Streptococcus Competence Induction
Natural Transformation with pNZ8048 plasmid w/antibiotic resistance ONLY 1. Grow S. suis strain in an overnight culture containing Todd-Hewitt broth (THB) at 37 °C. 2. When ready to start the transformation experiment, dilute overnight culture 1:40 in pre-warmed media (37°C with no shaking). 3. After 1 hour, measure the optical density of the culture using the spectrophotometer (refer to the protocol to use this machine on the Laboratory WIKI). Assuming favorable O.D, remove 100 uL of sample from the main culture and place in 1.5 mL epi tube. a. Competence Development occurs with the following ranges of bacterial densities (O.D. 0.035 to 0.058) MAX EFFICIENCY (O.C. 0.042). MAKE SURE THE BACTERIA FALLS WITHIN THIS RANGE 4. Add 1.2 mg of pNZ8048 plasmid in EB buffer (10 mM Tris-Cl, pH 8.5) to the epi tube. 5. Immediately afterwards, add 5 uL of synthetic peptide [FINAL - 250 mM]. 6. Incubate at 37 °C for 2 hours. 7. Prepare solid agar media by adding 12 g/L to the medium. Add either chloramphenicol (5mg/mL) or spectinomycin (100 mg/mL) to the medium. 8. Following incubation, add dilute samples to the THB/agar/antibiotic plates. a. Make sure to also dilute and add bacteria to an Antibiotic (-) control plate 9. Count the number of colonies in one quadrant each plate. Multiply by 4 to get an estimate of the total number of bacteria on each plate.
Natural Transformation with pNZ8048 plasmid w/antibiotic resistance and red fluorescence genes 1. Inoculate S. suis strain in an overnight culture containing Todd-Hewitt broth (THB) at 37 °C. 2. When ready to start the transformation experiment, dilute overnight culture 1:40 in pre-warmed media (37°C with no shaking). 3. After 1 hour, measure the optical density of the culture using the spectrophotometer (refer to the protocol to use this machine on the Laboratory WIKI). Assuming favorable O.D, remove 100 uL of sample from the main culture and place in 1.5 mL epi tube. a. Competence Development occurs with the following ranges of bacterial densities (O.D. 0.035 to 0.058) MAX EFFICIENCY (O.C. 0.042). MAKE SURE THE BACTERIA FALLS WITHIN THIS RANGE 4. Add 1.2 mg of pNZ8048 plasmid in EB buffer (10 mM Tris-Cl, pH 8.5) to the epi tube as well as 5 uL of synthetic peptide [FINAL - 250 mM]. 5. Incubate at 37 °C for 2 hours.