Construction of the mycobacterium phage Giles genome through transformation‐associated recombination (TAR) cloning in Saccharomyces cerevisiae

Abstract

The rise of antibiotic resistance in pathogenic bacteria has lead to a renewed interest in bacteriophages. These naturally occurring antimicrobial agents can potentially be exploited as novel therapeutic agents. Existing limitations such as bacterial resistance and narrow host range; however, are problematic. The advent of new techniques in genome science and synthetic biology have provided mechanisms to build unique phage genomes that confer new functions that may overcome these difficulties. Building upon methods developed in the Build‐a‐Genome course at Johns Hopkins University and the Synthetic Yeast Project ( www.syntheticyeast.org), we present the complete synthetic genome of the mycobacterium phage Giles. We adapted the novel assembly line approach introduced by Oldfield et al in 2017. We set out to construct the complete genome from 12 overlapping DNA fragments of the phage using the yeast based transformation‐associated recombination (TAR) system. Individual mini‐chunks of DNA were incorporated via polymerase chain reaction (PCR) with a TAR cloning vector using specifically designed primers. The DNA fragments were then assembled into the full length phage genome inside yeast. Genetic modifications were attempted by inserting novel DNA sequences into one or more fragments. The final objective of this project is to transform the synthetic phage into bacterial cells and measure its infectivity. The results of this project are expected to show successfully engineered and infectious clone of phage Giles. The significance of which will be to demonstrate the optimization of novel methods to construct a synthetic phage genome.