Establishment of a genetic toolbox for ammonia-oxidising archaea

Klein, Timothy Matsiko Ninsiima (2024) Establishment of a genetic toolbox for ammonia-oxidising archaea. Doctoral thesis, University of East Anglia.

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A genetic system is an essential tool to decipher the complex molecular mechanisms that dictate the physiology of an organism. The primary goal of this study was to establish a foundation for the development of a genetic system for ammonia-oxidising archaea (AOA) using ‘Ca. Nitrosocosmicus franklandus C13’ as a model. To facilitate mutant selection, a novel cultivation method (Liquid-Solid method) was developed that enables the growth of ammonia-oxidising archaea on solid medium as single colonies. The growth of ‘Ca. N. franklandus C13’ and N. viennensis EN76 on solid medium was dependent on the use of Phytagel™ (gellan gum) as the gelling agent as agar-based gels were inhibitory. The antibiotics puromycin and hygromycin B were identified as potential candidates for use as selective agents. Both antibiotics strongly
inhibited nitrite production at concentrations of > 27 μg/ml. Addressing the lack of native plasmids for the AOA, three E. coli - ‘Ca. N. franklandus C13’ shuttle vectors based on the chromosomal replication origin of ‘Ca. N. franklandus C13’ were assembled. Plasmid pfrank-CRISPR-amoB, contains the necessary elements needed for CRISPR-Cas9-based genome editing. In contrast, the plasmids pfrank-mCherry- Cdc-orb and pfrank-mCherry-orb are mCherry reporter gene-based plasmids. In addition to containing a reporter gene, the latter plasmids differ in the presence or absence of a cis or trans Orc1/Cdc6 encoding cdc gene respectively. All three plasmids contain the puromycin resistance gene (pac). The transformation attempts of ‘Ca. N. franklandus C13’ using either CaCl2 heat-shock, electroporation and polyethylene glycol were unsuccessful. This work serves as a much needed reference for future efforts aiming to establish a genetic system for the AOA.

Key words: Ammonia-oxidising archaea (AOA), genetic system, Phytagel™, solid medium,
colonies, antibiotics, electroporation, polyethylene glycol, CaCl2 heat-shock.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Zoe White
Date Deposited: 27 Jun 2024 15:22
Last Modified: 27 Jun 2024 15:22

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