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 Abs (O.D. 0.035 to 0.058) MAX EFFICIENCY (O.C. 0.042). MAKE 
           SURE THE BACTERIA FALLS WITHIN THIS RANGE

4. Add 20 ng/mL 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. WE can reduce the number of colonies on a plate by using a serial dilution.

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.

Competence Induction and Transformation Protocol (without peptide. What I am currently using in Lab)

1.) Grow Strains overnight in Todd-Hewitt Broth (THB)

2.) Pipette 100 uL of the strain into 3 mL of THB in a “fresh culture tube. a. Measure the Abs and place in the 37 Celsius

3.) Wait one hour to allow the Abs to grow up to about 0.05ish (Starting Abs).

4.) Once at 0.05ish, pipette 100 uL out of the “fresh culture” tube and place this into a 1.5 mL Epi Tube

5.) Add 2 ng of plasmid (or 1.027 uL from a 1:100 dilution in water). a. Remember to also add a control sample with 1.027 uL of water.

6.) Place Epi tube back into the 37-degree Celsius incubator.

7.) Plate onto TSA only and TSA + Choloramphenicol plates at the following time increments (10, 20,30, 40, 60, 90, and 120 minutes) a. Plate 10 uL directly from the epitube onto the TSA and TSA + Chloramphenicol plates b. To look at a dilution series for each strain, pipette 50uL of the strain into 450 uL of 1x PBS (this creates a 10^-1 dilution). c. To create subsequent dilutions, pipette 100 uL from the 10^-1 dilution and place into 900 uL of 1xPBS. This will create a 10^-2 dilution).

8.) Place all the blood plates back into the 37-degree incubator overnight. Return tomorrow to count the CFUs that formed on the plate.