Want, Andrew James (2010)
Ph.D. thesis, University of Birmingham.
Two staining protocols were formulated that enabled the detection of cellular stress at the single-cell level for Bacillus cereus. Both DiOC6(3) and RedoxSensor Green™ can be employed to detect perturbations in the energetic status of the cell at concentrations of 0.30 \mug.mL-1 and 3.0 \muM respectively. These methods can be employed for sensitive analysis of bacteria of both industrial and clinical interest. Flow cytometry was used throughout this work in order to assess the quality of recombinant Escherichia coli populations present within an agitated bioreactor. It was demonstrated in shake-flask culture that the cells could be grown to moderate cell densities (OD600nm 7 25) whilst producing measurable levels of antibody fragment. Despite being described in a patent which claims invention of a 100 % effective repression system (Hodgson et al., 2006), there was extensive evidence of promoter leakiness. Fab production was usually synonymous with cellular breakdown, however, a strategy based on simultaneous feeding and induction, before the exhaustion of the primary carbon source, yielded the highest concentration of Fab, 105 mg.L-1, with more than 50 mg.L-1 successfully targeted to the extracellular environment. Unlike all the previous cultures, this attainment also preceded the breakdown in the cellular structure.
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