Abstract 2130 Construction of a synthetic mycobacterium phage genome using gene editing and transformation-associated recombination (TAR) cloning in Saccharomyces cerevisiae

The rise of antibiotic resistance in pathogenic bacteria is driving 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. Recently, an yeast strain was engineered with over 50% synthetic DNA, with editing across 6 chromosomes. 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 construction of a synthetic genome of the mycobacterium phage Giles. With assembly and editing that provides proof of concept that synthetic phage genomes can be generated. Using exiting phage genome editing techniques 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 adapting Bacteriophage Recombineering of Electroporated DNA (BRED) and the yeast based transformation-associated recombination (TAR) system. Individual mini-chunks of DNA are incorporated via polymerase chain reaction (PCR) with a TAR cloning vector using specifically designed primers. Unique Phage DNA fragments are subjected to both deletion of nonessential genes or insertion of gain of function genes and then assembled into the full length phage genome inside yeast. The final objective of this project is to transform the synthetic phage into bacterial cells and measure its infectivity. The results of this project will show a 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. NSF Research Experiences for Undergraduates (REU): REU in Synthetic Biology