Measuring Lysis Timing of T7 Phage

Reagents / Materials:

  • Phage lysate
  • 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 ~ 0.2-0.3)
  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) (may need to adjust volumes?)
  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

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Topic revision: r2 - 13 Sep 2017 - 13:49:32 - Main.ColinBrown
 
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