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:
- Add 500uL of an overnight culture of the E. coli host to 9.5mL of LB in a 250mL flask
- Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.5 ?)
- Add 5e7 phages and incubate 5min without shaking at 37C (may need to be shorter for some strains?)
- Transfer 1uL of culture to fresh 10mL (10000x dilution) of LB pre-warmed to 37C (may need to adjust volumes?)
- 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 (?)
- 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
Topic revision: r1 - 2017-06-14 - 18:19:04 - Main.ColinBrown