Mar 15, 2023
Phage infection and timed harvest of E. coli and B. subtilis cells
- Januka Athukoralage1,
- Adair Borges1
- 1Arcadia Science
- Arcadia Science
Protocol Citation: Januka Athukoralage, Adair Borges 2023. Phage infection and timed harvest of E. coli and B. subtilis cells . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw2d5l5r/v1
Manuscript citation:
Athukoralage J, Borges A, Reiter T. (2023). Challenges of isolating bacteriophage mRNA for chemical analysis. https://doi.org/10.57844/arcadia-j03a-mz33
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 22, 2023
Last Modified: January 30, 2024
Protocol Integer ID: 77401
Keywords: phage, bacteriophage, SPO1, T4, phages, bacteriophages, E. coli, B. subtilis, Escherichia coli, Bacillus subtilis, infection, infect, harvest, grow, produce, bacteriophage transcript, harvesting cells at different timepoint, harvest cell, harvesting cell, phage t4, infection, timed harvest, cell, enrichment for downstream analysis, rna
Abstract
This protocol outlines how to infect E. coli and B. subtilis with phage T4 and SPO1, respectively, and harvest cells at set time points.
Harvesting cells at different timepoints during infection allows you to evaluate when bacteriophage transcripts are most abundant in cells and maximize their enrichment for downstream analyses such as chemical analysis and RNA sequencing.
Materials
50 mL sterile Erlenmeyer flasks
5 mL sterile and capped test tubes
1.5 mL microcentrifuge tubes
Bechtop centrifuge capable of cooling
Shaking incubator
Shaking mixer
Liquid nitrogen and cryogenic container for safe handling
Freezer at –70 °C or above
Troubleshooting
Bacteriophage infection
Prepare 30 mL subcultures of E. coli and B. subtilis at 1:100 dilution (300 µL in 30 mL) and grow to a target OD600 of 0.4 in a shaking incubator (180 rpm). For both E. coli and B. subtilis this is ~2 h at 37 °C. Supplement the subculture media (LB) with 1 mM CaCl2 and 1 mM MgCl2.
Aliquot 500 µL of cell culture into several 1.5 mL microcentrifuge tubes and inoculate with 50 µL of phage at the correct dilution to reach your target MOI (see our protocol on MOI calculations, linked below).
Here, we are infecting E. coli with T4 and and B. subtilis with SPO1. Be sure to simultaneously carry out a control experiment where you mock-infect 500 µL E. coli and B. subtilis cultures with 50 µL sterile SM buffer. Once inoculated, incubate the microcentrifuge tubes at 37 °C on a heated shaker block, shaking at 300 rpm.
Plate out phage and bacteria to calculate the actual MOI used in the experiment.
Cell harvest
Pre-cool a bench-top centrifuge to 4 °C and at desired time points, harvest cells by centrifuging at 5000 × g for 3 min. For example, cells can be harvested at 5 min and 15 min in the hope of capturing both earlier and later infection stages. This timing will be highly phage-dependent!
Remove the supernatant from the pellet and immediately flash-freeze the pellets in liquid nitrogen. You must be speedy during this process to effectively halt phage infection. You can store bacterial pellets at –80 °C and use later for downstream applications such as RNA extraction.