CONSTRUCTION OF BACTERIOPHAGE GENOMIC DNA FRAGMENT LIBRARIES

The arms race between bacteria and their viruses (bacteriophages) is a millions-of-year-long struggle that has led to the mutual evolution of microorganisms [1]. One of the defense strategies used by bacteria against bacteriophages is abortive infection (Abi). Abi is a group of different systems that work by the same mechanism, where phage infection initiates cell death or metabolic slowdown to prevent the completion of phage replication cycle and infection of neighboring cells [2]. Many mechanisms of defense systems underlying abortive infection remain poorly understood and identifying the factors that activate abortive infection remains a major challenge [3]. So far, one approach to identifying genes that activate abortive infection has been the isolation and sequencing of bacteriophage mutants that escape the effects of defense systems. However, isolation of such phage mutants is successful only for a smaller part of the systems, and other assays are slower and of low-throughput [4]. Our study seeks to overcome the limitations of phage mutant assays and introduce a faster method based on high-throughput phage genes testing. This method is based on the construction of bacteriophage genomic DNA fragment libraries. To construct these phage libraries, first we amplified phage genomic DNA using Oxford Nanopore PCR Barcoding Kit, which allowed us to obtain 3000 kb and longer DNA fragments. These DNA fragments were then cloned into high-copy and low-copy cloning vectors to test whether toxic phage genes are better retained in the low-copy vector. As expected, the number of colonies differed between high-copy plasmid and low-copy plasmid phage libraries. The quality of constructed libraries was then assessed using the Oxford Nanopore sequencing method. The sequences of the phage library were then aligned to the reference phage genome. The reference phage genome was almost completely and evenly overlaid, which showed that the phage libraries were constructed successfully. The libraries will be further screened in cells, coding Abi systems of interest. The constructed phage library is transformed together with the Abi system, and library genes lost due to activated Abi proteins potentially encoding activators would be identified by sequencing.