19- Lysogeny broth (LB): 10 g/L tryptone, 5 g/L NaCl, 5 g/L
yeast extract. Supplement with 15 g/L agar to make plates.
Mix together with water and autoclave, then cool to approxi-
mately 45 °C prior to adding appropriate antibiotics. - Antibiotics for selection of plasmid-containing cells.
- Premixed brain heart infusion (BHI) media made according to
manufacturer’s instructions. Supplement with 15 g/L agar to
make plates. Mix together with water and autoclave, then cool to
approximately 55 °C prior to adding appropriate antibiotics. - Antibiotics for selection of integrates.
- Suitable electroporation machine (e.g., Bio-Rad Gene Pulser).
- Standard laboratory centrifuge (appropriate for 50 mL tubes at
~ 4000 × g). - Sterile phosphate buffered saline (PBS); commonly made by
dissolving tablets in water then autoclaving. - Sterile 10% (v/v) glycerol solution; either autoclaved or
filtered. - Sterile conical flasks of an appropriate size for growth of 10 mL
and 500 mL bacterial culture (at least fourfold larger ves-
sel volume is recommended for efficient aeration). - Suitable sterile tubes for bacterial aliquots and centrifugation
(1.5 mL, 30 mL, 50 mL). - Premixed BHI media made according to manufacturer’s
instructions. - LK medium: 10 g/L tryptone, 5 g/L KCl, 5 g/L yeast extract.
Supplement with 15 g/L agar and 0.5 g/L sodium citrate to
make “phage agar” plates. Mix together with water and auto-
clave, then cool to approximately 45 °C prior to adding appro-
priate antibiotics. - Phage buffer: 2 mM MgSO 4 , 5 mM CaCl 2 , 0.1 M NaCl,
2.5 mM Tris–HCl (pH 7.8), 0.1% (w/v) gelatin. Autoclave to
dissolve and sterilize. - 1 M CaCl 2 solution. Autoclave or filter to sterilize.
- 20 mM sodium citrate. Autoclave or filter to sterilize.
- φ11 bacteriophage lysate harvested from a previous
preparation. - Suitable sterile tubes for transduction and centrifugation
(30 mL, 50 mL). - Standard laboratory centrifuge (appropriate for 50 mL tubes at
~ 4000 × g).
2.2 Strain
Construction:
Integration of Suicide
Vectors into S. aureus
RN4220
2.3 Strain
Construction:
Bacteriophage
Transduction
of Mutations
Between S. aureus
Strains
Construction and Use of Staphylococcus aureus Strains to Study Within-Host...