Bacteriophage Transduction

Updated 9/13/2018 02:02 PM

by OlsonLaboratory

Introduction

The genetic manipulation of Staphylococcus aureus for molecular experimentation is a valuable tool for assessing gene function and virulence. Genetic variability between strains coupled with difficult laboratory techniques for strain construction are frequent roadblocks in S. aureus research. Bacteriophage transduction greatly increases the speed and ease of S. aureus studies by allowing movement of chromosomal markers and plasmids between strains. This technique enables the S. aureus research community to focus investigations on clinically relevant isolates

Materials

13x100 mm Tryptic soy agar (TSA)αα/Brain Heart Infusion (BHI) slants

Tryptic soy broth (TSB) + 5mM CaCl₂

Petri plates

15mL Falcon tubes (BD Biosciences) or equivalent

TSA (1.5% agar) + 5mM CaCl₂

TSA (1.5% agar) + 500mg/L NaCitrate + antibiotic of choice

Soft Agar TSA (0.5% agar) + 5mM CaCl₂

0.5M CaCl₂

0.02M NaCitrate

Antibiotic of choice; typical antibiotics and concentrations include erythromycin 10 μg/mL, chloramphenicol 10 μg/mL, trimethoprim 10 μg/mL, tetracycline 2-10 μg/mL, kanamycin 50 μg/mL.

Procedure

Bacteriophage Propagation

Transduction of plasmid and chromosomal markers of interest requires a phage titer of approximately 1010 pfu/ml. As phage titers gradually decrease during storage at 4°C, it is appropriate to propagate phage to acquire a high titer (1010) before the transducing lysate is generated.

Grow S. aureus propagation strain on 13x100 mm TSA slant overnight at 37°C. Ensure that a plasmid free strain of S. aureus is used that is susceptible to the phage of choice. Strain RN4220 is recommended as it is both a good recipient and propagation strain for either phage 80α or Φ11 and allows for optimal phage titers (1010 PFU -Plaque Forming Units; see note 1).

Resuspend S. aureus propagation strain in 1mL TSB + 5mM CaCl₂ (see note 2).

Add 4mL TSA soft agar to 10 Falcon tubes (15mL). Hold in 50°C water bath to prevent agar from solidifying.

Serially dilute bacteriophage stock 10 fold to 10-10 in TSB + 5mM CaCl₂.

Combine 10µL S. aureus cells and 100µL bacteriophage dilution in soft agar. Gently mix (do not vortex) and pour onto TSA + 5mM CaCl₂ plates. Repeat for all 10 bacteriophage dilutions. Fresh TSA plates work best to prevent soft agar from excess drying during phage propagation.

Incubate overnight (plates right side up) at 37°C. Do not invert plates to ensure soft agar is maintained on the agar surface.

Harvest Bacteriophage and Titer Determination

Select up to three plates for bacteriophage harvest. Optimal plates will show near confluent lysis and minimal bacterial growth (Ideally the 10-3-10-5 plates, but this depends upon the original titer of the bacteriophage stock).

Add 3mL TSB to plates. Harvest bacteriophage by breaking up and scraping off soft agar with a plate spreader. Transfer resulting agar/TSB mixture to a 50mL tube.

Disrupt agar as much as possible by gently pipetting up and down. Avoid bubbles, vortexing, and sonication as they mechanically sheer bacteriophage tails. This step facilitates the release of bacteriophage particles from agar encasement.

Centrifuge 10 min at 10,000 × g.

Filter supernatant through 0.22 µm filter.

Store bacteriophage at 4°C.

The titer of the resulting bacteriophage lysate should be determined by repeating the experiment outlined in 3.1; optimal bacteriophage titer should be approximately 1010 pfu/ml. In some cases, when the titer of the original bacteriophage stock is low, multiple propagation experiments may be required to acquire the desired titer of 1010 pfu/ml.

Preparation of Transducing Lysate

Repeat bacteriophage propagation and harvest protocol (3.1 and 3.2) using S. aureus strain of interest (either plasmid or chromosomal marker). Note that overnight growth may require 30°C if using temperature sensitive plasmid (i.e. pE194ts-derived). 1010 pfu/ml of the transducing lysate should be achieved to ensure an appropriate transduction frequency (~10-8)

Transduction

Grow the strain to be transduction recipient overnight on a 13x100mm TSA slant.

Resuspend recipient strain in 1mL TSB + 5mM CaCl₂.

Add 500 μL of the recipient strain suspension to a 50mL tube.

Add 1.5mL TSB + 5mM CaCl₂.

Add 500µL bacteriophage lysate (1010 pfu/ml) to tube

(see note 3).

Shake at 225 RPM for exactly 20 min at 37ºC (30ºC if transducing a temperature sensitive plasmid).

Add 1mL cold (4°C) 0.02M NaCitrate.

Centrifuge at 3000 RPM, 4°C for 10 min.

Resuspend pellet in 1mL cold (4°C) 0.02M NaCitrate.

Plate 10 aliquots (100 μL/plate) of transduced cells to TSA + 500mg/L NaCitrate + antibiotic plates.

Incubate at 37°C (30ºC if transducing a temperature sensitive plasmid).

Pick single colonies to streak for isolation on the TSA + 500mg/L NaCitrate + antibiotic plates (see note 4)

Confirm movement of plasmid/chromosomal marker by standard methods including: plasmid analysis, PCR or Southern Blot.

Notes

1. Determine of strain of choice is phage susceptible by first streaking the strain on TSA containing 5mM CaCl₂. 10mL of phage stock (1010 pfu/ml) is then spotted on the plate in the first quadrant and allowed to dry. The plate is then incubated at 37°C for 24 hours; an area of clearing (lysis) will be evident in susceptible strains.

2. Calcium chloride (CaCl₂) is added to the media to facilitate bacteriophage attachment to S. aureus. NaCitrate chelates the calcium, arresting the bacteriophage infectious cycle and preventing reinfection.

3. As a negative control to test for contaminated lysate, perform the transduction experiments using all components except the bacteriophage.

4. NaCitrate is required in these plates to chelate residual calcium in the TSA. Although individual transductant colonies are picked, bacteriophage titer on the plate is high enough that phage can be transferred to subsequent TSA plates resulting in partial lysis of colony growth if NaCitrate is not added.