Measuring Adsorption Rate of T7 Phage

Reagents / Materials:

  • *Fresh* phage lysate (no more than *one day* old)
  • 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)
  3. Add 1e6 phages (from fresh lysate) and incubate 5min without shaking at 37C
  4. Remove 2 1mL aliquots, centrifuge one sample to pellet adsorbed phage (4000g, 1min, RT) and let the other sit at RT
  5. Plate dilutions of supernatant from pelleted sample (unadsorbed phage) and the non-centrifuged sample (total phage)
  6. Calculate Ntotal and Nfree from plate counts, by rate equation Nfree = Ntot*e^(-5a). Adsorption rate a = ln(Ntot/Nfree)/5

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 - 15 Jun 2017 - 15:35:27 - Main.ColinBrown
 
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