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
• 2-3x 250mL flasks
• Shaking water bath
• 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
  • 30C or 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 1.5h @ 30C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.2-0.3)
3. Add 5e7 phages and incubate 5min without shaking at 37C* (may need to be shorter for some strains?) *30C for nsAA evolved strains
4. 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)
5. Plate samples at 1min timepoints from 5-15min (may depend on strain)
   • plate 10uL 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