Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.781450
Title: An ultra-high throughput droplet microfluidics platform for the generation and screening of SICLOPPS cyclic peptide libraries in vitro
Author: Sohrabi, Catrin
ISNI:       0000 0004 7967 0735
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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Abstract:
In vitro compartmentalisation (IVC) has emerged as an attractive cell-free based technological platform for the miniaturisation and mass parallelisation of biochemical reactions in microscopic aqueous-in-oil emulsion droplets. In implementing this emulsion-based technology, this thesis describes the development of an ultra-high throughput drug discovery platform for the IVC of split-intein based SICLOPPS cyclic peptide libraries in femtolitre-sized aqueous compartments for the rapid identification of novel protein-protein interaction (PPI) inhibitors. In utilising the unique thermoresponsive properties of agarose, the efficient encapsulation and isothermally-mediated pre-DNA amplification of single plasmid DNA copies in approximately 113 fL agarose-in-oil femtodroplets is performed, yielding porous agarose beads comprising highly condensed concentrations of DNA. Solidified beads are thereafter employed in in vitro transcription/translation (IVTT) of the encoded cyclic peptide genes alongside a pETDuet-1 coencoded Aβ42-GFP fusion assay, whereby the aggregation state of the Aβ42 Alzheimer's disease peptide is linked to the correct folding of GFP, by co-encapsulating individual agarose particles alongside PURExpress IVTT machinery. Fluorescence-activated cell sorting (FACS) is subsequently used to demonstrate the feasibility through which beads exhibiting increased levels of GFP fluorescence, and thus of those comprising cyclic peptides with the ability to modulate the aggregation state of Aβ42, may be efficiently and selectively recovered at rates exceeding 107 per hour. Collectively, the isolation and recovery of a diverse Npu SICLOPPS cyclic peptide library via FACS is achieved, demonstrating the value and widespread utility of the droplet microfluidics screening platform as described herein for the prompt discovery of novel cyclic peptide inhibitors of challenging PPIs.
Supervisor: Tavassoli, Ali Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.781450  DOI: Not available
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