• 2-3x 250mL flasks
• Shaking water bath

• • 30C or 37C Incubator

1. Allow to grow for 1.5h @ 30C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2-0.3)

1. Dilute 100uL of culture into 900uL of media in an eppendorf tube, then transfer 10uL into 10mL fresh media pre-warmed to 37C* (10000x dilution total)

1. Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2-0.3)
2. Add 5e7 phages and incubate 5min without shaking at 37C* (may need to be shorter for some strains?) *30C for nsAA evolved strains
3. Dilute 100uL of culture into 900uL of media in an eppendorf tube, then transfer 10uL into 10mL fresh media pre-warmed to 37C* (10000x dilution total) (may need to adjust volumes?)

• • plate 10uL of each dilution as described in Determining Phage Titers

Measuring Lysis Timing of T7 Phage

Reagents / Materials:

• Phage lysate
  • see Preparing Phage Lysates for protocol
• Overnight stock of permissive bacterial host
• Make sure to grow in presence of 250uM nsAA if using nsAA-RS strains
• LB Media w/ appropriate supplements and antibiotics
• 250mL flasks
• For plating phages:
  • LB Plates with w/ appropriate supplements and antibiotics
  • LB Top Agar (LB + 0.8% Agar)
  • Test tubes
  • 55C Water bath or heat block
  • 37C Incubator

Procedure:

1. Add 500uL of an overnight culture of the E. coli host to 9.5mL of LB in a 250mL flask
2. Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.5 ?)
3. Add 5e7 phages and incubate 5min without shaking at 37C (may need to be shorter for some strains?)
4. Transfer 1uL of culture to fresh 10mL (10000x dilution) of LB pre-warmed to 37C (may need to adjust volumes?)
5. Plate samples at 1min timepoints from 5-15min (may depend on strain)
   • plate 100uL of each dilution as described in Determining Phage Titers
   • Probably don't need to set up dilutions for these (?)
6. Plot titers over timepoints; 'lysis time' is timepoint just before titer begins to increase

References:

[1] Heineman, Richard H, and James J Bull. "Testing Optimality with Experimental Evolution: Lysis Time in a Bacteriophage." Evolution 61, no. 7 (2007): doi:10.1111/j.1558-5646.2007.00132.x.

-- Main.ColinBrown - 14 Jun 2017