Table 8.

Requirements to Consider When Applying This Protocol to Other Phage:Bacterium:Mammalian Cell Combinations

Step	Requirement(s)
Before You Begin - Primer Design	•The bacteriophage genome is sequenced, and the relevant bacterial attB and phage attP sites are known •The phage genome region of interest to modify (for example, the lysis operon) is well annotated
Before You Begin – Prepare PCR Products	•A lysogenization protocol exists for the bacteriophage / bacterium pair •The bacterial genomic DNA can be easily extracted and purified to be used as PCR template
Recombinant Phage Particle Formation	•The bacterial strain of interest can be made highly competent for transformation of large DNA constructs •The bacteriophage of interest can both integrate into the bacterial genome and form infectious particles •The phage receptor or mechanisms to stimulate receptor expression is known for the bacterial strain of interest, such that an efficient infection can occur to form lysogens •A colony PCR protocol exists such that the lysogenic bacterial genome can be validated
Lysis reporter Functional Validation	•A stimulus (for example, UV or DNA-damaging antibiotics) is known for prophage induction for testing the functionality of the phage lysis reporter •The doubling rate of the bacterial strain of interest is compatible with live-cell imaging
Bacterial Lysogen Infection of RAW264.7 Cells	•The bacterial strain can be transformed with a plasmid constitutively expressing a fluorescent protein marker •The mammalian cell line of interest can stably express a nuclear, fluorescent protein marker •An infection protocol exists for the bacterial/mammalian cell pair
Live-Cell Imaging Conditions	•The mammalian cell line can adhere to the imaging plate and survive on the microscope for the duration of the bacterial infection (~24 h)