High resolution mapping of the model bacteriophage φX174 transcriptome and the rational elimination and creation of cryptic promoters
Creators: name: Logel, Dominic Yves
Publisher: name: Macquarie University
Year: 2023
Subjects: Other education not elsewhere classified

description: The modification and systematic control of bacteriophage transcriptional networks requires an in-depth knowledge of the specific bacteriophage transcriptome and the associated host effects. With this knowledge as a base, rational design choices can be made to modify the transcriptome to place it under greater control. In this thesis I have made multiple original contributions to knowledge, such as the generation of a high-resolution transcriptome for the bacteriophage φX174, the first in-depth transcriptional analysis of <em>Escherichia coli </em>C122 during infection, and the formulation of a rational engineering system to create and eliminate promoters internal to coding domains through synonymous codon changes. These findings have filled in the critical gaps in our knowledge of this phage-host relationship and generated the first rational system for eliminating internal promoter sequences. In this thesis I performed high-resolution RNA-sequencing of the model bacteriophage φX174 captured at lysis and have characterised the full transcriptomic profile of the phage. I confirmed the general canonical transcription model along with a new terminator, TB, and a potential new sense promoter, and two antisense promoters which are the first evidence of antisense transcription in a ssDNA virus. This has provided φX174 with an up-to-date and highly characterised transcriptome expanding our understanding of how this relatively simple transcriptional network functions. I further explored the bacteriophage-host relationship between φX174 and its host, <em>E. coli </em>C122, by characterising the host’s transcriptomic response to infection for the first time. This revealed large-scale effects on the host membrane components such as the activation of the phage shock protein membrane response. This occurred along with a general upregulation in the heat shock response components and large decreases in the expression of multiple transport systems. This analysis provides the first transcriptional evidence of what occurs within this laboratory strain during infection. Synthetic biology seeks to reimagine complex biological systems as a series of predictable controllable genetic circuits. However, this reimagining is complicated by the presence of internal regulatory elements. Standard design approaches fail to account for internal regulatory elements without resorting to large-scale codon usage changes to eliminate sequence motifs, however this can have severe deleterious effects on an organism. To both address this gap in ongoing φX174 refactoring designs, I develop a rational engineering system for eliminating internal promoter sequences through localised synonymous codon changes named COdon Restrained Promoter SilEncing (CORPSE). Utilising CORPSE I have eliminated the φX174 promoter <em>pB </em>activity in a reporter by 99 % through synonymous codons choices weakening the DNA-σ factor interaction. The CORPSE system was also inverted (iCORPSE) to create promoters internal to coding domains by selecting for synonymous codons strengthening the DNA-σ factor interaction. A single iCORPSE variant (BPROM-01) led to a &gt;10,000 % increase in sfGFP expression by actively functioning as a promoter. This leaves the CORPSE and iCORPSE methods as potentially crucial tools in the future synthetic biology applications to complex genomes by both eliminating internal promoter sequences in decompressed and refactored genomes, and creating new promoters in novel compressed circuits.