Bacteria can quickly adapt to changing environmental conditions by activating alternative sigma factors. It has been shown previously that single cell approaches can reveal hidden dynamics in sigma factor activation. Here, we investigate the single cell response dynamics of the B. subtilis extracytoplasmic function sigma factors, which are an important part of the cell envelope stress response, under their specific stresses. To do this we use transcriptional reporters of sigma factors, quantitative single cell snapshots, time-lapse microscopy, and microfluidics. By developing an improved microfluidics setup for single cell time-lapse microscopy, as well as improved single cell analysis code, we are able to observe new sigma factor dynamics. First, we observe heterogeneous entry into a higher $\sigma^{V}$ activity state in response to lysozyme, which displays a memory, as the heterogeneity is lost on removal and reapplication of the stress. Next, we observe a pulse amplitude and duration modulated sigma factor response of $\sigma^{M}$ to bacitracin. Finally, for $\sigma^{M}$ under ethanol and acidic stress, and for $\sigma^{Y}$ under ethanol stress, we observe a noisy increase in activity to a new steady state level, where the degree of variability between cells depends on the stress condition. This thesis also discusses efforts on building a single cell microfluidic device based on the ”mother machine” design, for the rod-shaped cyanobacterium, S. elongatus, which forces the cells to grow in a straight line. Growing this organism in a traditional mother machine device has, so far, proved challenging. To adapt the mother machine for cyanobacteria we modify the channel geometry using electron beam lithography, and improve the loading protocol. The research presented here reveals the range of regulatory dynamics possible for ECF sigma factors in B. subtilis, and provides improved microfluidics and analysis code that will enable easier quantification of bacterial gene circuits at the single cell level in the future.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:753484 |
| Date | January 2018 |
| Creators | Schwall, Christian Philipp |
| Contributors | Locke, James |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/280309 |
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