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，如温度、渗透应力和力。后者发生在多个大小尺度上，DNA修复蛋白容易与其他与DNA结合的蛋白质发生碰撞，免疫细胞迁移到感染部位必须冲破障碍，肌肉因负荷而收缩。这些都是力对生物系统影响的例子。目前，我们对这些进程缺乏清楚的了解，因为没有足够的机会使用能够研究力的影响的工具。在这个提案中，我们的目标是通过在肯特大学安装一个被称为Lumicks C-trap的光学捕获系统来填补这一空白。光捕获（也被称为激光镊子）是一种技术，它的发现者去年获得了诺贝尔奖，它是一种允许珠子或其他物体，包括囊泡、细胞或细胞器被聚焦的光束捕获的技术。这种类似“牵引波束”的技术使研究人员能够对感兴趣的生物系统进行物理操纵。这种实验的结构可以包括将单个DNA分子悬浮在两个小珠之间，然后将DNA浸入不同的溶液中，在DNA上组装蛋白质复合物，这种溶液是由Lumicks C-trap微流控室制造的。使用荧光检查组装，然后可以研究系统的力依赖性。通过在体外施加这些力，我们了解了系统的特性，以及它在细胞内的自然环境中如何反应，目前还不可能在细胞内进行此类测量。或者，我们可以捕获致病酵母细胞，测量其对流室表面物质的粘附，直接测量其对影响粘附的药物的反应。该系统的用途是广泛的，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，微流体，光学捕获，TIRF，宽视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动的实验中，使用IRM克服了这一限制。事实上，该提案中的一个项目试图使用IRM来测量膜涂层表面蛋白质复合物的形成。通过结合这些功能，生成的系统非常强大，而且是独一无二的。在英国，没有具备这种能力的系统，全世界也只有两个。我们希望确保这项技术的广泛使用，因此我们保留了25%的仪器时间供外部使用。我们将把调查人员带到肯特郡，对该系统进行培训，并尝试在他们的生物系统上进行强制实验。肯特大学的环境非常适合c -阱，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光捕获专家。此外，在肯特大学，我们有各种各样的研究人员，他们将接触到这个系统的功能，因此我们将实现更快的应用多样化，这反过来将吸引更多的研究人员到肯特使用C-trap。最后，这个系统不是对现有系统的附加，也不是对我们能力的增量推进。C-trap为全英国的研究人员的能力提供了一个真正的步骤变化，这是正确的时间和正确的研究小组来支持这样的工具。

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