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Title: Monitoring bacterial physiology during recombinant protein production using reporter gene technology
Author: Vizcaino Caston, Isaac
ISNI:       0000 0004 2713 7138
Awarding Body: University of Birmingham
Current Institution: University of Birmingham
Date of Award: 2012
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This work presents an evaluation of the applicability of gene reporter technology to monitor Escherichia coli stress in industrial conditions with special interest in recombinant protein production. Different reporter plasmids containing promoter sequences of genes of the heatshock response were utilized to monitor chaperone expression upon different sources of stress such as exposure to chemicals, temperature and anaerobic growth. Activation of the heat shock response was monitored by \(\beta\)-galactosidase activity from the reporter plasmid pQF50groE. Cultures responded to heat-shock, anaerobiosis and \(\beta\)-mercaptoethanol by increasing the expression of \(\sigma\)\(^{32}\)-related genes. The performance of fluorescence reporters containing varieties of GFP was measured by fluorimetry and flow cytometry. Low copy number plasmids were demonstrated to be better suited than medium-high copy plasmids to report stress in industrial conditions. Reporter plasmids containing the promoters of the chaperones DnaK and GroES were utilized to measure E. coli stress in reducing environments and during recombinant protein production. It was demonstrated that the production strategy caused an impact in the host physiology which determined the outcome of the process. Flow cytometry showed excellent potential to obtain reliable measurements providing data of reporter activity cell death and cell morphology.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: tics ; QR Microbiology ; TP Chemical technology