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Title: Engineering a biological nanopore for protein sensing
Author: Ionescu, Sandra
ISNI:       0000 0004 8507 8690
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2020
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Protein nanopores have been engineered with chemical or genetic sensing elements for the stochastic detection of analytes at the single-molecule level. Upon insertion into a lipid bilayer, a nanopore allows an ionic current to flow across the bilayer under an applied transmembrane potential. Interactions between the sensing element and an analyte molecule modulate the ionic current, generating a unique electrical signature useful for analyte identification and characterisation. This thesis describes approaches to engineer the E. coli porin OmpF as a novel nanopore for stochastic sensing. In contrast to previously reported monomeric nanopore sensors, OmpF is a trimer composed of three identical β-barrels. As the most abundant porin in the bacterial outer membrane, it forms a major route for antibiotic permeation and is therefore of clinical interest. First, the orientation of OmpF in synthetic lipid bilayers was determined by covalent modification of introduced cysteine residues. This is a necessary step for dissecting periplasmic from extracellular interactions in a physiological context. The targeted covalent modification approach can be generally applied to determine the orientation of other porins and toxins. Alongside this, a protocol was developed for in vitro expression of OmpF to facilitate rapid production of mutants for studying bacterial physiology or prototyping stochastic sensors. The protocol was applied to construct heterotrimers, in which one or two of the three identical OmpF barrels were differently modified. This enables the production of nanopores with only one sensing element, eliminating the possibility of multivalent interactions that would complicate analysis of the sensing output. Finally, peptide libraries were engineered into the peripheral loops of OmpF and screened by bacterial display against an antibody protein target. Although initial attempts did not yield viable nanopore sensors, a foundation was established for the screening and testing of nanopore libraries based on their interactions with a target of interest. The ultimate goal is to produce sensors for any protein target, including disease biomarkers for which no suitable ligand exists. Future improvements for successful implementation of this technology, which would enable highly selective and multiplexed protein detection, are discussed.
Supervisor: Bayley, Hagan Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available