Streptococcus Competence Induction

Day 1: Streaking Bacteria From Freezer Stocks

• Refer to the strain database to determine which strain you plan to remove from the -80 degree Celsius freezer stock.

1.) Practicing Bio safety 2 protocols, bring an ice box to the -80 degree Celsius freezer (located in S223 instrument room). Obtain desired strains and put in the ice box.

2.) Under a sterile hood, use an inoculating loop to pick the desired strain. Streak out onto pre-labeled blood plate (date, strain number, and strain code).

3.) Dispose of contaminated inoculating look in 5% virkon S solution.

4.) Place streaked blood plates face down in the 37 degree Celcius incubator and allow them to incubate overnight.

Day 2: Overnight Culture

• All work done in the hood while practicing sterile technique.

1.) Obtain autoclaved empty culture tubes and Todd Hewitt Broth (THB).

2.) Pipette 3ml of THB into culture tubes. One tube will be used as a control/blank and all other tubes will be used to culture individual strains overnight.

3.) Remove culture plate from the 37-degree Celsius incubator. Place under a sterile hood.

4.) Label each culture tube with the corresponding strain number on the blood plate.

5.) Using an inoculating loop, pick a single colony from the blood plate. Place loop in corresponding culture tube and move loop up and down in the THB media to mix. Place loop in the Virkon S for disposal.

6.) Repeat step 5 for all other samples.

7.) Place culture tubes/ plates back into the 37-degree Celsius incubator and allow to sit overnight.

Day 3 : Part A- Preliminary Natural Transformation Experiments

Competence Induction and Transformation Protocol (Without Competence-Inducing Peptide)

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.

Day 3: Part B - Revised Natural Transformation Experiments

Extreme Overnight

1. Set up an overnight liquid culture in the incubator.

2. The next day, put 1 uL of this into 100 uL of THB within an eppendorf tube.

3. Immediately add the Donor DNA (2 ng of plasmid or 1.027 uL from a 1:100 dilution in water); vortex and put back into the 37 degree Celsius incubator.

• Do not put samples in the water bath as it needs to be incubated overnight.

4. The next day, plate onto TSA only and TSA + Chloramphenicol plates.

5. Use a dilution series (including undiluted samples) as 10uL dots on both of these plates. Make sure to plate strain + water samples (negative control) as well.

Serial Dilution and Plating

1. Remove extreme overnight culture from the 37 degree Celsius incubator.

2. Pipette 10 uL from the extreme overnight directly onto the TSA only and TSA + Chlorampheniol plates (This represents the 10^0 dilution).

3. To create subsequent dilutions in a 1:10 series, pipette 50 uL from the extreme overnight into 450 uL of 1 x PBS (This represents the 10-1 dilution). All additional dilutions created by taking 100 uL from the previous dilution and placing into 900 uL of 1 x PBS. Remember to go up to the 10^-6 dilution.

4. Once dry, flip TSA only and TSA + Chloramphenicol plates upside down and place them back into the 37 degree Celsius incubator.

Competence Induction and Natural Transformation with pNZ8048 plasmid (Chloramphenicol resistance and RFP)

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.

Day 3: Part C - Updated Competence Induction and Natural Transformation with pNZ8048 plasmid ('Without Competence-Inducing Peptide)

• Natural Transformation Experiment Modeled off of the Zaccaria Paper.

1. Grow an overnight culture for the strain(s) under investigation.

2. Following overnight incubation, return to lab the next day to begin transformation procedures

3. Fill up roughly 6 sterile culture tubes (13 mm) with 2.7 uL of THB.

4. To create a dilution series, pipette, 300 uL from the prior dilution and place into a new tube.

5. Within the 6ish tube, measure the Abs. Convert this to O.D ( Remember: Abs /path length (cm) = O.D. Our tubes are 13 mm so O.D = Abs/ 1.3)

6. Do dilution to get calculated O.D of 0.01 in a new 13mm test tube [or microcentriguge]. Ex: if the 6th tube has an O.D of 0.1, do a 1:10 dilution to bring the O.D down to 0.01.

7. Aliquot 100 uL into six microtubes and incubate in the 37 degree celcius incubator.

8. Wait till points 0, 60, 120, 180, 240, 300, 360 minutes.

9. At each time increment, add 1.128 uL of pNZ8048 plasmid (from the 886.1 ng/uL donor DNA) and incubate at 37 degrees Celsius for 5 minutes

10. Plate

       a. TSA Only Plates     : 20 uL of cels / 180 u< of 1 x PBS
       b. TSA and CAM Plates  : 50 uL and 50  uL of sample. 

11. Incubate all plates in the 37 degree Celsius incubator overnight. Return tomorrow to count CFUs that grew overnight.

Day 4: Counting CFUs

1.) Obtain strains from the 37 degree Celsius hood.

2.) Place all plates under a sterile hood.

3.) Open the lid to one of the blood plates and count the number of CFUs. Record on the Laboratory Ipad.

a. For CFUs plated onto individual blood plates, an ideal number of CFUs is in the range of 20-200 CFUs. For anything above ~200 CFUs, you can count the CFUs by counting one quadrant and multiplying by 4. 
b. If cells are too abundant, record as TMTC (Too Many to Count). 

4.) Repeat step 3 until ALL CFUs have been recorded for each sample.

5.) Dispose of blood plates in the red bio-safety containers.