Controlling bacterial gene induction with mechanical forces and applying the technology for cell preservation in industrial biotechnology

机械力控制细菌基因诱导及细胞保存技术在工业生物技术中的应用

• 批准号: 2248631
• 金额: --
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2248631
• 关键词: Controlling; bacterial; gene; induction; mechanical

Bacterial cells are exposed to a range of mechanical forces during processing for industrial biotechnology purposes. For example, our partner Chr. Hansen prepares probiotic bacteria in a form that keeps them viable for several years, and to achieve it uses a range of preservation processing methods, e.g. freeze-drying. The mechanical forces that the cells are exposed to during these processes, as well as shape changes that they undergo are not fully characterized, and consequently, the changes in cell responses are not well understood. Similarly, the influence of the starting shape and pressure of the bacteria, which to an extent can be controlled with the media bacteria are grown in, on the subsequent response to mechanical forces are also not understood. For example, both mechanical forces and shape changes could be detrimental or enhance the cell survival, and if better characterized could be either avoided or employed during the industrial process. With this in mind in this project we will proceed along the following aims: (1) we will characterize shape changes and mechanical forces cells are exposed to during each of the steps in Chr. Hansen's preservation process and we will test how altering the response (by, for example, adjusting the osmolarity of the media) influences the cell survival; (2) we will test how the initial cell shape and size, which can be controlled with the available carbon sources, pH or temperature of the media cells are grown in, influences their response and survival to the mechanical forces and (3) we will use our recently developed technology to allow us to decouple the bacterial response to mechanical stress and directly asses the influence of mechanical forces on gene expression in bacteria. Bacteria regulate the expression of their genome in response to a wide range of environmental conditions, including temperature, external osmolarity, presence or absence of certain chemical species and density of neighbouring cells. However, and in contrast to mammalian cells, there remain relatively few examples of changes in bacterial gene expression directly in response to external mechanical forces. To achieve genetic expression of a fluorescent reporter protein in response to controlled mechanical compression, we will work with a model bacterium Escherichia coli, and use a part of its osmoregulatory network that is up-regulated at higher osmolarities and produces trehalose to help maintain E. coli's volume and osmotic pressure. In addition, custom microscopy and microfluidic platform we developed allows us to control the application of mechanical force to single E. coli's cells simultaneously with imaging. Using the platform and the reporter fluorescent protein, expressed both constitutively on the chromosome and on a plasmid, we will investigate the link between mechanical forces and gene expression. The knowledge gained will be coupled with our characterization of cell shape changes during the industrial biotechnology processing steps, to both understand the bacterial response to mechanical forces and immediately employ our understanding. The results will also be of wider applicability to the entire synthetic biology field who will be interested in mechanical induction and control of gene expression. Furthermore, even before processing steps for preservation, and during large scale fermentation cells are continuously exposed to shear forces. Understanding their response to it can be used to alter the type and speed of agitation during fermentation, e.g. to induce expression of genes that can help with cells' survival during the post-fermentation preservation processing steps.

在用于工业生物技术目的的加工过程中，细菌细胞暴露于一系列机械力。例如，我们的合作伙伴科汉汉森将益生菌制成可保持数年活力的形式，并使用一系列保存加工方法（如冷冻干燥）来实现这一目标。细胞在这些过程中所受到的机械力以及它们所经历的形状变化尚未完全表征，因此，细胞反应的变化尚未得到很好的理解。类似地，细菌的初始形状和压力对随后对机械力的响应的影响也不清楚，所述细菌的初始形状和压力在一定程度上可以用细菌生长的培养基来控制。例如，机械力和形状变化都可能是有害的或增强细胞存活，并且如果更好地表征，则可以在工业过程中避免或使用。考虑到这一点，在本项目中，我们将继续沿着以下目标：（1）我们将表征形状变化和机械力细胞暴露在每一个步骤中的Chr.汉森的保存过程中，我们将测试如何改变反应（例如，通过调节培养基的渗透压）影响细胞存活;（2）我们将测试初始细胞形状和大小如何生长，其可以用培养基细胞的可用碳源、pH或温度来控制，影响其对机械力的反应和存活;（3）我们将使用我们最近开发的技术，使我们能够分离细菌对机械应力的反应，并直接评估机械力对细菌基因表达的影响。细菌调节其基因组的表达以响应广泛的环境条件，包括温度、外部渗透压、某些化学物质的存在或不存在以及邻近细胞的密度。然而，与哺乳动物细胞相反，细菌基因表达直接响应外部机械力的变化的例子相对较少。为了实现响应于受控机械压缩的荧光报告蛋白的基因表达，我们将与模式细菌大肠杆菌合作，并使用其在较高渗透压下上调并产生海藻糖以帮助维持E.大肠杆菌的体积和渗透压。此外，我们开发的定制显微镜和微流控平台允许我们控制机械力对单个E.大肠杆菌的细胞同时成像。使用该平台和报告荧光蛋白，在染色体和质粒上组成型表达，我们将研究机械力和基因表达之间的联系。所获得的知识将与我们在工业生物技术加工步骤中对细胞形状变化的表征相结合，以了解细菌对机械力的反应并立即使用我们的理解。这些结果也将对整个合成生物学领域具有更广泛的适用性，这些领域将对基因表达的机械诱导和控制感兴趣。此外，甚至在用于保存的加工步骤之前，以及在大规模发酵期间，细胞连续地暴露于剪切力。了解它们对它的反应可以用于改变发酵期间搅拌的类型和速度，例如诱导可以在发酵后保存处理步骤期间帮助细胞存活的基因的表达。