Engineering leave-one-out GFP based biosensors

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Authors
Fraser, Keith
Issue Date
2016-08
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Electronic thesis
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Language
ENG
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Biology
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Abstract
To this end we proposed the development of a biosensor platform based on the green fluorescent protein (GFP) called leave-one-out GFP (LOO-GFP) biosensors. This platform is built on the principle that when one of the 11 β-strands in GFP is excised (LOO-GFP) there is a loss in the native fluorescence of the molecule that can be reconstituted upon the addition of the missing peptide. Additionally, the pocket created as a result of the omission of any of the β-strands can be redesigned to accommodate an exogenous peptide sequence, allowing for LOO-GFPs to function as designable biosensors. Since this approach is a redesign of the existing molecule, we conducted a mechanistic study of the structure function relationship in GFP so as to identify the best engineering strategies. This work focuses on how the production of the fluorescent para-hydroxbenzylidene imidazolinone chromophore is regulated, the identification of optimal design scaffolds and developing methods that can be used to design LOO-GFP biosensors for diagnostics and environment surveillance.
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August 2016
School of Science
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Rensselaer Polytechnic Institute, Troy, NY
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