8. Repeat for step 7.
9. Add ~10 mL ice-cold sterile 10% glycerol and resuspend pellet.
Add an additional 30 mL ice-cold sterile 10% glycerol. Centrifuge at 4600 Â g for 10 min at 4
C. Remove supernatant.
10. Repeat step 9.
11. Resuspend the cell pellet in 750 μL ice-cold sterile 10%
glycerol. The final cell concentration should be !1 Â 10
10
CFU/mL.
12. Working on ice, distribute 50 μL aliquots of cell suspension
into sterile, prechilled 1.5 mL tubes and freeze immediately at
À80
C (see Note 2).
3.4 Transformation
of E. coli Plasmid
Artificial Modification
Strains
All plasmids which will be introduced into S. aureus must first be
passage through an appropriate E. coli plasmid artificial modification strain in order to bypass the S. aureus restriction barrier
[7, 24]. If the clonal complex and/or sequence type of the
S. aureus strain of interest is not known in advance, we recommend
empirically testing plasmid modified using all three of the E. coli
plasmid artificial modification strains (DC10B, SA08B, and
SA30B) to empirically determine which of them results in the
highest transformation efficiency. This protocol essentially follows
that of Ausubel et al. [31].
1. Thaw the appropriate electrocompetent E. coli cells on ice (~5
to 10 min). Simultaneously, prechill a 0.1 cm electroporation
cuvette on ice.
2. Add up to 2 μL of plasmid DNA to thawed cell suspension.
Pipet three times to mix, then incubate on ice for 1 min.
Transfer the mixture to the electroporation cuvette, keeping
on ice.
3. Place the cuvette in the sample chamber of the electroporator
and immediately pulse once at 1.8 kV without using a fixed
time constant.
4. Immediately add 1 mL SOC medium to the cuvette, and
quickly but gently resuspend cells using a pipette.
5. Transfer the cell suspension to a sterile culture tube, and incubate at 37
C for 1 h with constant aeration (shaking at
250 rpm).
6. Plate dilutions of the cells on LB containing the appropriate
selective medium. Artificially modified plasmids can subsequently be extracted from the transformed cells.
Genome Editing in Staphylococcus aureus by Conditional. . .
135
9. Add ~10 mL ice-cold sterile 10% glycerol and resuspend pellet.
Add an additional 30 mL ice-cold sterile 10% glycerol. Centrifuge at 4600 Â g for 10 min at 4
C. Remove supernatant.
10. Repeat step 9.
11. Resuspend the cell pellet in 750 μL ice-cold sterile 10%
glycerol. The final cell concentration should be !1 Â 10
10
CFU/mL.
12. Working on ice, distribute 50 μL aliquots of cell suspension
into sterile, prechilled 1.5 mL tubes and freeze immediately at
À80
C (see Note 2).
3.4 Transformation
of E. coli Plasmid
Artificial Modification
Strains
All plasmids which will be introduced into S. aureus must first be
passage through an appropriate E. coli plasmid artificial modification strain in order to bypass the S. aureus restriction barrier
[7, 24]. If the clonal complex and/or sequence type of the
S. aureus strain of interest is not known in advance, we recommend
empirically testing plasmid modified using all three of the E. coli
plasmid artificial modification strains (DC10B, SA08B, and
SA30B) to empirically determine which of them results in the
highest transformation efficiency. This protocol essentially follows
that of Ausubel et al. [31].
1. Thaw the appropriate electrocompetent E. coli cells on ice (~5
to 10 min). Simultaneously, prechill a 0.1 cm electroporation
cuvette on ice.
2. Add up to 2 μL of plasmid DNA to thawed cell suspension.
Pipet three times to mix, then incubate on ice for 1 min.
Transfer the mixture to the electroporation cuvette, keeping
on ice.
3. Place the cuvette in the sample chamber of the electroporator
and immediately pulse once at 1.8 kV without using a fixed
time constant.
4. Immediately add 1 mL SOC medium to the cuvette, and
quickly but gently resuspend cells using a pipette.
5. Transfer the cell suspension to a sterile culture tube, and incubate at 37
C for 1 h with constant aeration (shaking at
250 rpm).
6. Plate dilutions of the cells on LB containing the appropriate
selective medium. Artificially modified plasmids can subsequently be extracted from the transformed cells.
Genome Editing in Staphylococcus aureus by Conditional. . .
135