Monitoring bacterial physiology during recombinant protein production using reporter gene technology

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http://eprints.org/relation/isVersionOf http://etheses.bham.ac.uk/id/document/19526 http://eprints.org/relation/isVolatileVersionOf http://etheses.bham.ac.uk/id/document/19526 http://eprints.org/relation/isIndexCodesVersionOf http://etheses.bham.ac.uk/id/document/19526 archive 4031 disk0/00/00/33/09 2012-05-04 13:38:52 2019-01-23 09:55:59 2012-05-04 13:38:52 thesis show 0 Vizcaino Caston Isaac Overton Tim University of Birmingham Monitoring bacterial physiology during recombinant protein production using reporter gene technology unpub QH426 QR TP 10col_ephy 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providing data of reporter activity cell death and cell morphology. 2012-07 graduation eng author unchecked This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. public 319 University of Birmingham School of Chemical Engineering d_ph na 2012-07 graduation