. Also, at the University of Kent we have a diverse range of investigators that will be exposed to the capabilities of this system and therefore we will achieve more rapid diversification of application, which in turn will bring more investigators to Kent to use the C-trap. Finally, this system is not an add-on to an existing system, nor is it an incremental advance in our capabilities. The C-trap offers a genuine step change in the capabilities of researchers across the UK, and this is the right time and right group of investigators to support such an instrument.

生物系统受到许多外部因素的影响，例如温度、渗透压和力。后者发生在多个尺寸尺度上，DNA 修复蛋白会与其他与 DNA 结合的蛋白发生碰撞，迁移到感染部位的免疫细胞必须越过障碍物，并且肌肉会收缩以抵抗负载。这些都是力对生物系统影响的例子。目前，由于缺乏能够研究武力影响的仪器，我们对这些过程缺乏清晰的了解。在本提案中，我们的目标是通过在肯特大学安装称为 Lumicks C-trap 的光学捕获系统来填补这一空白。光学捕获（也称为激光镊子）的发现者去年获得了诺贝尔奖，这是一种允许聚焦光束捕获珠子或其他物体（包括囊泡、细胞或细胞器）的技术。这种类似“牵引光束”的技术使研究人员能够物理操纵感兴趣的生物系统。此类测定的架构可能包括将单个 DNA 分子悬浮在两个珠子之间，然后通过将 DNA 浸入由 Lumicks C-trap 微流体室提供的不同溶液中，在 DNA 上组装蛋白质复合物。使用荧光来检查装配，然后可以研究系统的力依赖性。通过在体外应用这些力，我们了解系统的特性以及它在细胞的天然环境中如何响应，目前无法进行此类测量。或者，我们可以捕获致病酵母细胞并测量它们对流动室表面材料的粘附，并且可以直接测量它们对影响粘附的药物的反应。该系统的用途广泛，在本提案中，我们提出了七个具有不同应用范围的项目。我们建议安装的系统具有多种组合功能，包括微流体、光捕获、TIRF、宽场荧光和干涉反射显微镜 (IRM)。后一种技术利用光的干涉来检测没有任何标签的物体。这意味着在一些标记影响活性的实验中，使用 IRM 克服了这一限制。事实上，该提案中的一个项目旨在使用 IRM 来测量膜涂层表面中蛋白质复合物的形成。通过结合这些功能，所得系统非常强大且独特。在英国，没有具有此功能的系统，而全世界也只有两个。我们希望确保这项技术的广泛使用，因此我们保留 25% 的仪器时间供外部使用。我们将带研究人员到肯特郡接受系统培训，并在他们的生物系统上尝试力实验。肯特大学的环境非常适合 C-trap，PI 是光捕获技术使用和开发方面的专家，该项目还包括来自诺丁汉大学的第二位光捕获专家。此外，在肯特大学，我们有各种各样的研究人员将接触到该系统的功能，因此我们将实现更快速的应用多样化，这反过来又会吸引更多的研究人员到肯特使用 C-trap。最后，该系统不是现有系统的附加组件，也不是我们能力的增量进步。 C-trap 为英国研究人员的能力带来了真正的进步，现在是支持这种仪器的正确时机和正确的研究人员群体。

项目成果

Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC.

• DOI: 10.1042/bsr20220403
• 发表时间: 2022-06-30
• 期刊: Bioscience reports
• 影响因子: 4

Single-molecule analysis of DNA-binding proteins from nuclear extracts (SMADNE).

• DOI: 10.1093/nar/gkad095
• 发表时间: 2023-04-24
• 期刊: Nucleic acids research
• 影响因子: 14.9

Combining cancer chemotherapeutics with bacterial DNA repair inhibitors to develop novel antimicrobials

• DOI: 10.1101/2023.03.17.532951
• 发表时间: 2023-03
• 期刊: bioRxiv
• 作者: L. Bernacchia;Arya Gupta;A. Paris;Alexandra A. Moores;N. Kad