Sauer:P1vir phage transduction

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[[P1vir phage transduction|Back to P1vir phage transduction]]
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contributed by [[Sean Moore]]
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==Background==
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Phage transduction is used to move selectable genetic markers from one "donor" strain to another "recipient" strain.  Nat Sternberg, among others, pioneered the use of phage P1 to move genetic elements in <i>E. coli</i> and the use of the Cre/Lox system from P1 for controlled recombination.  Today, phage P1 is commonly used as a transducing agent because it is a generalized tranducer (it can package random sections of the host chromosome instead of its own genome) giving rise to "transducing particles".  P1<i>vir</i> is a mutant phage that enters the lytic cycle upon infection (ensuring replication and lysis).  During the replication and lysis of the phage in a culture of bacteria, a small percentage of the phage particles will contain a genome segment that contains your gene of interest.  P1 packages approximately 90 kb of DNA, so you can transduce genes that are linked to a selectable marker.
 +
 +
Once a phage population has been generated from a donor host, the phage are used to infect a recipient host.  Most of the bacteria are lysed by phage that packaged P1 genomes, but a fraction of the phage inject a genome segment derived from the donor host. Homologous recombination then allows the incoming genomic segment to replace the existing homolgous segment.  The infected recipient bacteria are plated on a medium that selects for the genome segment of the donor bacteria (antibiotic resistance, prototrophy, etc.)
 +
 +
All of this would not work if the infectivity of the phage could not be controlled.  Otherwise, phage released from neighboring cells would infect and lyse the bacteria that had been infected with transducing particles.  Someone really smart discovered that phage P1 requires calcium for infectivity.  Therefore, you can control P1 infectivity by growing in the presence and absence of calcium.  The calcium chelator citrate is usually used because it lowers the concentration of free calcium (by forming Ca-citrate) low enough to prevent P1 infection, but not so low as to starve the cells for calcium.
 +
==Lysate preparation==
==Lysate preparation==
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1. Dilute an overnight culture (LB medium) of donor strain 1:100 in fresh LB + 5 mM CaCl2 and 0.2% glucose (2.5 mL should be enough). Grow with aeration at 37 ˚C for 1 hr. Add 100 µL of P1 phage lysate to the culture, continue growing at 37 ˚C. Monitor for 1–3 hr until the culture has lysed completely.
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1. Dilute an overnight culture of donor strain <u>grown with selection</u> for the marker to be transduced 1:100 in fresh LB supplemented with 10-25 mM MgCl2, 5 mM CaCl2, and 0.1-0.2% glucose (1 mL per lysate). DO NOT ADD ANTIBIOTIC TO THIS CULTURE. Grow with aeration at 37 ˚C for 1-2 hr. When the cells are in early log phase (slightly turbid, but noticeable growth) Add 40 µL of P1 phage lysate to the culture, continue growing at 37 ˚C. Monitor for 1–3 hr until the culture has lysed (you'll see cellular debris in the tube and the culture will have significantly lessened in its turbidity).
-
2. Add several drops of chloroform to the lysate and vortex. Centrifuge away the debris (14,000 rpm, 1–2 min) and transfer the supernatent to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.
+
NOTE: In my experience less phage the best, since you must have a moi of about 1 pfu/cel. The best option is try different amounts (from 5 to 100 µL) and take for transduction the lysate got with less P1 inocule.
 +
--[[User:Modesto Redrejo Rodriguez|zurdo]] 12:38, 2 September 2010 (EDT)
 +
 
 +
2. Add several drops (50-100 uL) of chloroform to the lysate and vortex. Centrifuge away the debris (14,000 rpm, 1–2 min) and transfer the supernatant to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.
==Transduction==
==Transduction==
Line 9: Line 26:
1. Grow recipient strain overnight in LB medium (2 mL culture is plenty).
1. Grow recipient strain overnight in LB medium (2 mL culture is plenty).
-
2. On the next day, harvest the cells by centrifugation (6000 rpm, 2 min) and resuspend in original culture volume in fresh LB + 100 mM MgSO4 + 5 mM CaCl2. (note: 10 mM MgSO4 works fine, too, so you can use the 0.1 M MgSO4 the kitchen makes.)
+
2. On the next day, harvest the cells by centrifugation (6000 rpm, 2 min) and resuspend in 1/5-1/3 the harvested culture volume in fresh LB + 100 mM MgSO4 + 5 mM CaCl2. (note: 10 mM MgSO4 works fine, too, so you can use the 0.1 M MgSO4 the kitchen makes.) For example, if you're planning 2 transductions, resuspend 1 mL of overnight in 300 uL of supplemented LB.
-
3. Set up four "reactions":
+
2b.(optional) Transfer 100 uL of transducing P1 lysate into a 1.5 mL microfuge tube for each transduction and incubate them with the caps opened at 37 ˚C for ~30 minutes. This step allows excess chloroform to evaporate from the phage stock. You can place your resuspended recipient strain in the incubator as well during this time to help them wake up from their nap.
 +
 
 +
3. Set up four "reactions" by adding recipient bacteria to the tubes with phage, mix rapidly after addition, close the caps:
A. 100 µL undiluted P1 lysate + 100 µL recipient cells
A. 100 µL undiluted P1 lysate + 100 µL recipient cells
Line 22: Line 41:
4. Incubate tubes at 37 ˚C for 30 min.
4. Incubate tubes at 37 ˚C for 30 min.
-
5. Add 200 µL Na-Citrate (pH 5.5), then add 1 mL LB (the real thing this time) and incubate at 37 ˚C for 1 hr to allow expression of the antibiotic resistance marker.
+
5. Add 200 µL 1 M Na-Citrate (pH 5.5), then add 1 mL LB (the real thing this time) and incubate at 37 ˚C for 1 hr to allow expression of the antibiotic resistance marker. If you are working with a marker or recipient that needs to grow at 30 ˚C, double the recovery time.
 +
 
 +
6. Spin cells at 6000 rpm for 5 min.
 +
 
 +
7. Resuspend each in 100 µL LB supplemented with 100 mM Na-Citrate (pH 5.5), vortex well to disperse cells, and plate all of it on an appropriate antibiotic-containing plate.
 +
 
 +
8. You should get anywhere from ~ 10 to 2000 colonies.  These colonies are growing on a plate that is covered with P1 phage.  If you simply pick a colony from this plate and prepare a freezer stock, you will most likely have phage contamination that will manifest when a culture is grown up in the absence of a calcium chelator.  Therefore, prepare a plate spread with the selection antibiotic mixed in 100 µL of 1 M citrate (pH 5.5).  Then, use a toothpick to touch the top of a few colonies and re-streak on the new plate for isolated colonies.
 +
 
 +
9.  Test a colony from each re-streak for the presence of the mutant gene you intended to transduce using diagnostic PCR or Southern blotting.
 +
 
 +
==Anecdotes==
 +
 
 +
*The chloroform used to sterilize the phage lysates, well, sterilizes.  If you have visible chloroform drops in the lysate stock, don't add this to your recipient cells directly because you can kill a decent number of bacteria.  Instead, aliquot your phage into microfuge tubes and incubate with the caps open at 37 ˚C for about 30 minutes to allow the chloroform to evaporate (step 2b).  Then add the recipient cells to the tubes with the phage.
 +
 
 +
*When preparing the donor phage lysate, there is a huge variability in the titer of phage obtained at this step which makes transduction performance unpredictable.  Some donor cells are slow "wake up" from stationary phase and 3 hours will not be enough.  If it is obvious that there was no culture development in the tube, let it shake overnight.  The next morning, you will have a culture of cells and, perhaps, noticeable cell debris.  Treating this with chloroform and preparing it as a phage lysate usually works.
 +
 
 +
*P1 lysis is accelerated under reducing conditions (Ryland Young's Lab).  Adding 1 mM DTT to the top agar allows P1 to develope better plaques.  It follows that reducing agents may help the donor lysate develope and help the recipients infecected with infectious P1 to lyse before plating.  If you're having trouble getting a high titer of donor phage, try &beta;-mercaptoethanol at 1/1000 culture volume.
 +
 
 +
*P1 replicates poorly in <i>recA</i>- hosts.  Moreover, RecA is required for the homologous recombination needed to integrate the donor DNA.  Therefore, I have not been able to transduce into <i>recA</i> bacteria.  In cases that I needed to have <i>recA</i>- bacteria following a transduction, I first transduced my desired marker into a <i>recA</i>+ recipient, then subsequently transduced <i>recA::kan</i> from a specialized <i>recA+</i> donor strain generated by Barry Wanner (BW 26,547 <i>recA::kan</i> Lambda <i>recA</i>+).
 +
 
 +
*As mentioned above, P1 packages ~90 kb of DNA.  This means that genes in the vicinity of your target gene in the recipient will most likely be replaced with copies of the neighboring genes from the donor.  Therefore, you can't easily transduce a marker close to an existing marker in the recipient unless you have good selection for both markers and there is enough space between the genes to allow significant recombination between them. It is a good idea to be aware of the relative distances of genes of interest in your strains when transducing to avoid accidental curing of relevent markers.
 +
 
 +
*Be mindful some E. coli are "B" strains and your current knockouts may be in a "K" strain (or visa versa).  You may have noticed that when you move plasmids from a "cloning strain" like DH5-alpha into BL-21 for expression, you don't get as many transformants as you would expect (restriction).  The same will happen with P1, only worse because the genome is larger than a plasmid.  I have transduced into a BL-21 derivative called ER2566 (sold by NEB in the past), but not tried into BL-21.  If we need complex expression strains, we generally use a K strain lineage that has the DE3 lysogen or some other T7 expression system. If you need to transduce a strain with an active restriction system, I recommend including a control using the same phage to transduce the marker into a strain that does not have that restriction.
 +
 
 +
*We have also found that titering the phage can really help solve problems.  We plate 10 uL spots of serial dilutions on a lawn of recipient bacteria in the presence of 1-5 mM DTT.  In some cases, the titer of the phage is too high relative to the amount of bacteria in the 1:1 mix used for the transduction infection.  You are aiming for a multiplicity of infection (MOI) of 0.5-1.  If you put too many phage in the infection, you will kill every cell whether or not it gets a transducing particle.  We solved this by simply resuspending the overnight of the recipient strain in less volume to make the concentration of bacteria higher (3-5X) when we have a high-titer phage stock.
-
6. Spin cells at 6000 rpm for 2-3 min.
+
==References==
 +
Reference <cite>1</cite> is the first report of allelic exchange by "transduction" in enteric bacteria. Ref. <cite>2</cite> is the earliest report I know of bacteriophage-dependent transduction in E. coli. This report is notable because it describes the K12-infective variant of P1 that has since been considered the "wild-type" P1.  Ref. <cite>3</cite> is a beautiful and detailed experimental demonstration that the transducing particles responsible for transduction co-elute with infecting particles in ultracentrifugation, and thus are fully-formed, intact virions, but carry DNA exclusively of bacterial origin. It also is an early (first?) report of the use of virulent mutant of P1, which is defective for lysogeny (i.e. P1vir). Ref. <cite>4</cite> Describes transduction across species boundaries.  Refs <cite>5 6</cite> provide additional reading about the development of the modern protocol.  Refs. <cite>7,8</cite> give good overviews of the molecular biology of P1 and practical protocols for use in transduction.  However, the agar plate method for preparation of phage lysates given in <cite>8</cite> is more laborious than the simple liquid culture method given in this protocol.  Resulting phage titers are often higher with the plate based protocol, but liquid lysate preparations also given sufficient titer to effect transduction.
-
7. Resuspend each in 100 µL LB + 100 mM Na-Citrate (pH 5.5) and plate all of it on an appropriate antibiotic-containing plate.
+
<biblio>
 +
#1 pmid=12999698
 +
#2 pmid=13267987
 +
#3 pmid=5883909
 +
#4 pmid=3494
 +
#5 pmid=4598005
 +
#6 pmid=4598709
 +
#7 pmid=6364958
 +
#8 [[A Short Course in Bacterial Genetics]]
-
=Who to ask about this protocol=
 
-
Chris, Sean, Kathleen
 
-
[[Sauer Lab | Back to the Sauer lab]]
+
</biblio>

Current revision

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Back to P1vir phage transduction

contributed by Sean Moore

Contents

Background

Phage transduction is used to move selectable genetic markers from one "donor" strain to another "recipient" strain. Nat Sternberg, among others, pioneered the use of phage P1 to move genetic elements in E. coli and the use of the Cre/Lox system from P1 for controlled recombination. Today, phage P1 is commonly used as a transducing agent because it is a generalized tranducer (it can package random sections of the host chromosome instead of its own genome) giving rise to "transducing particles". P1vir is a mutant phage that enters the lytic cycle upon infection (ensuring replication and lysis). During the replication and lysis of the phage in a culture of bacteria, a small percentage of the phage particles will contain a genome segment that contains your gene of interest. P1 packages approximately 90 kb of DNA, so you can transduce genes that are linked to a selectable marker.

Once a phage population has been generated from a donor host, the phage are used to infect a recipient host. Most of the bacteria are lysed by phage that packaged P1 genomes, but a fraction of the phage inject a genome segment derived from the donor host. Homologous recombination then allows the incoming genomic segment to replace the existing homolgous segment. The infected recipient bacteria are plated on a medium that selects for the genome segment of the donor bacteria (antibiotic resistance, prototrophy, etc.)

All of this would not work if the infectivity of the phage could not be controlled. Otherwise, phage released from neighboring cells would infect and lyse the bacteria that had been infected with transducing particles. Someone really smart discovered that phage P1 requires calcium for infectivity. Therefore, you can control P1 infectivity by growing in the presence and absence of calcium. The calcium chelator citrate is usually used because it lowers the concentration of free calcium (by forming Ca-citrate) low enough to prevent P1 infection, but not so low as to starve the cells for calcium.

Lysate preparation

1. Dilute an overnight culture of donor strain grown with selection for the marker to be transduced 1:100 in fresh LB supplemented with 10-25 mM MgCl2, 5 mM CaCl2, and 0.1-0.2% glucose (1 mL per lysate). DO NOT ADD ANTIBIOTIC TO THIS CULTURE. Grow with aeration at 37 ˚C for 1-2 hr. When the cells are in early log phase (slightly turbid, but noticeable growth) Add 40 µL of P1 phage lysate to the culture, continue growing at 37 ˚C. Monitor for 1–3 hr until the culture has lysed (you'll see cellular debris in the tube and the culture will have significantly lessened in its turbidity).

NOTE: In my experience less phage the best, since you must have a moi of about 1 pfu/cel. The best option is try different amounts (from 5 to 100 µL) and take for transduction the lysate got with less P1 inocule. --zurdo 12:38, 2 September 2010 (EDT)

2. Add several drops (50-100 uL) of chloroform to the lysate and vortex. Centrifuge away the debris (14,000 rpm, 1–2 min) and transfer the supernatant to a fresh tube. Add a few drops of chloroform and store at 4 ˚C.

Transduction

1. Grow recipient strain overnight in LB medium (2 mL culture is plenty).

2. On the next day, harvest the cells by centrifugation (6000 rpm, 2 min) and resuspend in 1/5-1/3 the harvested culture volume in fresh LB + 100 mM MgSO4 + 5 mM CaCl2. (note: 10 mM MgSO4 works fine, too, so you can use the 0.1 M MgSO4 the kitchen makes.) For example, if you're planning 2 transductions, resuspend 1 mL of overnight in 300 uL of supplemented LB.

2b.(optional) Transfer 100 uL of transducing P1 lysate into a 1.5 mL microfuge tube for each transduction and incubate them with the caps opened at 37 ˚C for ~30 minutes. This step allows excess chloroform to evaporate from the phage stock. You can place your resuspended recipient strain in the incubator as well during this time to help them wake up from their nap.

3. Set up four "reactions" by adding recipient bacteria to the tubes with phage, mix rapidly after addition, close the caps:

A. 100 µL undiluted P1 lysate + 100 µL recipient cells B. 100 µL 1:10 diluted P1 lysate + 100 µL recipient cells C. 100 µL LB + 100 µL recipient cells D. 100 µL undiluted P1 lysate + 100 µL LB

(note for step 3: LB = LB + 100 mM MgSO4 + 5 mM CaCl2; dilute your P1 lysate in this as well)

4. Incubate tubes at 37 ˚C for 30 min.

5. Add 200 µL 1 M Na-Citrate (pH 5.5), then add 1 mL LB (the real thing this time) and incubate at 37 ˚C for 1 hr to allow expression of the antibiotic resistance marker. If you are working with a marker or recipient that needs to grow at 30 ˚C, double the recovery time.

6. Spin cells at 6000 rpm for 5 min.

7. Resuspend each in 100 µL LB supplemented with 100 mM Na-Citrate (pH 5.5), vortex well to disperse cells, and plate all of it on an appropriate antibiotic-containing plate.

8. You should get anywhere from ~ 10 to 2000 colonies. These colonies are growing on a plate that is covered with P1 phage. If you simply pick a colony from this plate and prepare a freezer stock, you will most likely have phage contamination that will manifest when a culture is grown up in the absence of a calcium chelator. Therefore, prepare a plate spread with the selection antibiotic mixed in 100 µL of 1 M citrate (pH 5.5). Then, use a toothpick to touch the top of a few colonies and re-streak on the new plate for isolated colonies.

9. Test a colony from each re-streak for the presence of the mutant gene you intended to transduce using diagnostic PCR or Southern blotting.

Anecdotes

  • The chloroform used to sterilize the phage lysates, well, sterilizes. If you have visible chloroform drops in the lysate stock, don't add this to your recipient cells directly because you can kill a decent number of bacteria. Instead, aliquot your phage into microfuge tubes and incubate with the caps open at 37 ˚C for about 30 minutes to allow the chloroform to evaporate (step 2b). Then add the recipient cells to the tubes with the phage.
  • When preparing the donor phage lysate, there is a huge variability in the titer of phage obtained at this step which makes transduction performance unpredictable. Some donor cells are slow "wake up" from stationary phase and 3 hours will not be enough. If it is obvious that there was no culture development in the tube, let it shake overnight. The next morning, you will have a culture of cells and, perhaps, noticeable cell debris. Treating this with chloroform and preparing it as a phage lysate usually works.
  • P1 lysis is accelerated under reducing conditions (Ryland Young's Lab). Adding 1 mM DTT to the top agar allows P1 to develope better plaques. It follows that reducing agents may help the donor lysate develope and help the recipients infecected with infectious P1 to lyse before plating. If you're having trouble getting a high titer of donor phage, try β-mercaptoethanol at 1/1000 culture volume.
  • P1 replicates poorly in recA- hosts. Moreover, RecA is required for the homologous recombination needed to integrate the donor DNA. Therefore, I have not been able to transduce into recA bacteria. In cases that I needed to have recA- bacteria following a transduction, I first transduced my desired marker into a recA+ recipient, then subsequently transduced recA::kan from a specialized recA+ donor strain generated by Barry Wanner (BW 26,547 recA::kan Lambda recA+).
  • As mentioned above, P1 packages ~90 kb of DNA. This means that genes in the vicinity of your target gene in the recipient will most likely be replaced with copies of the neighboring genes from the donor. Therefore, you can't easily transduce a marker close to an existing marker in the recipient unless you have good selection for both markers and there is enough space between the genes to allow significant recombination between them. It is a good idea to be aware of the relative distances of genes of interest in your strains when transducing to avoid accidental curing of relevent markers.
  • Be mindful some E. coli are "B" strains and your current knockouts may be in a "K" strain (or visa versa). You may have noticed that when you move plasmids from a "cloning strain" like DH5-alpha into BL-21 for expression, you don't get as many transformants as you would expect (restriction). The same will happen with P1, only worse because the genome is larger than a plasmid. I have transduced into a BL-21 derivative called ER2566 (sold by NEB in the past), but not tried into BL-21. If we need complex expression strains, we generally use a K strain lineage that has the DE3 lysogen or some other T7 expression system. If you need to transduce a strain with an active restriction system, I recommend including a control using the same phage to transduce the marker into a strain that does not have that restriction.
  • We have also found that titering the phage can really help solve problems. We plate 10 uL spots of serial dilutions on a lawn of recipient bacteria in the presence of 1-5 mM DTT. In some cases, the titer of the phage is too high relative to the amount of bacteria in the 1:1 mix used for the transduction infection. You are aiming for a multiplicity of infection (MOI) of 0.5-1. If you put too many phage in the infection, you will kill every cell whether or not it gets a transducing particle. We solved this by simply resuspending the overnight of the recipient strain in less volume to make the concentration of bacteria higher (3-5X) when we have a high-titer phage stock.

References

Reference [1] is the first report of allelic exchange by "transduction" in enteric bacteria. Ref. [2] is the earliest report I know of bacteriophage-dependent transduction in E. coli. This report is notable because it describes the K12-infective variant of P1 that has since been considered the "wild-type" P1. Ref. [3] is a beautiful and detailed experimental demonstration that the transducing particles responsible for transduction co-elute with infecting particles in ultracentrifugation, and thus are fully-formed, intact virions, but carry DNA exclusively of bacterial origin. It also is an early (first?) report of the use of virulent mutant of P1, which is defective for lysogeny (i.e. P1vir). Ref. [4] Describes transduction across species boundaries. Refs [5, 6] provide additional reading about the development of the modern protocol. Refs. [7, 8] give good overviews of the molecular biology of P1 and practical protocols for use in transduction. However, the agar plate method for preparation of phage lysates given in [8] is more laborious than the simple liquid culture method given in this protocol. Resulting phage titers are often higher with the plate based protocol, but liquid lysate preparations also given sufficient titer to effect transduction.

  1. ZINDER ND and LEDERBERG J. . pmid:12999698. PubMed HubMed [1]
  2. LENNOX ES. . pmid:13267987. PubMed HubMed [2]
  3. Ikeda H and Tomizawa JI. . pmid:5883909. PubMed HubMed [3]
  4. Tyler BM and Goldberg RB. . pmid:3494. PubMed HubMed [4]
  5. Goldberg RB, Bender RA, and Streicher SL. . pmid:4598005. PubMed HubMed [5]
  6. Wall JD and Harriman PD. . pmid:4598709. PubMed HubMed [6]
  7. Sternberg N and Hoess R. . pmid:6364958. PubMed HubMed [7]
  8. A Short Course in Bacterial Genetics [8]
All Medline abstracts: PubMed HubMed
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