21
- Obtain vector plasmid DNA (see Note 3) using a commercial
mini-prep kit. - Digest approximately 1–2 μg of PCR and plasmid DNA and
purify the relevant insert and vector fragments using an aga-
rose gel purification kit. - Ligate your insert into the vector using a standard ligation pro-
tocol. Include a positive (e.g., single-enzyme digested vector)
and negative (e.g., water instead of insert DNA) control. - Transform ligation mixtures into a high-efficiency E. coli clon-
ing strain such as DH5α or TOP10 using a standard heat shock
or electroporation protocol. Plate onto appropriate antibiotic
agar to select for successful transformants. - Recover colonies and verify the sequence of ligated plasmids
(see Note 4).
For all plating/growth steps, use BHI supplemented with
antibiotics as indicated.
- Grow 10 mL overnight culture of S. aureus RN4220 (a com-
mon restriction-deficient cloning intermediate) at 37 °C with
rapid aeration (e.g., 250 rpm). - Subculture into 500 mL prewarmed medium such that
OD 600 = 0.1. Grow as above for approximately 1 h until the
culture reaches OD 600 = 0.4–0.6 (see Note 5). - Aliquot 4 × 50 mL samples and pellet cells in a centrifuge for
10 min at approximately ~ 4000 × g). Discard the supernatant
then add a further 50 mL culture to each tube and repeat the
centrifugation. Discard the second supernatant. - Resuspend each pellet in 25 mL sterile, room temperature
water. Repeat centrifugation. Discard supernatant. Repeat this
step twice more. - Resuspend each pellet in 20 mL sterile, room temperature 10%
(v/v) glycerol. Repeat centrifugation. Discard supernatant. - Resuspend each pellet in 10 mL sterile, room temperature 10%
(v/v) glycerol. Combine into a single tube.
3.2 Strain
Construction:
Integration of Suicide
Vectors into S. aureus
RN4220
Identify &
clone target
locus
Construct
antibiotic-
resistant
suicide vector
Integrate
vector into
chromosome
Transduce
marker
between
strains
Ready for
biological
assays
Fig. 1 The workflow presented in this chapter for construction of antibiotic-resistant strains
Construction and Use of Staphylococcus aureus Strains to Study Within-Host...