Difference: ProtocolPhageLysate (1 vs. 6)

Revision 62021-06-08 - JeffreyBarrick

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META TOPICPARENT name="ProtocolsPhageAdsorptionRate"

Phage Lysate Preparation

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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.
>
>
We have used this protocol with phages T4, T5, T6 and T7. The general procedure should work with most lytic 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.
 

Materials

  • 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)
Changed:
<
<
  • 50 mL flasks (recommended) or test tubes
>
>
  • 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.)

Revision 52020-02-25 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolsPhageAdsorptionRate"

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.
Line: 23 to 23
 *REL606 is resistant to phage T6
Changed:
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<

Procedure:

>
>

Procedure

 
T7-lysate.png
T7 lysate showing small amount of chloroform that has settled to the bottom of the tube.

Revision 42020-02-24 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolsPhageAdsorptionRate"

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.
Line: 7 to 7
 
  • 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)
Changed:
<
<
  • 50 mL flasks (recommended) or test tubes
>
>
  • 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.)
Line: 24 to 25
 

Procedure:

Added:
>
>
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)
Line: 32 to 36
 
  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
Changed:
<
<
  1. Spin 5min @ 5000 &multiply; g (RCF)
>
>
  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
Line: 46 to 50
 
  • T5 and T6 lysates (107-109 PFU/ml)
  • T7 lysates (???? PFU/ml)
Added:
>
>
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.
Line: 55 to 62
 

Sources

Elements of this protocol were adapted from the Lenski Lab Phage-pharming Protocol Brendan Bohannan and Neerja Hajela.
Added:
>
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META FILEATTACHMENT attachment="T7-lysate.png" attr="h" comment="" date="1582571908" name="T7-lysate.png" path="T7-lysate.png" size="692277" stream="T7-lysate.png" tmpFilename="/usr/tmp/CGItemp56326" user="JeffreyBarrick" version="1"

Revision 32020-02-24 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolsPhageAdsorptionRate"

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.
Line: 7 to 7
 
  • 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)
Changed:
<
<
  • 50 mL flasks or test tubes
>
>
  • 50 mL flasks (recommended) or test 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.)
Changed:
<
<
phage host strain freezer stock
T4 REL373 JEB1010
T5 REL373 JEB1011
T6 REL373 JEB1012
>
>
phage host strain phage stock
T4 REL606 JEB1010
T5 REL606 JEB1011
T6 REL373* JEB1012
 
T7 BL21 MJH158
T7Δ2 BL21 MJH159
λ-vir REL606 JEB1066
Added:
>
>
*REL606 is resistant to phage T6
 

Procedure:

  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)
Changed:
<
<
    • Can be useful to start an no-phage control (i.e. diluted host w/ no phage) for turbidity comparison w/ lysed cultures
>
>
    • It's highly recommended that you start a no-phage control at the same time (i.e., an extra flask of diluted host w/no phage added) for turbidity comparison w/ lysed cultures
 
  1. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~106 PFU.
  2. 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.

Revision 22020-02-23 - JeffreyBarrick

Line: 1 to 1
 
META TOPICPARENT name="ProtocolsPhageAdsorptionRate"

Phage Lysate Preparation

Changed:
<
<
  • Definitely works with T4 and T7, should work with most E. coli phages (although lysis times and final titers may differ)
>
>
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.
 
Changed:
<
<

Reagents / Materials:

  • Overnight culture of a permissive host
  • BL21 usually works well for T7 and T7Δ2 (a.k.a mutator); MJH116(Amberless) with appropriate nsAA-aaRS for nsAA evolution strains
  • Frozen Stock of Phage (or fresh plating)
  • LB Media (w/ antibiotics or supplements appropriate for host)
>
>

Materials

  • 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)
 
  • 50mL flasks or test tubes
Changed:
<
<
  • Shaking incubator at appropriate temp (37 or 30) *Chloroform (optional but recommended)
>
>
  • 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.)

phage host strain freezer stock
T4 REL373 JEB1010
T5 REL373 JEB1011
T6 REL373 JEB1012
T7 BL21 MJH158
T7Δ2 BL21 MJH159
λ-vir REL606 JEB1066
 

Procedure:

Changed:
<
<
  1. Prepare overnight culture of permissive host (Amberless aaRS strains should be grown w/ 250μM nsAA in media and may need up to 24h to reach saturation)
  2. Dilute 1mL of overnight culture into 10mL fresh media (1:10 dilution), grow for 30-60min (OD ~0.2)
    • Some finicky strains (e.g. nsAA evolved strains) apparently lyse stochastically, so best to set up 8-10 lysis cultures (lyses?)
>
>
  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)
 
    • Can be useful to start an no-phage control (i.e. diluted host w/ no phage) for turbidity comparison w/ lysed cultures
Changed:
<
<
  1. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip.
>
>
  1. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip. Ideally, you are aiming for an inoculum of ~106 PFU.
 
  1. Shake @~200RPM at the appropriate temperature until culture lyses completely
Changed:
<
<
    • i.e. little or no turbidity compared to no-phage control; will usually see bits of aggregated cell debris also
  1. Add 50-100uL of chloroform and vortex
  2. Spin 5min @ 5000RPM
>
>
    • It should have little or no turbidity compared to the no-phage control. You will usually see bits of aggregated cell debris also.
  1. Add 50-100 L of chloroform and vortex
  2. Spin 5min @ 5000 &multiply; g (RCF)
 
  1. Transfer lysate (supernatant) to a clean tube
Changed:
<
<
    • For long-term storage @ 4C, add 50μL chloroform to prevent bacterial growth
>
>
    • 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
  1. Determine phage titer Protocol: Measuring Phage Titers
Changed:
<
<
-- Main.ColinBrown - 14 Dec 2017
>
>

Expected Results

If you are starting from a freezer stock or old lysate, you may need to perform this procedure multiple times to get the titer of your lysate up. Expected ranges of "good" lysate that can be achieved with this procedure should be in these ranges:

  • T4 lysates (1010-1012 PFU/ml)
  • T5 and T6 lysates (107-109 PFU/ml)
  • T7 lysates (???? PFU/ml)

Variants

nsAA evolution strains

  • Use the host strain MJH116(Amberless) transformed with a plasmid encoding the desired nsAA-aaRS.
  • Supplement media with antibiotics and 250μM nsAA.
  • Cultures of this strain may need up to 24h to reach saturation.
  • Some evolved strains may lyse stochastically. It may help to set up 8-10 lysis cultures and use the clearest one.

Sources

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

Revision 12017-12-14 - ColinBrown

Line: 1 to 1
Added:
>
>
META TOPICPARENT name="ProtocolsPhageAdsorptionRate"

Phage Lysate Preparation

  • Definitely works with T4 and T7, should work with most E. coli phages (although lysis times and final titers may differ)

Reagents / Materials:

  • Overnight culture of a permissive host
  • BL21 usually works well for T7 and T7Δ2 (a.k.a mutator); MJH116(Amberless) with appropriate nsAA-aaRS for nsAA evolution strains
  • Frozen Stock of Phage (or fresh plating)
  • LB Media (w/ antibiotics or supplements appropriate for host)
  • 50mL flasks or test tubes
  • Shaking incubator at appropriate temp (37 or 30) *Chloroform (optional but recommended)

Procedure:

  1. Prepare overnight culture of permissive host (Amberless aaRS strains should be grown w/ 250μM nsAA in media and may need up to 24h to reach saturation)
  2. Dilute 1mL of overnight culture into 10mL fresh media (1:10 dilution), grow for 30-60min (OD ~0.2)
    • Some finicky strains (e.g. nsAA evolved strains) apparently lyse stochastically, so best to set up 8-10 lysis cultures (lyses?)
    • Can be useful to start an no-phage control (i.e. diluted host w/ no phage) for turbidity comparison w/ lysed cultures
  3. Pick a plaque from a fresh overnight plate (for clonal lysate) or scrape frozen stock with a sterile toothpick or pipet tip.
  4. Shake @~200RPM at the appropriate temperature until culture lyses completely
    • i.e. little or no turbidity compared to no-phage control; will usually see bits of aggregated cell debris also
  5. Add 50-100uL of chloroform and vortex
  6. Spin 5min @ 5000RPM
  7. Transfer lysate (supernatant) to a clean tube
    • For long-term storage @ 4C, add 50μL chloroform to prevent bacterial growth
    • For long-term storage @ -80C, add glycerol to 20% and mix well
  8. Determine phage titer Protocol: Measuring Phage Titers

-- Main.ColinBrown - 14 Dec 2017

 
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