A sensor exploiting the properties of Green Fluorescent Protein (GFP) was developed and was used to optimise fennentation conditions for production of the soluble fusion protein CheYGFP. A 9-fold increase in yield was achieved. Genes encoding CheY, Defl and DXR were fused in-frame to the 5' and 3' end ofthe GFP gene to detennine whether this affected optimised production of soluble GFP fusion proteins. Different conditions were required for optimal accumulation of the recombinant protein and the optimised conditions for CheYGFP and DXRGFP were identical to those for CheY and DXR respectively. Conditions optimal for the accumulation of Defl, DeflGFP, GFPDefl, GFPCheY, GFPDXR and GFP were the same. During several fennentations the production of soluble recombinant protein did not increase after 12 h. This was concomitant with a rapid increase in the rate of carbon dio~ide production by the culture. Cell pellets after centrifugation contained a layer of bacteria that were not fluorescent, indicating the presence of a non-productive population of bacteria. Restriction mapping and sequence analysis confinned the presence after 12 h of bacteria carrying a plasmid in which a frameshift mutation had occurred. Improvement in the maintenance of the original plasmid was achieved by further manipulation offennentation conditions.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:487493 |
| Date | January 2007 |
| Creators | Jones, Joanna Jane |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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