Protocols for gene deletion in Staphylococcus aureus Nov. 1, 2007
Preparation of competent Staphylococcus aureus cells
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1. Remove Staphylococcus aureus cells from the vial with a sterile toothpick or inoculation loop, and streak it out on LB agar.
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2. Incubate at 37°C overnight.
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3. Pick a single colony and inoculate it in 5-10 ml of LB. Grow at 37°C overnight.
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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).
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5. Cool the culture on ice for 5 min, and transfer the culture to a sterile, round-bottom centrifuge tube.
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6. Collect the cells by centrifugation at low speed (5-10 min, 2500 x g, 4°C).
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7. Discard the supernatant carefully. Always keep the cells on ice.
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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.
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9. Collect the cells by centrifugation (5 min, 2500 x g, 4°C).
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10. Discard the supernatant carefully. Repeat step 8 and 9.
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11. Resuspend the cells carefully in 1 ml ice-cold 0.5 M sucrose and keep the suspension on ice for 15 min.
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12. Prepare aliquots of 100–200 μl in sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at –70°C.
Construction of deletion vector
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1. PCR amplify a 400 bp fragment upstream and a 400 bp fragment downstream of the target gene.
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2. PCR amplify the ermB (Em resistance marker) from pECI.
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3. Digest the three fragments, ligate, and PCR amplify the ligated product.
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4. Purify the PCR product, double digest it, and ligate it into pBT2.
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5. Transform the ligated product into E. coli.
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6. Pick clones that can grow on the LB plate containing Em (100 mg/ml), purify the plasmid and digest it.
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7. If the result of enzyme digestion is correct, get further confirmation by sequencing.
Procedure for electroporation
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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.
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2. The settings for electroporation are as follows: Voltage, 2.5 kV; capacitor, 50 μF; resistance, 200 ohms.
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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.
Modify deletion vector
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1. Before transform the plasmid into Staphylococcus aureus NCTC8325, the plasmid should be transformed into Staphylococcus aureus RN4220.
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2. Pick clones, after overnight growth, extract the plasmid.
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3. Then the plasmid is modified and can’t be digested by restriction enzyme system of NCTC8325.
Deletion of target gene
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1. Extract plasmid from RN4220, transform it into NCTC8325.
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2. Pick up clones, incubate in B-medium, 30°C, grow to late-stationary phase, then change temperature to 40°C, and grow overnight.
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3. 1: 100 dilute the culture into fresh B-medium, and grow overnight.
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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.
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5. Repeat step 4 until Em-resistant, Cm-sensitive clones are found.
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6. Extract genome DNA of these clones, use PCR for further check.
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