Mar 24, 2026
Propagation and Stock Preparation of Temperate Bacteriophage ΦV10::NanoLuc in Escherichia coli O157:H7 C7927
- Caleb Waddell1
- 1Purdue University
Protocol Citation: Caleb Waddell 2026. Propagation and Stock Preparation of Temperate Bacteriophage ΦV10::NanoLuc in Escherichia coli O157:H7 C7927. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld6do9g5b/v1
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: In development
We are still developing and optimizing this protocol
Created: November 22, 2025
Last Modified: March 24, 2026
Protocol Integer ID: 233259
Keywords: escherichia coli o157, apple cider isolate, efficient laboratory host for this phage, productive infection, phage, standard host for this phage, h7 antigen, known efficient laboratory host, overnight growth of c7927, selection of kanamycin, infection, background reporter phage stock, single lysogenic colony
Abstract
This protocol describes the propagation, lysogen selection, induction, harvest, and concentration of the NanoLuc reporter bacteriophage ΦV10::NanoLuc in Escherichia coli O157:H7 strain C7927, an apple cider isolate that is widely used as the standard host for this phage. Productive infection appears to require both the O157 and H7 antigens; C7927 is currently the only known efficient laboratory host for this phage.
The workflow begins with overnight growth of C7927, infection with ΦV10::NanoLuc, selection of kanamycin-resistant lysogens, and isolation of a single lysogenic colony. A 2×YT culture of the lysogen is then induced with mitomycin C, allowed to lyse, clarified, treated with chloroform, and concentrated over a sucrose cushion. The recovered pellet is resuspended in SM buffer and further processed using 100 kDa MWCO centrifugal ultrafiltration devices (e.g., Amicon Ultra-15) to reduce free NanoLuc, yielding a concentrated, low-background reporter phage stock.
Materials
Biological materials
- E. coli O157:H7 C7927 strain.
- ΦV10::NanoLuc phage stock (initial working stock or lab reference).
- ΦV10::NanoLuc lysogen of C7927 (generated within this protocol).
Media supplements
- LB broth (lab formulation)
- 10 g tryptone
- 10 g NaCl
- 5 g yeast extract
- Bring to 1 L with distilled water; autoclave.
- LB agar plates with kanamycin
- LB base as above
- 1.4% (w/v) agar
- Kanamycin 50 μg/mL (added after autoclaving and cooling to ~55 °C).
- 2×YT broth
- 16 g tryptone
- 10 g yeast extract
- 5 g NaCl
- Bring to 1 L with distilled water; autoclave.
- Soft agar (top agar) for plaque assays
- LB or 2×YT base
- 0.7% (w/v) agar
- Optional but recommended: supplement to 10 mM MgSO4 and 5 mM CaCl2 final in soft agar and/or bottom agar.
Antibiotic
- Kanamycin sulfate stock solution
- 2,500 mg kanamycin sulfate in 50 mL sterile water → 50 mg/mL.
- Filter-sterilize and store aliquots at -20 °C or 4 °C according to local practice.
- Working concentration in this protocol:
- 100 μL of 50 mg/mL stock added to 100 mL culture → 5 mg total → 50 μg/mL final.
Inducing agent
- Mitomycin C
- Prepare a stock (e.g., 1–2 mg/mL in sterile water, protected from light).
- Store in small aliquots at -20 °C.
- Use to achieve 1 μg/mL final concentration in exponential-phase lysogen culture.
Buffers and solutions
- SM phage buffer (no gelatin during processing)
- 5.8 g NaCl
- 2.0 g MgSO4•7H2O
- 50 mL of 1 M Tris-HCl, pH 7.5
- No gelatin for all steps prior to Amicon filtration
- Bring to 1 L with distilled water
- Sterilize by autoclaving or 0.22 μm filtration
- Store at 4 °C
- 30% (w/v) sucrose solution
- 30 g sucrose in 100 mL distilled water
- Gently warm to dissolve; do not caramelize
- Filter-sterilize; store at 4 °C.
- 5 M NaCl solution
- 292.2 g NaCl in 1 L distilled water
- Filter-sterilize or autoclave as appropriate.
Other reagents
- Chloroform (molecular biology grade).
- Sterile deionized water.
- NanoGlo or equivalent NanoLuc substrate.
Equipment
- Shaking incubator at 37 °C, capable of 100 rpm.
- Static 37 °C incubator or non-shaking shelf (for plates).
- 100 mL Erlenmeyer flasks for initial cultures and infections.
- 1 L storage flasks (baffled or non-baffled) for induction cultures.
- Spectrophotometer for OD600.
- Centrifuges:
- Large capacity centrifuge capable of spinning 300 mL HDPE bottles at 8,000 × g.
- High-speed centrifuge capable of spinning 50 mL HDPE tubes at 48,000 × g.
- All centrifugations in this protocol are performed at 4 °C.
- 300 mL HDPE centrifuge bottles.
- 50 mL HDPE centrifuge tubes (for sucrose cushions).
- 50 mL conical tubes for pooling concentrated phage.
- 0.22 μm filters (bottle-top or syringe; low-protein-binding).
- Amicon Ultra-15 centrifugal filter devices, 100 kDa MWCO.
- Luminometer or plate reader for NanoLuc luminescence.
Troubleshooting
Problem
Little or no visible clearing after induction
Solution
- Cause: Heavily lysogenic phage; induction still occurs but OD drop is subtle.
- Solution: Rely on NanoLuc luminescence and plaque titer after 24 h rather than OD alone to judge induction.
Problem
Low phage titer after sucrose cushion
Solution
- Cause: Insufficient NaCl; incorrect g-force or time; pellet loss.
- Solution: Confirm NaCl at ~1 M before spinning; verify rotor speed and run time; mark tube orientation and look carefully for small pellets.
Problem
Filter failure during 0.22 µm filtration
Solution
- Cause: Residual chloroform damaging membrane.
- Solution: Ensure thorough phase separation; discard any layer touching chloroform; if in doubt, let lysate stand, then re-spin before filtration.
Problem
High NanoLuc background in final stock
Solution
- Cause: Too few diafiltration cycles; membrane fouling.
- Solution: Increase number of Amicon cycles; avoid gelatin in SM before filtration; consider multiple devices to reduce overloading.
Problem
Poor plaque formation
Solution
- Cause: Suboptimal Mg2+/Ca2+, soft agar too stiff or too soft.
- Solution: Adjust MgSO4/CaCl2 to 5–10 mM range; keep soft agar ~0.4–0.7%; incubate at 37 °C and check host lawn density.
Before start
Biosafety ethics
- Work with E. coli O157:H7 and its phages must be performed under BSL-2 containment with appropriate institutional approvals.
- Mitomycin C is a DNA-crosslinking chemotherapeutic; handle with cytotoxic reagent PPE and dispose of waste according to institutional hazardous chemical policies.
- Chloroform is volatile and toxic; use in a fume hood and collect waste in appropriate organic waste containers.
- Treat all phage-containing materials as potentially infectious. Decontaminate surfaces and liquids with appropriate disinfectants and/or autoclaving.
Experimental design overview
- Host strain: E. coli O157:H7 C7927 (apple cider isolate).
- Phage: ΦV10::NanoLuc reporter phage.
- Working bacterial form: ΦV10::NanoLuc lysogen of C7927, denoted E. coli O157:H7 C7927(ΦV10::NanoLuc).
- Primary induction: Mitomycin C at 1 μg/mL final during exponential growth in 2×YT.
- Amplification medium: 2×YT broth (richer than LB for robust growth and higher yields; LB also supports growth but may give lower titers).
- Downstream use: High-titer reporter phage stock for NanoLuc-based detection, titration, and infection dynamics assays in C7927.
Note: ΦV10::NanoLuc is heavily lysogenic. Unlike many strictly lytic phages, you may not see a dramatic clearing of the culture or sharp OD drop after mitomycin C addition. Successful induction can instead be confirmed by NanoLuc luminescence in the lysate after 24 h.
SECTION A — Preparation of C7927 Overnight Culture
Inoculate 100 mL LB (10 g tryptone, 10 g NaCl, 5 g yeast extract per L) in a 250–500 mL Erlenmeyer flask with 100 µL of a frozen or working culture of E. coli O157:H7
C7927.
Incubate overnight at 37 °C with shaking at 100 rpm.
SECTION B — Initial Infection and Lysogen Selection
1h 30m
Infection to enrich for ΦV10::NanoLuc-infected cells
In a fresh 100 mL LB flask, add:
- 100 µL of the overnight C7927 culture (from step 2), and
- 100 µL of ΦV10::NanoLuc stock phage.
Incubate100 rpm, 37°C, 01:30:00 to allow infection and some bacterial growth.
1h 30m
Kanamycin selection for lysogens
Add 100 µL of 50 mg/mL kanamycin stock to the 100 mL culture (final
concentration 50 µg/mL).
Continue incubation at 37 °C, 100 rpm overnight.
Note: Kanamycin selects for cells that have acquired the ΦV10::NanoLuc prophage, which encodes
kanamycin resistance, thereby enriching for lysogens.
Isolation of single lysogenic colonies
The next day, streak a loopful of the kanamycin-selected culture onto LB agar plates
containing:
- 1.4% (w/v) agar
- Kanamycin 50 µg/mL.
Incubate plates overnight at 37 °C.
Select a single, well-isolated colony; this is presumed to be an E. coli O157:H7
C7927[ΦV10::NanoLuc] lysogen.
SECTION C — Growth of Lysogen in 2×YT and Mitomycin C Induction
Inoculate a sterile 1 L storage flask containing 2×YT broth (no antibiotic) with the single
C7927[ΦV10::NanoLuc] colony (from step 5.3).
Incubate at 37 °C, 100 rpm until the culture reaches an OD600 of 0.2–0.3.
At OD600 ≈ 0.2–0.3, add mitomycin C to a final concentration of 1 µg/mL.
Continue incubation at 37 °C, 100 rpm for up to 24 h.
SECTION D — Harvest of Crude Lysate and Chloroform Treatment
First clarification spin
After 24 h of post-induction incubation (or when you decide to harvest), distribute the culture into 300 mL HDPE centrifuge bottles.
Centrifuge at 8,000 × g for 15 min at 4 °C to pellet bacteria and debris.
Carefully decant or pipette the supernatant (crude lysate) into a clean bottle, avoiding the pellet.
Chloroform treatment
To the clarified lysate, add 10 mL chloroform per ~1 L lysate (scale proportionally if needed).
Gently invert several times to mix; do not shake vigorously and do not use a stir bar.
Incubate the chloroform–lysate mixture open/unlidded at 37 °C with mild shaking (~60 rpm) to promote phase separation and to kill remaining bacteria.
Once phases are clearly separated, gently pour off the upper, aqueous phage-containing phase without disturbing the chloroform layer.
If needed, transfer remaining mixture to a narrow graduated cylinder to facilitate separation, then carefully remove only the aqueous phase with a pipette.
Second clarification spin
Pool the chloroform-treated aqueous lysate and centrifuge again at 8,000 × g for 15 min at 4 °C.
Carefully collect the supernatant.
Sterile filtration
Filter the lysate through a 0.22 µm filter into a sterile 1 L glass bottle.
SECTION E — Salt Adjustment and Sucrose Cushion Concentration
Estimate the total lysate volume and add 5 M NaCl to bring the NaCl concentration to 1
M, taking into account NaCl already present from the medium.
Sucrose cushion setup
Prepare 50 mL HDPE centrifuge tubes compatible with a high-speed rotor.
For each tube, either: - Add 10 mL of sterile 30% sucrose to the bottom, then gently layer 30 mL lysate on top, or - Start with 30 mL lysate and gently inject 10 mL 30% sucrose beneath it using a needle, forming a lower cushion.
High-speed sucrose cushion spin
Centrifuge the tubes at 48,000 × g for 1 h at 4 °C.
After centrifugation, carefully pour off the supernatant and sucrose layer without disturbing the small pellet at the bottom of each tube.
Gently rinse each pellet with a small volume of chilled sterile deionized water, taking care not to dislodge it.
Resuspend each pellet in ~2 mL SM phage buffer (no gelatin) and transfer to a common 50 mL conical tube, combining all resuspensions.
SECTION F — Optional Diafiltration / Cleanup with Amicon Ultra-15 (100 kDa)
Priming the Amicon filters
For each Amicon Ultra-15 100 kDa device, add 1 mL SM buffer to the membrane and centrifuge at ~4,000 × g for ~3 min (within the manufacturer’s recommended limits) to wet/prime the membrane and reduce non-specific binding.
Discard the flow-through and proceed immediately to loading the phage suspension.
Concentration and buffer exchange cycles
Load up to 15 mL of the SM-resuspended phage into each Ultra-15 device.
Centrifuge at up to 6,000 × g (or per validated conditions for your device) until the retained volume is approximately 100–200 µL. A starting spin time of ~8 min is reasonable; adjust based on observed behavior.
Optionally recover the concentrate or simply add fresh SM buffer to the top chamber to bring the volume back up to 15 mL for diafiltration.
Repeat the concentration (step 18.2) and dilution (step 18.3) multiple times (e.g., ≥4 cycles) until NanoLuc luminescence in the permeate and/or stock is reduced to the desired level.
After the final cycle, recover the concentrated phage from the Amicon device into an appropriate low-binding tube.
SECTION G — Titration and NanoLuc QC
Plaque assay (double-layer)
Prepare soft agar (0.7% agar) in LB or 2×YT, optionally supplemented with 10 mM MgSO4 and 5 mM CaCl2.
Prepare a fresh overnight C7927 culture and dilute into log-phase as needed for plating.
Make 10-fold serial dilutions of the phage stock in SM buffer.
For each dilution to be plated, mix: - A fixed volume of phage dilution (e.g., 100 µL) with - A defined volume of host culture (e.g., 100–200 µL log-phase C7927).
Add the phage–host mixture to ~3–4 mL molten soft agar held at ~45–50 °C, mix gently, and overlay onto pre-warmed bottom agar plates.
Allow top agar to solidify, then incubate plates overnight at 37 °C.
Count plaques and calculate phage titer in PFU/mL.
NanoLuc luminescence QC
Mix an aliquot of phage stock with NanoGlo or equivalent NanoLuc substrate, following the manufacturer’s instructions.
Measure luminescence (RLUs) on a luminometer or plate reader.
Record titer (PFU/mL), luminescence values, and the number of Amicon diafiltration cycles performed.
SECTION H — Aliquoting and Storage
Aliquot the final concentrated ΦV10::NanoLuc stock into sterile, labeled cryovials (e.g., 0.2–1 mL per aliquot, depending on usage).
Store:
- Short term: 4 °C (days to weeks, as validated).
- Long term: −80 °C (months to years, as validated).
Label each vial with:
- Phage: ΦV10::NanoLuc
- Host: C7927
- Date
- PFU/mL
- Number of Amicon cycles
- Storage buffer (SM ± gelatin if used for long-term only)
- Operator initials
Protocol references
1. Perna NT et al. Genome sequence of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Nature. 2001;409(6819):529–533. (Background on E. coli O157:H7 genomics.)
2. Sharma VK, Bearson BL. Evaluation of Escherichia coli O157:H7 strain C7927 for survival in acidic environments and apple cider. Journal of Food Protection. 2013;76(7):1178–1185. (Example reference for C7927 as an apple cider isolate; adjust to your preferred primary C7927 source.)
3. Hagens S, Loessner MJ. Application of bacteriophages for detection and control of foodborne pathogens. Applied Microbiology and Biotechnology. 2007;76(3):513–519. (General phage methods and host range considerations.)
4. O'Flynn G, Ross RP, Fitzgerald GF, Coffey A. Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. Applied and Environmental Microbiology. 2004;70(6):3417–3424. (Example plaque assay and phage propagation methods for E. coli O157:H7 phages.)
5. Loessner MJ. Bacteriophage endolysins—current state of research and applications. Current Opinion in Microbiology. 2005;8(4):480–487. (General phage biology and lytic/lysogenic considerations.)
6. Little JW. Mechanism of specific LexA cleavage: autodigestion and the role of RecA coprotease. Biochimie. 1991;73(4):411–421. (Background on SOS response and mitomycin C–mediated prophage induction.)
7. Goh S, Klaenhammer TR. Bacteriophage distribution and prophage induction in lactic acid bacteria. Journal of Applied Microbiology. 2000;89(3):442–452. (Example of mitomycin C ranges and prophage induction in Gram-positive bacteria.)
8. Adams MH. Bacteriophages. Interscience Publishers, New York; 1959. (Classic reference for phage propagation, plaque assays, and purification methods.)
9. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001. (Standard reference for media recipes, phage buffer formulations, and general molecular biology techniques.)
10. Promega Corporation. Nano-Glo® Luciferase Assay System Technical Manual. (Reference for NanoLuc luminescence measurement and assay conditions; use the current version number and URL as appropriate.)