Bacteria-Yeast Communication
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Bacteria-Yeast Communication Protocol
Yeast
1. Grow cells overnight, 20 hours in 6 mL media with 120 uL of PCB (50x stock) (!!need to grow total of 18 mL cells, 360 uL of PCB!!), in leu-/trp- media supplemented with extra histidine. Use a little scraping of cells from the aliquot. (Take stuff from aliquot B, has a mL of frozen culture, use end of a bacterial spreader)
2. At 16-20 hours of growth, want cell concentration at 1x10^6 cells/mL. On the hemocytometer, if you count two diagonal blocks there should be 200 cells total. If you take the OD, the OD of 2E7 cells is 0.8.
3. Spin down the 6 mL of cells (at top speed, 8000rpm, 1 minute should pellet all of the yeast). Resuspend in 200 uL of fresh medium with PCB (4 uL 50x stock).
Bacteria
1. Grow luciferase bacteria in LB amp overnight , 10 mL.
2. Grow bacteria overnight, induce 5 hours ahead of time with IPTG, 10 mMolar, 0.238 g per 100 mL, dump powder in directly into media, should dissolve well. Use 2x YT, we have 300 mL, use 100 mL.
3. Centrifuge the bacteria down at max (4400 rpm for 10 minutes), and resuspend in 5 mL sodium citrate buffer, with 10 mMol luciferin, so 0.032 g in 10 mL sodium citrate.
4. ONLY SPIN DOWN AND RESUSPEND IMMEDIATELY BEFORE EXPOSING THE YEAST
Filter Lifts
1. Get the liquid nitrogen
2. Prepare the Z buffer
3. Lay filter paper down on the yeast plate, rough side down, and lift up. Place filter on foil boat on liquid nitrogen, then submerge in liquid nitrogen for 2-5 seconds.
4. Place in small dish with Z buffer
3 plates: Experimental, negative control in bioreactor (no light), laser control. All PCB treated, grown overnight.
3.5 mm radius for a well in the 96 well plate, 27 mm radius for miniplate, so we need 6 mL cells for whole plate as opposed to 100 uL per well for same cell density.
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