A Protocol to Engineer Bacteriophages for Live-Cell Imaging of Bacterial Prophage Induction Inside Mammalian Cells

The gut microbiome is dominated by lysogens, bacteria that carry bacterial viruses (phages). Uncovering the function of phages in the microbiome and observing interactions between phages, bacteria, and mammalian cells in real time in specific cell types are limited by the difficulty of engineering fluorescent markers into large, lysogenic phage genomes. Here, we present a method to multiplex the engineering of life-cycle reporters into lysogenic phages and how to infect macrophages with engineered lysogens to study these interactions at the single-cell level. For complete details on the use and execution of this protocol, please refer to Bodner et al. (2020).