Bacterial Growth and Light Exposure Protocol
Introduction
Materials
- Equipment
- Flow cytometer
- Calibration beads for flow cytometer (?)
- 8 24-well LPA instruments
- Burner (?)
- 2 black-walled clear-bottomed 96-well plates
- 8 multichannel-ready tube racks
- 37°C Water bath
- Micropipette
- Pipette tips
- Vortexer
- 250mL Beaker
- Flow cytometry tubes
- Plate reader (X)
- Microcentrifuge
- Reagents
70% EtOHDI water10% casamino acids20% glucose1m MgO41m CaCl25x M9 salts- M9 Media with Casamino Acids
- 10% bleach
- Other
- Foil
- -80°C culture aliquot
- PBS solution (pH7-7.2) (phosphate-buffer saline)
- Rifampicin
- Ice
- Appropriate antibiotics
Procedure
Experiment Initialization
- Prepare LPAs.
- Start preparing 24-well plates for use by soaking the 24-well plates for 15 minutes in 70% EtOH.
- Rinse the plates using DI water 3 times and place rinsed plates on foil.
- Dry plates near lit burner until interior of the wells are dry (about 45 minutes).
- Wipe the bottom of the plate and the foil, and wrap the plate in the foil.
- Make a 25mL aliquot of M9 medium, add appropriate antibiotics, and shake the container.
- Remove -80°C culture aliquot from the freezer and allow it to thaw. Briefly vortex thawed culture aliquot and spin down in a microcentrifuge.
- Add appropriate culture volume to the 25mL media.
NOTE: inoculation densities for the strains used in Tabor's paper for Cph8-OmpR is 5E-6.
- Shake inoculated media and pipet 500 μL of media into each well of the 24-well plates. Cover each plate with foil seals.
- Load the plates onto each LPA and allow the cells to grow in the LPAs at 37°C for 8 hours.
Experiment Completion and Data Collection
- Prepare a PBS + rifampicin solution by adding 62.5mg of rifampicin to 125mL of a PBS solution in the pH7-7.2 range in a 250mL beaker. Cover the solution with foil.
NOTE: COVER the solution AS SOON AS POSSIBLE because rifampicin is light-sensitive.
- Put the beaker on a stir-plate and add the rifampicin to the middle of the vortex (that doesn't reach down to the bar), and then slide the beaker slightly off-center on the plate. Remove the plate after 10 minutes, and filter the rifampicin solution using a 0.22 μm filter.
NOTE: The solution should be a vivid, bright orange after 10 minutes. If it is dark-colored, there was a light leakage, vigorous stirring, or the saline was not in the pH range of 7-7.2. In that case, the solution should be remade.
- Prepare 1 autoclave tray with an ice-water bath.
NOTE: Make sure water level is near the surface of the ice.
- Load 48 wells of a 96-well plate with 500µL of rifampicin each, then place the plate in the ice-water bath.
- When there is 2 minutes remaining on the LPA programs, open the incubator with the LPAs.
- When the program is complete, remove the enclosed plates and immediately submerge them at least halfway up the side of the plate in the ice-water bath.
- Transfer 100 μL volumes of the cultures into the corresponding wells on the 96-well plate. Repeat until all 48 samples have been transferred to the 96-well plate.
- To homogenize the cytometry tubes, tilt the racks nearly horizontally and gently shake the rack. Rotate the rack 180° and repeat.
- Transfer the rifampicin+culture tube racks into a 37°C water bath for 1 hour for fluorescence maturation. After it is complete, place the tube racks into an ice-water bath for at least 30 minutes.
- Prepare a bead sample for cytometry calibration.
- Pipette 500 μL of PBS into a cytometry tube and add one drop of calibration beads in to the PBS. Cap the tube and place it on ice.
- Transport the rifampicin+culture samples and the bead sample to the cytometer.
- Analyze the bead sample. Collect atleast 5,000 events.
They should be tightly-clustered in the center of the FSC vs. SSC scatter plot. The gain setting on the fluorescence channels should match the gain used to acquire the cell samples. NOTE: if measuring a strain for the first time, it is wise to measure the cultures anticipated to have the highest and lowest fluorescence levels.
- Run the cell samples through the cytometer. Collect at least 20,000 events per sample.
They should be clearly visible on the FSC vs. SSC scatter plot. NOTE: Make sure the fluorescence histograms are fully contained within the measurement range at the current gain settings.
- Clean the cytometer by running the clean protocol on it.
This is the Appendix Supplementary Method S1 protocol from Dr. Tabor's paper that details gene expression characterization experiments to train the optogenetic model.