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

在工业生物技术加工过程中，细菌细胞暴露在一系列机械力下。例如，我们的合作伙伴Chr。汉森将益生菌制成一种能使其存活数年的形式，并使用一系列保存处理方法来实现这一目标，例如冷冻干燥。在这些过程中，细胞所受到的机械力以及它们所经历的形状变化并没有得到充分的表征，因此，细胞反应的变化也没有得到很好的理解。同样，细菌的初始形状和压力（在一定程度上可以通过细菌生长的培养基来控制）对随后对机械力的响应的影响也不清楚。例如，机械力和形状变化都可能是有害的，或提高细胞存活率，如果更好地表征，可以避免或在工业过程中使用。考虑到这一点，在这个项目中，我们将沿着以下目标进行：(1)我们将描述形状变化和细胞在Chr的每个步骤中暴露的机械力。汉森的保存过程，我们将测试如何改变反应（例如，通过调整介质的渗透压）影响细胞存活；(2)我们将测试细胞的初始形状和大小是如何影响它们对机械力的反应和存活的，这可以用可用的碳源、pH值或培养基的温度来控制。(3)我们将使用我们最近开发的技术，使我们能够解耦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缺乏以及邻近细胞的密度。然而，与哺乳动物细胞相比，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。为了在受控的机械压缩下实现荧光报告蛋白的基因表达，我们将与大肠杆菌模型细菌合作，并使用其渗透调节网络的一部分，该网络在较高的渗透压下上调并产生海藻糖，以帮助维持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使我们能够在成像的同时控制机械力对单个大肠杆菌细胞的应用。利用该平台和在染色体和质粒上均组成表达的报告荧光蛋白，我们将研究机械力与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处理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应用我们的理解。结果也将广泛适用于整个合成生物学领域谁将感兴趣的机械诱导和基因表达的控制。此外，即使在保存的加工步骤之前，以及在大规模发酵期间，细胞也不断地暴露在剪切力下。了解它们对搅拌的反应可以用来改变发酵过程中搅拌的类型和速度，例如，在发酵后保存处理步骤中诱导有助于细胞存活的基因表达。

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.