Exploring the fitness effects of transgene insertions throughout the phage T7 genome using randomly-barcoded transposon insertion sequencing

Bacteriophages have immense potential for applications in fields such as phage therapy and microbiome engineering. Synthetic biology approaches can be used to generate phages with greater effectiveness and wider applicability. However, we currently lack tools for systematically exploring how inserting transgenes into phage genomes in different locations impacts phage function, stability, and viability. This thesis describes the development of a randomly-barcoded transposon insertion sequencing (RB-TnSeq) approach for simultaneously characterizing the effects of many transgene insertions into the phage T7 genome. To use this method, one first generates a transposon mutant library by selecting for insertion of a trxA gene cassette into the genomes of phages in an Escherichia coli host that does not supply this essential host factor. After associating individual barcodes with trxA insertion locations, barcode sequencing is used to measure changes in the frequencies of each insertion during further infection cycles under different conditions. While transposon insertions were found at locations throughout most of the T7 genome in the initial library generated in this work, there was a bias towards more insertions and higher frequencies for those insertions in the early region of the T7 genome that was not explained by their fitness effects. Using this library in experiments in E. coli host strains in which different T7 genes are essential demonstrated that this method can detect changes in gene essentiality under different conditions. In the future, this RB-TnSeq approach could be used to characterize the most effective and stable transgene insertion sites in phage T7 and other phages for which appropriate selectable markers can be discovered or engineered.