Edinburgh Research Archive logo

Edinburgh Research Archive

University of Edinburgh homecrest
View Item 
  •   ERA Home
  • Biological Sciences, School of
  • Biological Sciences thesis and dissertation collection
  • View Item
  •   ERA Home
  • Biological Sciences, School of
  • Biological Sciences thesis and dissertation collection
  • View Item
  • Login
JavaScript is disabled for your browser. Some features of this site may not work without it.

P1 phagemid-based delivery of Cas9 antimicrobial into Shigella flexneri

View/Open
Huan2022.pdf (4.550Mb)
Date
22/12/2022
Author
Huan, Yang Wei
Metadata
Show full item record
Abstract
A bacteriophage P1 phagemid system allowed efficient packaging of a DNA-sequence specific, Cas9 antimicrobial into transducing particles, which could be used to infect and kill pathogenic bacteria, such as Shigella flexneri. Although the transduction of a cas9 phagemid yielded significant antimicrobial effect on S. flexneri, the phagemid lysates produced a significant non-Cas9-mediated cytotoxic effect, which might be attributed to the presence of wildtype P1 phage and/or other cytotoxic factors in phagemid lysates. Treatment of phagemid lysates with a low concentration of polythene glycol (PEG) reduced the titre of wildtype P1 phage and led to a lower cytotoxicity of phagemid lysates on S. flexneri cells. Mutations introduced into the P1 lytic origin of replication (oriL) inhibited its lytic stage DNA replication, yet the mutant was incapable of producing phagemid transducing particles. Deletion of the DNA packaging site, pac, from the P1 genome significantly reduced the titre of wildtype P1 phage. A Δpac mutation, however, did not reduce the cytotoxicity of phagemid lysates significantly, when used for treatment of S. flexneri in vitro and in a zebrafish larvae infection model. An alternative P4 cosmid system allowed efficient packaging of cas9 construct into transducing units without the contamination of host and/or phage DNA. Contrastingly, treatment of S. flexneri with P4 cosmid lysates gave negligible cytotoxic killing of the bacterial cells, suggesting that the non-Cas9-mediated killing effect might be attributed to impurities associated with the P1 phagemid transducing units and/or due to inherent properties of P1 transduction. Nevertheless, the versatility of the P1 phagemid system allowed an inducible Cpf1-mediated, vector curing function to be incorporated into the phagemid while retaining its Cas9-mediated antimicrobial effect on S. flexneri cells. Furthermore, the P1 cas9 phagemid was compatible with the ΔnadC autotrophy complementation system, providing antibiotic-free maintenance and selection of the modified vector in ΔnadC E. coli. Taken together, the phagemid and/or cosmid system that produces pure transducing particles might be the key to achieving a high Cas9 antimicrobial effect on S. flexneri cells with negligible lysate cytotoxicity.
URI
https://hdl.handle.net/1842/39659

http://dx.doi.org/10.7488/era/2908
Collections
  • Biological Sciences thesis and dissertation collection

Library & University Collections HomeUniversity of Edinburgh Information Services Home
Privacy & Cookies | Takedown Policy | Accessibility | Contact
Privacy & Cookies
Takedown Policy
Accessibility
Contact
feed RSS Feeds

RSS Feed not available for this page

 

 

All of ERACommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsPublication TypeSponsorSupervisorsThis CollectionBy Issue DateAuthorsTitlesSubjectsPublication TypeSponsorSupervisors
LoginRegister

Library & University Collections HomeUniversity of Edinburgh Information Services Home
Privacy & Cookies | Takedown Policy | Accessibility | Contact
Privacy & Cookies
Takedown Policy
Accessibility
Contact
feed RSS Feeds

RSS Feed not available for this page