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Title: Assembly of modified and non-native proteins in Rhodobacter sphaeroides and Escherichia coli
Author: Grayson, Katie
ISNI:       0000 0004 5370 6576
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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Photosynthesis is a highly efficient and productive process that converts solar energy into chemical energy. Much of the visible and near-infrared radiation falling on the surface of the Earth is not absorbed by photosynthesising organisms, which occupy particular spectral niches depending on the absorption of the particular pigments they synthesise. For synthetic biology applications it would be worthwhile to design and construct bacteria that could utilise a greater range of wavelengths than naturally-evolved photosynthetic bacteria. Although the incorporation of synthetic chromophores to complement native light-harvesting proteins is promising, the approach generally involves in vitro reassembly. In order to create tailor-made light harvesting antennas in vivo, we must make use of the toolbox of proteins and pigments available in nature, or create synthetic elements that are able to be created by the host organism. To investigate the possibility of creating artificial light-harvesting antennas in vivo, the yellow fluorescent protein, YFP, was incorporated as a chromophore into the photosynthetic apparatus of the purple photosynthetic bacterium Rhodobacter (Rba.) sphaeroides. It is shown that energy absorbed by YFP can transfer to the native reaction centre and LH1 proteins, sufficient to enhance the photosynthetic growth rate in a Rba. sphaeroides carotenoidless mutant. The light-driven proton pump, proteorhodopsin (PR), also has potential to augment the proton motive force in Rba. sphaeroides and drive downstream metabolism. Rba. sphaeroides was engineered to express PR and its chromophore retinal. The gene for a transmembrane synthetic peptide maquette was designed and expressed in Rba. sphaeroides. This work forms the basis of the bottom-up redesign of photosystem components with the aim of augmenting photosynthesis in Rba. sphaeroides and in the long term to create new photosynthetic complexes and membrane assemblies. In addition, the plasticity of the E. coli Tat pathway for the export of maquettes was investigated, as the quality control mechanism of the Tat transporter makes it a desirable system for efficient large-scale protein production to facilitate further characterisation of maquette proteins.
Supervisor: Hunter, C. N. ; Leggett, G. J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available