Villanueva, Hugo Fernand Jean-Frédéric (2020) Refactoring bacteriophages as repurposed nanomachines. Doctoral thesis, Birkbeck, University of London.
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Abstract
Bacteriophages (phages) are viruses infecting both bacteria and archaea and are thought to be the most numerous biological entities on earth. The recent rise of antibiotic-resistant bacteria has engendered a renewed interest in the therapeutic uses of bacteriophages: phage therapy [1]. Advancements in molecular and synthetic biology have rendered possible phage engineering both at the structural level and at the genome level, enabling the phage to target different hosts [2] and deliver DNA packages [3], respectively. In Picovirinae, of the tailed phages order (Caudovirales), the connector is central to both capsid (head) formation and tail assembly. Engineering the clip domain of the connector could enable a Picovirinae to assemble a heterologous tail targeting a different host. In order to engineer the connector of Bacillus subtilis phage φ29, the structures of the connectors of phage GA-1 (B. pumilus host) and ΦCPV4 (Clostridium perfringens host) were determined via cryo-electron microscope at a resolution of 3.2 Å and 2.8 Å (FSC=0.143), respectively. This high resolution permits insightful analysis of conserved features of Picovirinae connectors. Conserved regions at the clip domain were used to create two chimeric connectors, the first steps towards redirecting a Bacillus phage to a Clostridium host. Furthermore, the development of a novel method for the delivery of multiple plasmids in one transformation event is presented. This has the potential to be delivered via refactored bacteriophages. Since directed evolution of large complexes and gene pathways may require libraries to be split over several plasmids, the MPD method overcomes a bottleneck in the transformation of multiple plasmids, allowing the delivery of multiple plasmids without losing library-generated diversity.
Metadata
| Item Type: | Thesis |
|---|---|
| Copyright Holders: | The copyright of this thesis rests with the author, who asserts his/her right to be known as such according to the Copyright Designs and Patents Act 1988. No dealing with the thesis contrary to the copyright or moral rights of the author is permitted. |
| Depositing User: | Acquisitions And Metadata |
| Date Deposited: | 23 Jan 2024 16:07 |
| Last Modified: | 24 Jan 2024 04:29 |
| URI: | https://eprints.bbk.ac.uk/id/eprint/52898 |
| DOI: | https://doi.org/10.18743/PUB.00052898 |
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