Team:LASATX/Experiments




Materials/Methods

T7-GFP in pCDF, pCooF-GFP in pCDF, and T7-CooA in pCR2.1 were constructed by Gibson Assembly. E. coli BL21 (DE3) cells (New England Biolabs) were transformed with pCooF-GFP+T7-CooA, pCooF-GFP, or T7-GFP. A crude anaerobic chamber was created from a radiation waste container: Inlet and outlet adaptors were drilled into the walls of the radiation waste container, the lid sealed with vaseline, and the presence of oxygen determined with anaerobic indicator strips. Cultures were grown in LB Broth to OD600, then the cells resuspended with antibiotics and IPTG. CORM-2 (Sigma) was added at a concentration of 100 µM. After 2 hr incubation in the anaerobic chamber, or 2 hr aerobic incubation for controls, cells were resuspended in PBS and their fluorescence measured at 600 nm for 25 flashes.



Step-by-Step Instructions

Day 1:

Make four ON cultures: 2 mL LB, antibiotic (AMP = 2 uL/tube, SPEC = 4 uL/tube)

DE3 cells: N/A

  • T7-gfp: SPEC
  • pCoof-gfp: SPEC
  • pCoof-gfp + cow1: AMP + SPEC

Day 2:

  1. Use 2 mL deep well plate (37 shaking incubator room, left wall, top shelf, left side, white box labeled “2 mL”).
  2. Want 60 samples (four constructs x 5 CORM concentrations x triplicates of each condition).
  3. In each well, add: 10 uL cells (1:100 dilution), 1 mL LB, antibiotic (AMP = 1 uL/tube, SPEC = 2 uL/tube), 100 uL of 20% glucose.
Part II:
  1. Place the plate in the top 37°C shaking incubator (the one covered with foil). Make sure the rpm is 275. The plate should fit perfectly in one of the plastic 1 mL tip boxes screwed to the bottom of the incubator.
  2. Grow the cells to OD 0.6. This should take ~2 hours. In the meantime, flush the anaerobic chamber with argon.
  3. To measure OD, take 10 uL from at least 4 wells (one of each sample). Put the 10 uL of cells in a clear 96-well plate (brown box, shelf below the box of deep well plates). Add 90 uL LB (for a 1:10 dilution). Add 100 uL LB for a blank in another well. Using the plate reader, measure absorbance, multiplying each sample by 10 and subtracting by LB-only absorbance.
  4. Once OD is at 0.6, centrifuge the plate for 15-20 min, 3000 x g, 4C in the plate centrifuge.
  5. Resuspend with 1 uL of 1M IPTG, 1 mL LB, antibiotic. Add 976.5 uL DMSO to .01 g CORM (making 20 mM CORM). Add 20 mM CORM to samples for (0, 50, 100, 200, 500 uM).
    • 0 uM = 0 uL
    • 50 uM = 2.5 uL
    • 100 uM = 5 uL
    • 200 uM = 10 uL
    • 500 uM = 25 uL
  6. Grow under anaerobic conditions for 2 hours.
Part III:

Spin down cells (20 min, 3000 x g, 4°C). Resuspend in 1 mL PBS. Wash two more times, centrifuging for 3 min instead.

Read absorbance and fluorescence intensity:

Transfer 200 uL to 96-well COSTAR clear plate for fluorescence reading. In another well, have 200 uL PBS only (for a blank). lid off.

Absorbance

wavelength measurement at 600 nm. Read 25 flashes.

Fluorescence Intensity

wavelength excitation 480 nm, emission 520 nm. Z-position calculated from well. Read 25 flashes. Optimal gain.