pBT2

Protocols for gene deletion in Staphylococcus aureus   Nov. 1, 2007


Preparation of competent Staphylococcus aureus cells

1. 1. Remove Staphylococcus aureus cells from the vial with a sterile toothpick or inoculation loop, and streak it out on LB agar.
2. 2. Incubate at 37°C overnight.
3. 3. Pick a single colony and inoculate it in 5-10 ml of LB. Grow at 37°C overnight.
4. 4. Add 1 ml overnight culture to 100 ml LB medium in a 500 ml flask, and shake at 37°C until an OD600 of 0.4 is reached (approximately 90–120 min).
5. 5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.
6. 6. Collect the cells by centrifugation at low speed (5-10 min, 2500 x g, 4°C).
7. 7. Discard the supernatant carefully. Always keep the cells on ice.
8. 8. Resuspend the cells gently in 0.5 M sucrose (10-15 ml for a 100 ml culture) at 4°C and keep the suspension on ice for additional 5 min.
9. 9. Collect the cells by centrifugation (5 min, 2500 x g, 4°C).
10. 10. Discard the supernatant carefully. Repeat step 8 and 9.
11. 11. Resuspend the cells carefully in 1 ml ice-cold 0.5 M sucrose and keep the suspension on ice for 15 min.
12. 12. Prepare aliquots of 100–200 μl in sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at –70°C.

1. 1. PCR amplify a 400 bp fragment upstream and a 400 bp fragment downstream of the target gene.
2. 2. PCR amplify the ermB (Em resistance marker) from pECI.
3. 3. Digest the three fragments, ligate, and PCR amplify the ligated product.
4. 4. Purify the PCR product, double digest it, and ligate it into pBT2.
5. 5. Transform the ligated product into E. coli.
6. 6. Pick clones that can grow on the LB plate containing Em (100 mg/ml), purify the plasmid and digest it.
7. 7. If the result of enzyme digestion is correct, get further confirmation by sequencing. 　

1. 1. Mix 500 ng of plasmid DNA with electrocompetent Staphylococcus aureus cells and place them in a Gene Pulser cuvette with a 0.2 cm electrode gap.
2. 2. The settings for electroporation are as follows: Voltage, 2.5 kV; capacitor, 50 μF; resistance, 200 ohms.
3. 3. After electroporation the cells are immediately placed in 400 μl of TSB with shaking (200-220 rpm, 37°C) for 1h. Plate the cells on Em-containing medium and incubate at 37°C.

1. 1. Before transform the plasmid into Staphylococcus aureus NCTC8325, the plasmid should be transformed into Staphylococcus aureus RN4220.
2. 2. Pick clones, after overnight growth, extract the plasmid.
3. 3. Then the plasmid is modified and can’t be digested by restriction enzyme system of NCTC8325.

1. 1. Extract plasmid from RN4220, transform it into NCTC8325.
2. 2. Pick up clones, incubate in B-medium, 30°C, grow to late-stationary phase, then change temperature to 40°C, and grow overnight.
3. 3. 1: 100 dilute the culture into fresh B-medium, and grow overnight.
4. 4. Follow step 3, spread 1 μl overnight culture (diluted into 100 μl) on agar plate (containing Em 2.5 mg/ml). Screen clones which are Em-resistant and Cm-sensitive.
5. 5. Repeat step 4 until Em-resistant, Cm-sensitive clones are found.
6. 6. Extract genome DNA of these clones, use PCR for further check.