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An ultra-high throughput droplet microfluidics platform for the generation and screening of SICLOPPS cyclic peptide libraries in vitro

An ultra-high throughput droplet microfluidics platform for the generation and screening of SICLOPPS cyclic peptide libraries in vitro
An ultra-high throughput droplet microfluidics platform for the generation and screening of SICLOPPS cyclic peptide libraries in vitro
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.
University of Southampton
Sohrabi, Catrin
2b783876-baf5-4f48-bfea-8a9d463b12ce
Sohrabi, Catrin
2b783876-baf5-4f48-bfea-8a9d463b12ce
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2

Sohrabi, Catrin (2018) An ultra-high throughput droplet microfluidics platform for the generation and screening of SICLOPPS cyclic peptide libraries in vitro. University of Southampton, Doctoral Thesis, 196pp.

Record type: Thesis (Doctoral)

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.

Text
Sohrabi Final Thesis for Award - Version of Record
Restricted to Repository staff only until 30 June 2022.
Available under License University of Southampton Thesis Licence.

More information

Published date: September 2018

Identifiers

Local EPrints ID: 433178
URI: http://eprints.soton.ac.uk/id/eprint/433178
PURE UUID: 015a55a6-0e54-482c-bb02-f2d7ea0cf8d8
ORCID for Ali Tavassoli: ORCID iD orcid.org/0000-0002-7420-5063

Catalogue record

Date deposited: 09 Aug 2019 16:30
Last modified: 10 Aug 2019 00:34

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Contributors

Author: Catrin Sohrabi
Thesis advisor: Ali Tavassoli ORCID iD

University divisions

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