22
- Leave at room temperature for 30 min, then repeat
centrifugation. - Resuspend in approximately 400 μL sterile, room temperature
10% (v/v) glycerol. Aliquot into 50 μL volumes (see Note 6). - To aliquots of competent cells, add 0, 5, 10, or 15 μL plasmid
mini-prep. - Transfer to an electroporation cuvette at room temperature.
- Electroporate with the following settings: 100 Ω, 2.3 kV,
25 μF. - Add 1 mL medium and transfer mixture to a 30 mL universal
tube. - Incubate for 3 h at 37 °C with rapid aeration (e.g., 250 rpm).
- Plate onto agar containing antibiotics at 0.2 × the standard
concentration (see Note 7). Incubate for 24–48 h at 37 °C
until healthy bacterial colonies appear. - Recover colonies and verify integration by colony PCR (see
Note 8). You may also restreak colonies onto the standard
antibiotic concentration to verify antibiotic resistance. - Grow 5 mL overnight culture of the donor strain (RN4220
containing your integrated antibiotic resistance cassette from
Subheading 3.2) in BHI at 37 °C with rapid aeration (e.g.,
250 rpm). - In a 30 mL tube, add 5 mL phage buffer, 5 mL BHI, 150 μL
overnight culture, and 100 μL phage lysate from a previous
preparation (see Notes 9 and 10 ). - Incubate the mixture at 25 °C without agitation until it clari-
fies (indicating complete lysis of bacterial cells) (see Note 11). - Filter-sterilize the lysate. Filtered bacteriophage lysates can be
stored for several years at 2–8 °C so long as they remain
uncontaminated. - Grow a 50 mL overnight culture of the recipient strain in LK
at 37 °C with rapid aeration (e.g., 250 rpm). - Centrifuge the culture at room temperature for 10 min at
approximately ~ 4000 × g). Discard the supernatant. - Resuspend the pellet in 3 mL LK.
- Prepare the following in 30 mL tubes:
(a) 500 μL donor phage lysate, 500 μL recipient culture,
1 mL LK, 10 μL 1 M CaCl 2.
(b) 500 μL recipient cells, 1.5 mL LK, 15 μL 1 M CaCl 2 (as
negative control). - Incubate for 25 min at 37 °C without agitation.
3.3 Strain
Construction:
Bacteriophage
Transduction
of Mutations
Between S. aureus
Strains
Gareth McVicker et al.