Media and Passaging

Minimal Media Recipe

LeMaster-Richards Minimal Media (LeMaster and Richards, 1982)

Final Volume	Water	4x Minimal Salts	2M MgSO4	Trace Minerals (4000x)	Glucose (if needed)
10ml	7.5ml	2.5ml	10ul	2.5ul	xxul
100ml	75ml	25ml	100ul	25ul	xxul
250ml	180ml	62.5ml	250ul	62.5ul	xxul

• Glycerol should be added to a final concentration of xx% (or xx% if using a double amount).

If using:

• • Black cells in 10ml: 1.25ul (for 10^7 cells total)
  • Green cells in 10ml: xxul (xxul for double amount)
  • Red cells in 10ml: xxul (xxul for double amount)
  • Green and Orange cells in 10ml: xxul (xxul for double amount)
  • Red and Blue cells in 10ml: xxul (xxul for double amount)

Cell Aliquots

Using E. coli Aliquots -- 10^7 total cells

Aliquot Color	For 10^7 cells, use:	Strain Number	Genotype	Grows in:	Fluorescence	Frozen Date
Black	1.25ul	S-734	Wild Type	All	None	Spring 2022
Green	1.83ul	S-735	Δppc	LB; glycerol; succinate; a-keto	sfGFP	Spring 2022
Red	1.42ul	S-736	Δppc	LB; glycerol; succinate; a-keto	DSRed-Express2	Spring 2022
Blue	5.13ul	S-737	Δfbp	LB; glucose; galactose	sfGFP	Spring 2022
Orange	1.16ul	S-738	Δfbp	LB; glucose; galactose	DSRed-Express2	Spring 2023

Phage Passaging Protocol

1. Prepare 10ml of appropriate media
   • For WT passaging, this is 10ml of MM + 0.3% glucose for '2x' lineages and 10ml of MM + 0.15% glucose for '1x' lineages.
2. Add cells
   • For WT passaging, this is 1.25ul of frozen, black, WT cell aliquots
3. Incubate in shaking incubator for 1 hour
4. Add 10^4 PFU of phage
5. Incubate in shaking incubator for 23 hours
6. Add additional sugars, if applicable
   • For WT passaging, add 389ul of 4% glucose
7. Note whether flasks are clear or cloudy; add this results to document on Notability on lab iPad.
8. Incubate in shaking incubator for 23-24 hours
9. Label 6 microcentrifuge tubes.
10. Add 125ul chloroform into a labeled microcentrifuge tube. Ensure picking chloroform from the bottom of the tube, as there is a water layer on top.
11. Add 1ml of incubated cells + phage into each tube.
12. Vortex for 15 seconds each.
13. Put in microcentrifuge and spin down, 5 minutes at max speed. You should see 2-3 layers of liquid, with the middle layer (if present) being a thin line of dead cells. If you only see 1 layer, something is off -- probably the top layer of water was added from the chloroform. Please start over.
14. Pipet out 800ul into the previously labeled, empty tube. Put into phage box in the refrigerator.
    • For WT passaging, create a 10^-2 dilution for the next phage passage by adding 990ul PBS to 10ul of collected, chloroform-purified phage.
15. If the passage is dividable by 5, do a plaque assay to correct the number of added PFU for the next passage.