Preparation of Bacteriophage Lysate for Generalized Transduction

Resumo

Source: Saragliadis, A., et al. Producing Gene Deletions in Escherichia coli by P1 Transduction with Excisable Antibiotic Resistance Cassettes. J. Vis. Exp.(2018)

This video demonstrates preparation of a bacteriophage lysate containing transducing particles. A donor strain is infected, plaques form, and the soft agar overlay is processed with chloroform to yield a clarified lysate for downstream analysis.

Protocolo

1. Infection of the donor strain
   1. Grow the donor strain JW4197 in 5 mL of lysogeny broth (LB) medium supplemented with 10 mM CaCl₂ and optionally with Kan (25 µg/mL) to an optical density at 600 nm (OD₆₀₀) of ~1.0. Measure the OD₆₀₀ value using a spectrophotometer.
   2. Make a dilution series of an existing P1 phage stock in the LB medium: recommended dilutions are between 10^-3 to 10^-7.
   3. Mix 200 μL of the bacterial suspension and 100 μL of a given phage dilution in a 15 mL centrifuge tube or equivalent. Prepare as many tubes as phage dilutions. Incubate the tubes for 20 min at 37 °C without shaking.
   4. Add ~3 mL of molten top agar (~50 °C) supplemented with 10 mM CaCl₂ to the tubes, mix the contents thoroughly by vortexing the tubes shortly, and pour the mixtures onto prewarmed LB plates to make even layers.
   5. Incubate the plates overnight at 37 °C.
2. Lysate preparation
   1. The following day choose a plate with a semi-confluent growth of phage plaques. On a semi-confluent plate, approximately half the surface area of the plate is clear (Figure 1).
   2. Scrape the top agar layer from such a plate using an inoculation loop or a similar tool and place the top agar in a centrifuge tube. Add 1–2 mL of LB and a drop of chloroform and vortex the tube vigorously for ~1 min. Add the chloroform in a fume hood.
   3. Centrifuge the tube for 15 min at 4,000 x g or faster to pellet the agar and bacterial cells.
   4. Move the supernatant to a fresh microcentrifuge tube, avoiding carrying over any debris from the pellet. Add 2 drops of chloroform and store the lysate at 4–10 °C. Add chloroform in a fume hood. Do not freeze the phage lysate as this will result in a significant reduction of the number of infectious particles.

Resultados

Figure 1: Examples of plates after a P1 infection. (A) This panel shows a plate with individual plaques. (B) This panel shows a semi-confluent plate. (C) This panel shows an over-infected plate...

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Strains
E. coli JW4179	NIG	JW4179-KC	tamA deletion mutant
P1 vir	NIG	HR16	Generally transducing bacteriophage
Agar	BD Bacto	214010
Calcium chloride	Merck	102382
Chloroform	Merck	102445