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===[[Streptococcus DNA Extraction]]===
===[[Streptococcus DNA Extraction]]===
===[[Streptococcus Competence Induction]]===
===[[Streptococcus Competence Induction]]===
 
===[[Measuring Competence : Fixation and Flow Cytometry]]===
Bacterial Transformation/Competence Measurements:
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.
 
Measuring Competence – Fixation and Flow Cytometry
The movement of Bio Safety 2 equipment/lab material MUST be well regulated. Since the Flow cytometer is located in _________, we need to take extra precautions to prevent contamination via infectious agents when transporting the S. suis samples out of lab.
Fixation
1. Centrifuge 1.5 ml epi tube to collect cells and aspirate supernatant
a. https://www.cellsignal.com/contents/resources-protocols/flow-cytometry-protocol-(flow)/flow
2. Resuspend the pelleted cells in 0.5-1 ml 1X PBS. Add formaldehydepe [FINAL 4%]
3. Allow mixture to sit for 15 minutes at room temperature to fix.
4. Wash via centrifugation with excess 1x PBS. Discard supernatant and resuspend in 0.5-1 ml 1X PBS.
Flow Cytometry  (Room : ______) 
5. Upon Fixation, run the Flow cytometer machine (Refer to the Flow cytometry protocal).
6. Record
a. Total number of particulates
b. Total number of red particulates


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Revision as of 13:15, 24 October 2018

Laboratory information

General microbiology protocols

Media Recipes

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Working with Antibiotics

Freezing -80 Stocks

Freezing Aliquots

Competition Assays

Phyllosphere protocols

Creating Sterile Agar Plates

Sterilization and Germination Protocol for ''Arabidopsis thaliana'' Seeds in Gnotobiotic Experiments

Germination Protocol for ''Arabidopsis thaliana'' Seeds in Non-Sterile Experiments

Growth Stage Phenotype Definitions

Growth Conditions for ''Arabidopsis thaliana''

Inoculation of ''Arabidopsis thaliana'' with Microbes

Removal and DNA Extraction of Phyllosphere Microbes

ARISA

Streptococcus pneumoniae protocols

Dual Layer Assays

Streptococcus DNA Extraction

Streptococcus suis protocols

Streptococcus DNA Extraction

Streptococcus Competence Induction

Measuring Competence : Fixation and Flow Cytometry

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