20
- Premixed 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 approxi-
mately 45 °C prior to adding appropriate antibiotics. - Sterile PBS; commonly made by dissolving tablets in water
then autoclaving. - Dry ice (for snap-freezing samples).
- Suitable sterile tubes for bacterial aliquots (15 mL).
- Standard laboratory centrifuge (appropriate for 50 mL tubes at
~ 4000 × g). - Standard laboratory vortex mixer.
- Premixed BHI media made according to manufacturer’s
instructions, supplemented 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. - Sterile PBS; commonly made by dissolving tablets in water
then autoclaving. - Suitable sterile tubes for mixing bacterial inoculation (7 mL).
- Suitable sterile needles and syringes for mouse tail vein
injection. - Sterile tools for extracting organs for quantification of bacterial
load. - Automatic homogenizer (e.g., Peqlab PreCellys 24-Dual).
- Sterile 7 mL tubes containing 2.8 mm ceramic beads, compat-
ible with homogenizer.
3 Methods
The workflow is summarized in Fig. 1.
For all plating/growth steps, use LB supplemented with anti-
biotics as indicated.- Using standard techniques or software, design oligonucleotide
primers to amplify the region of homology from the bacterial
chromosome into which you wish to insert your antibiotic cas-
sette (see Note 1). Include a unique restriction endonuclease
recognition sequence at the 5′ end of each primer for insertion
of the fragment into the vector. - Amplify the DNA fragment with a high-fidelity polymerase
and purify it using a commercial kit or similar technique (see
Note 2).
2.4 In Vivo Studies:
Preparation
of Bacteria for Mouse
Infection
2.5 In Vivo Studies:
Mouse Infection
and Quantification
of Bacterial Load
3.1 Strain
Construction: Suicide
Vectors
Gareth McVicker et al.