Antibiotic Resistance Protocols (Methods in Molecular Biology)

21

3. Obtain vector plasmid DNA (see Note 3) using a commercial
   mini-prep kit.


4. 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.


5. 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.


6. 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.


7. Recover colonies and verify the sequence of ligated plasmids
   (see Note 4).

For all plating/growth steps, use BHI supplemented with

antibiotics as indicated.

1. 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).


2. 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).


3. 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.


4. Resuspend each pellet in 25 mL sterile, room temperature
   water. Repeat centrifugation. Discard supernatant. Repeat this
   step twice more.


5. Resuspend each pellet in 20 mL sterile, room temperature 10%
   (v/v) glycerol. Repeat centrifugation. Discard supernatant.


6. 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...