Assad-Garcia N, D'Souza R, Buzzeo R, Tripathi A, Oldfield LM, Vashee S, Fouts DE

Cross-genus Boot-up of Synthetic Bacteriophage in Staphylococcus aureus Using a New and Efficient DNA Transformation Method.

Applied and environmental microbiology. 2021-11-24; AEM0148621.

is an opportunistic pathogen causing a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but methods are needed for efficient transformation of large DNA molecules into to boot-up (i.e., rescue) infectious phages. We present a new, efficient and reproducible DNA transformation method, NEST (Non-Electroporation Transformation), for to boot-up of purified phage genomic DNA (at least 150 kb in length tested) and whole yeast-assembled synthetic phage genomes. This method is a powerful new tool for transformation of DNA in and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. The continued emergence of antibiotic resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy, but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages, but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional non-model Siphophage from a constructed synthetic genome. This method will facilitate not only the engineering of and phages for therapeutic applications but also the engineering of strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

PMID: 34818102