This study outlines a method for transforming Staphylococcus aureus using chloramphenicol-resistant DNA. The process involves nutrient stress to enhance competence, allowing the bacterium to internalize foreign DNA and integrate it into its chromosome.
Take an S. aureus culture sensitive to the antibiotic chloramphenicol.
Centrifuge to pellet the bacteria and remove the supernatant.
Transfer the pellet to a tube containing nutrient-limiting media and incubate. Nutrient stress enhances competence-the bacterium’s ability to internalize foreign DNA.
Centrifuge again and remove the supernatant. Resuspend the pellet in fresh nutrient-limiting media.
Next, add DNA extracted from an S. aureus strain carrying a chloramphenicol-resistant gene. Incubate.
The DNA-uptake machinery processes the DNA into a single strand and translocates it to the cytoplasm, where it integrates into the bacterial chromosome.
Centrifuge and remove the supernatant. Resuspend the pellet in nutrient-rich media.
Transfer the suspension into molten nutrient-rich agar containing chloramphenicol.
Pour the mixture into a dish and incubate.
The growth of chloramphenicol-resistant colonies indicates successful DNA uptake.
Culture the recipient cell overnight, in five milliliters of TSB at 37 degrees celsius with shaking. The following morning, transfer 0.5 milliliters of the overnight culture into a 1.5 milliliter tube.
Precipitate the cells by centrifugation. Then suspend the cells with 10 milliliters of CS2 medium in a 50 milliliter tube. Next grow the bacteria at 37 degrees celsius with shaking at 180 rotations per minute for eight hours until the late exponential phase.
Harvest the cells by centrifugation. Discard the supernatant, and resuspend the cells in 10 milliliters of fresh CS2 medium. Add 10 micrograms of purified plasmid or genomic DNA to the cell suspension.
Shake the culture at 180 rotations per minute. Then collect the cells by centrifugation. Resuspend the cells in 10 milliliters of BHI medium.
Mix the cell suspension with 90 milliliters of melted BHI agar supplement With 32 milligrams per liter chloramphenicol, And five micrograms per liter tetracycline in the control experiment. Pour the mixture into the 90 millimeter petri dishes and swiftly cool and allow the agar to solidify. Replicate colonies using toothpicks to plates containing appropriate antibiotics to confirm their resistance characteristics.
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