An Integrated System for Geobacillus Protoplast Formation, Transfection, Recombinant Genomes Rebooting, Efficient Propagation, and Genomic DNA Isolation of the Thermophilic Bacteriophage TP‐84

ABSTRACT Protoplast transformation has been an effective method for introducing plasmid DNA into Bacillus species; however, its application to deliver complete bacteriophage genomes has not been demonstrated previously. Here, we describe an optimized and reproducible method for forming Geobacillus stearothermophilus protoplasts and efficiently transfecting them with purified recombinant or wild‐type genomic DNA from the thermophilic bacteriophage TP‐84. Systematic optimization of key parameters, including bacterial cell density, lysozyme concentration, polyethylene glycol type and concentration, and DNA input, enabled robust phage genomic DNA uptake and subsequent production of infectious phage particles. In addition, we optimized and scaled up a single‐layer solid‐state bacteriophage propagation procedure for integration into the TP‐84 genome‐engineering workflow, enabling the recovery of high‐titer lysates and sufficient genomic DNA for sequencing and subsequent genome manipulation from a single Petri dish. In our experimental system, solid‐state propagation also provided greater stability of recombinant phage variants than propagation in liquid bacterial cultures. Together, these methods establish an integrated protoplast‐based system for phage genomic DNA delivery, rebooting, propagation, and recovery, enabling controlled manipulation of bacteriophage genomes and facilitating the rapid generation and analysis of recombinant variants.