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

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.