Amplification and Recovery of Infectious Bacteriophages

Abstract

Source: Rustad, M. et al. Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J. Vis. Exp. (2017)

This video demonstrates the amplification and recovery of infectious bacteriophages from E. coli cultures. It outlines the steps for plaque isolation, phage replication, controlled lysis, and purification.

Protocol

1. Phage Amplification

NOTE: The single-plaque, multi-cycle (SPMC) phage production technique is well described for the T4 phage. The following phage amplification and deoxyribonucleic acid (DNA) extraction method is a generalization for use with Escherichia coli (E. coli) double-stranded DNA phages, e.g., T7, T4, or L. The ultimate success of the protocol heavily depends upon the selected host cell strain's ability to withstand superinfection conditions. If the host cells are unstable under superinfection, or superinfection is never reached, and lysis is never reached during this critical phase, it is best to proceed with a confluent lysis protocol. This includes separating the cellular debris through high-speed centrifugation and phage pelleting at ultracentrifugation speeds. All the following conditions and parameters are intended as a general starting point. The optimum conditions for a local host cell line may be different; determine and adhere to the appropriate conditions.

1. Prepare Inoculation Cells
   1. Inoculate 10 mL Luria-Bertani (LB) media in a 15 mL culture tube with E. coli host cells, e.g., B or K12.
   2. Place in a shaker set to 250 rpm and 37 °C. Leave overnight to grow to saturation.
2. Single-plaque, multi-cycle phage growth and concentration
   1. To prepare a plate of competent phage plaques, warm several LB-agar plates at 37 °C for 1 h, or overnight.
   2. Prepare several dilutions of phage stock (e.g., T7, T4, or L) of known concentration in LB media, aiming for ~10²-10³ phage/mL.
   3. Add 100 µL of overnight growth of concentrated host cells to each plate.
   4. Choose volumes of the prepared phage dilutions corresponding to a range from 10-100 phage/plate. Add the required volume of phage dilutions to each plate (e.g., 100 µL of 10³ phage/mL; this should produce ~100 plaques). Incubate plates at 37 °C and begin a count-up timer (+ 0 h). Incubate for 4-5 h.
   5. Prepare 49 mL LB media in a 250 mL flask and place in a rotary shaker set to 250 rpm and 37 °C.
   6. At + 2.5 h, dilute cells 50x by adding 1 mL of saturated cell culture from overnight growth into a pre-warmed flask containing 49 mL LB medium. Once the cells reach log growth (+ 4-5 h), measure absorbance at 600 nm (OD₆₀₀). Determine the concentration of cells using the conversion OD₆₀₀ of 1.0 = 8 x 10⁸ cell/mL.
   7. Dilute to 2 x 10⁷ cells/mL using additional LB growth media. Remove the agar plate containing the phage plaques from the incubator. Immediately use the end of a sterile Pasteur pipette to core and remove a single plaque. Blow the plaque into the diluted cells and incubate at 37 °C for an additional 2 h (+ 6-7 h).
   8. Test for full infection by taking a 500 µL sample into a 1.5 mL tube, adding 10 µL chloroform (CHCl₃), and immediately vortexing at high speed. Observe if the solution has clarified. Once cells are fully infected, incubate for another 2 h (+ 8-9 h).NOTE: If the cells are fully infected, they will rapidly lyse (<2 min) and clarify the solution. Other outcomes are considered in the Discussion Section below. The cells will be superinfected but will not lyse up to this point. If lysis begins, the addition of DNase and harvesting by centrifugation must begin immediately to prevent phage degradation.
   9. Add DNase to 5 µg/mL and centrifuge cells at 8,000 x g at 4 °C to pellet. Discard the supernatant. Resuspend the pellet in 10 mL of 1x TRIS magnesium chloride (TM) (50 mM Tris at pH 7.8, 10 mM MgCl₂) buffer with 5 µg/mL DNase. Add 500 µL of CHCl₃ and vortex at high speed to lyse the cells. Clarify by centrifugation at 12,000 x g for 10 min at 4 °C. Decant the supernatant into a 15 mL conical tube and store at 4 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ultracentrifugation tubes	Beckman Coulter	344057
Conical tubes	Falcon	352070
Culture tubes	Fischerbrand	14-961-33
LB agar plate recipe			25 g/L Luria-Bertani medium (LB Broth, Miller - Fisher BioReagents product number BP1426) and 15 g/L Bacto-Agar solid (Brenton, Dickenson and Company - product number 214010).