Difference: ProtocolPhageLysate (3 vs. 4)

Revision 42020-02-24 - JeffreyBarrick

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Phage Lysate Preparation

We have used this protocol with phages T4, T5, T6 and T7. The general procedure should work with most E. coli phages (although lysis times and final titers may differ). See the variants section for some ways to modify it for other hosts/phages.
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  • Overnight culture of a permissive E. coli host (see the table below)
  • Frozen stock of phage (or plate with fresh plaques)
  • LB-Miller Media (w/antibiotics or supplements appropriate for host E. coli)
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  • 50 mL flasks (recommended) or test tubes
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  • 50 mL flasks (recommended) or test tubes * 15 mL Falcon tubes
 
  • Shaking incubator at appropriate temperature (37 or 30)
  • Chloroform (Safety: All work with chloroform should be done in a fume hood with gloves, a lab coat, and eye protection.)
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Procedure:

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T7-lysate.png
T7 lysate showing small amount of chloroform that has settled to the bottom of the tube.
 
  1. Prepare overnight culture of permissive host
  2. Dilute 1 mL of overnight culture into 10 mL fresh media (1:10 dilution) for each phage lysate. Include an extra dilution as a no-phage control for turbidity comparison w/ lysed cultures
  3. Grow these cultures for 30-60 min (OD ~0.2)
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  1. Shake @ ~200 RPM at the appropriate temperature until culture lyses completely
    • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
  2. Add 50-100 L of chloroform and vortex
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  1. Spin 5min @ 5000 &multiply; g (RCF)
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  1. Spin 5min @ 5000 × g (RCF)
 
  1. Transfer lysate (supernatant) to a clean tube
    • For long-term storage @ 4C, add 50 L chloroform to prevent bacterial growth
    • For long-term storage @ -80C, add glycerol to 20% and mix well
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  • T5 and T6 lysates (107-109 PFU/ml)
  • T7 lysates (???? PFU/ml)
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BL21-TW-WT-inoculated.png Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) immediately after phage is added from a high-titer lysate.
BL21-TW-WT-lysed.png Blank (left), E. coli BL21 (center) and E. coli BL21 + Phage T7 (right) 3 hours later showing that lysis has occurred in the flask with phage but cell density has increased in the negative control with only E. coli
 

Variants

nsAA evolution strains

  • Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
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Sources

Elements of this protocol were adapted from the Lenski Lab Phage-pharming Protocol Brendan Bohannan and Neerja Hajela.
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