Spatial organisation and mechanical control of gene expression on the bacterial chromosome

在任何组织层次上，生命的一个关键条件是感知和正确应对动态环境的能力。在细胞水平上，感知这种环境的复杂信号机制最终导致染色体中基因表达的调节。细菌染色体线性压缩约1000倍，以适应称为类核的区域，支持基因调控和正确的DNA分离。了解基因调控的机制需要研究染色体组织和功能之间的联系，这是一个开放的问题，具有挑战性的动态性质的核。一个共同的假设是，传感和调控机制的运作是在纯粹的化学基础上。然而，在真核生物和原核生物中出现的证据表明，单独的化学不足以完全解释暗示生物化学和机械过程之间耦合的基因调控。基于细菌系统的现有知识和专业知识，我们工作的影响和目标将是进一步研究转录调控和组织，以及机械力在其中的作用。这项工作代表了牛津大学物理系两个生物物理研究小组之间的新合作：单分子“基因机器”小组由Alfrefs Kapananovich教授领导，他在体内和体外细菌转录和单分子超分辨率成像方面拥有丰富的专业知识。他们提供细胞力学、纳米级机械特性和材料操作方面的专业知识。 在这项工作中，我们将开发一种基于突变CRISPR dCas 9的新型遗传操作系统，与来自“基因机器”小组的Hafez El Sayyed博士密切合作-这可以用于荧光标记和生化询问。该新系统将能够同时对遗传基因座进行多色荧光成像，并将与单分子超分辨率显微镜一起用于研究染色体在3D空间中的结构。具体而言，我们将调查野生型物种中操纵子的相对接近度，以及突变株中剩余的操纵子数量;类核的潜在组织水平。另一个层次的组织可能是由液-液相分离的现象提供的，这一现象最近被认为与无膜细胞器的形成有关，最有趣的是真核细胞核仁。自然，这提高了在细菌类核中存在类似机制的可能性-受时间限制;这也可以使用多色荧光成像进行研究。 根据目标1的染色体组织成像还提供了将该工作与正在进行的工业相关项目整合的前景，该项目是在单细胞水平上快速检测抗菌素耐药性。在这里，我们将建立抗生素耐药表型的基础上的变化，类核结构响应抗菌剂，并补充与物种鉴定从靶向探针-无论是在单细胞水平。将其与宽视野显微镜相结合提供了建立临床测定的前景。我们将提供概念验证结果，首先使用同质样本，然后转向异质临床分离株。基因机器小组和牛津的约翰·拉德克利夫医院已经就这一倡议进行了合作。目的3机械力在染色体组织和调控中的作用机械力至少可以通过两种不同的方式影响基因调控。第一种是通过触发机械敏感的生化途径，

A crucial condition for life at any level of organisation is the ability to both perceive and correctly respond to a dynamic environment. At the cellular level, complex signalling mechanisms that sense this environment lead ultimately to the regulation of gene expression in the chromosome. The bacterial chromosome is linearly compacted by a factor of approximately 1000 to fit within a region called the nucleoid, supporting gene regulation and correct DNA segregation. Understanding the mechanisms of gene regulation requires the study of the link between chromosome organization and function an open question made challenging by the dynamic nature of the nucleoid.A common assumption is that the sensing and regulation machinery operates on a purely chemical basis. However, evidence is emerging across both eukaryotes and prokaryotes to suggest that chemistry alone is insufficient to fully explain gene regulation hinting at coupling between biochemical and mechanical processes. Building upon existing knowledge and expertise in bacterial systems, the impact and objective of our work will be further study of transcription regulation and organisation, and the role of mechanical forces on it. This work represents a novel collaboration between two Biological Physics research groups in the Department of Physics at the University of Oxford: The Single Molecule "Gene Machines" Group led by Prof. Achillefs Kapanidis, who have extensive expertise in both bacterial transcription and single-molecule super-resolution imaging, both in-vivo and in-vitro.The Nanoscience for Medicine Group, led by Prof. Sonia Contera, who offer expertise in cell mechanics, and nanoscale mechanical characterisation and manipulation of materials. Chromosomal organization in normal conditionsIn this work, we will develop a novel genetic manipulation system based on mutated CRISPR dCas9, working closely with Dr. Hafez El Sayyed from the "Gene Machines" group - this could be used for both fluorescent tagging and biochemical interrogation. The novel system will enable simultaneous multi-colour fluorescent imaging of genetic loci, and will be used with single-molecule super-resolution microscopy to investigate the structure of the chromosome in 3D space. Specifically we will investigate the relative proximity of operons in wild-type species, and of the remaining number of operons in mutant strains; the potential organisation levels of the nucleoid. Another level of organization may be provided by the phenomenon of liquid-liquid phase separation, which has recently been implicated in the formation of membrane-less organelles, most interestingly the eukaryotic nucleolus. Naturally, this raises the possibility of the existence of similar mechanism in the bacterial nucleoid - subject to time constraints; this could also investigated using multi-colour fluorescence imaging. Chromosomal organization under antibiotic-induced stress Imaging chromosomal organization as per Aim 1 also offers the prospect of integrating the work with an ongoing, industry-relevant project on rapid detection of antimicrobial resistance at the single cell level. Here, we will establish antibiotic resistance phenotypes based on changes to nucleoid structure in response to antimicrobial agents, and complement that with species identification from targeted probes - both at the single cell level. Combining that with wide field microscopy offers the prospect of establishing a clinical assay. We will provide proof-of -concept results, working firstly with homogenous samples and moving towards heterogeneous clinical isolates. Collaboration on this initiative is already in place between the Gene Machines group, and the John Radcliffe Hospital in Oxford. Aim 3 The role of mechanical forces in chromosomal organization and regulation Mechanical forces can affect gene regulation in at least two distinct ways. The first is by triggering of mechano-sensitive biochemical pathways a known t