3.5 Establishing
a Conditionally
Recombinogenic
S. aureus Strain
1. Extract plasmid pEF2132-tet from an E. coli plasmid artificial
modification strain using a commercial plasmid extraction kit.
1 μg of plasmid is required for optimal transformation
efficiency.
2. Prepare electrocompetent cells from the S. aureus strain of
interest (see Subheading 3.3).
3. Precipitate pEF2132-tet plasmid DNA.
(a) Bring Pellet Paint NF Co-Precipitant and 3 M Sodium
Acetate to room temperature. Invert several times until a
uniform suspension is achieved, but do not vortex.
(b) Add 2 μL Pellet Paint and 0.1 volume 3 M
Sodium Acetate to 1 μg plasmid DNA and mix briefly by
pipetting.
(c) Add 2 volumes ethanol and briefly vortex to mix.
(d) Incubate at room temperature for 2 min.
(e) Centrifuge at 14,000–16,000 Â g for 5 min. A pink pellet
should be visible at the bottom of the tube.
(f) Remove the supernatant by pipetting.
(g) Rinse pellet with two volumes 70% ethanol, then vortex
briefly and centrifuge at 14,000–16,000 Â g for 5 min.
(h) Remove supernatant and rinse pellet in 100% ethanol.
Spin at 14,000–16,000 Â g for 5 min, then remove supernatant by pipetting. Any residual ethanol should be
removed by air drying.
(i) Resuspend pellet in 2 μL TE buffer.
4. Transform S. aureus by electroporation.
(a) Thaw electrocompetent S. aureus cells on ice for 5 min,
then transfer to room temperature for an additional
5 min.
(b) Centrifuge cells at 5000 Â g for 1 min and resuspend in
50 μL 10% glycerol containing 500 mM sucrose.
(c) Transfer resuspended cells to the tube containing
precipitated DNA, incubate for 1 min, then transfer
the mixture to a 0.1 cm electrode gap electroporation
cuvette.
(d) Place the cuvette in the sample chamber of the electroporator and pulse once at 2.3 kV using a 2.5 ms time
constant.
(e) Immediately after electroporation, resuspend cells in
950 μL TSB containing 500 mM sucrose.
(f) Transfer the cell suspension to a sterile culture tube and
incubate at 32
C for 1 h with shaking at 250 rpm.
136
Kelsi Penewit and Stephen J. Salipante
a Conditionally
Recombinogenic
S. aureus Strain
1. Extract plasmid pEF2132-tet from an E. coli plasmid artificial
modification strain using a commercial plasmid extraction kit.
1 μg of plasmid is required for optimal transformation
efficiency.
2. Prepare electrocompetent cells from the S. aureus strain of
interest (see Subheading 3.3).
3. Precipitate pEF2132-tet plasmid DNA.
(a) Bring Pellet Paint NF Co-Precipitant and 3 M Sodium
Acetate to room temperature. Invert several times until a
uniform suspension is achieved, but do not vortex.
(b) Add 2 μL Pellet Paint and 0.1 volume 3 M
Sodium Acetate to 1 μg plasmid DNA and mix briefly by
pipetting.
(c) Add 2 volumes ethanol and briefly vortex to mix.
(d) Incubate at room temperature for 2 min.
(e) Centrifuge at 14,000–16,000 Â g for 5 min. A pink pellet
should be visible at the bottom of the tube.
(f) Remove the supernatant by pipetting.
(g) Rinse pellet with two volumes 70% ethanol, then vortex
briefly and centrifuge at 14,000–16,000 Â g for 5 min.
(h) Remove supernatant and rinse pellet in 100% ethanol.
Spin at 14,000–16,000 Â g for 5 min, then remove supernatant by pipetting. Any residual ethanol should be
removed by air drying.
(i) Resuspend pellet in 2 μL TE buffer.
4. Transform S. aureus by electroporation.
(a) Thaw electrocompetent S. aureus cells on ice for 5 min,
then transfer to room temperature for an additional
5 min.
(b) Centrifuge cells at 5000 Â g for 1 min and resuspend in
50 μL 10% glycerol containing 500 mM sucrose.
(c) Transfer resuspended cells to the tube containing
precipitated DNA, incubate for 1 min, then transfer
the mixture to a 0.1 cm electrode gap electroporation
cuvette.
(d) Place the cuvette in the sample chamber of the electroporator and pulse once at 2.3 kV using a 2.5 ms time
constant.
(e) Immediately after electroporation, resuspend cells in
950 μL TSB containing 500 mM sucrose.
(f) Transfer the cell suspension to a sterile culture tube and
incubate at 32
C for 1 h with shaking at 250 rpm.
136
Kelsi Penewit and Stephen J. Salipante