ActinoBase - User contributions [en-gb]

Isolation of phages from environmental samples

2020-05-05T12:22:26Z

Paulhoskisson: /* Isolation of phages from environmental samples */

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24
*[[Streptomyces coelicolor]] strain J1929 (pgl- strain)

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation (~1 x 109) from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from environmental samples

2020-05-05T12:22:03Z

Paulhoskisson: /* Protocol */

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24
*[[Streptomyces coelicolor]] strain J1929 (pgl negative strain)

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation (~1 x 109) from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from environmental samples

2020-05-05T12:21:45Z

Paulhoskisson: /* Protocol */

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24
*[[Streptomyces coelicolor]] strain J1929 (pgl negative strain)

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation (1 x 109) from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from environmental samples

2020-05-05T12:21:25Z

Paulhoskisson: /* Protocol */

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24
*[[Streptomyces coelicolor]] strain J1929 (pgl negative strain)

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation (1 x 10 9) from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from environmental samples

2020-05-05T12:20:33Z

Paulhoskisson: /* Isolation of phages from environmental samples */

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24
*[[Streptomyces coelicolor]] strain J1929 (pgl negative strain)

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation (1 x 10 9) from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from environmental samples

2020-05-05T12:19:55Z

Paulhoskisson:

=Isolation of phages from environmental samples=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces lividans]] strain TK24
*[[Streptomyces coelicolor]] strain J1929 (pgl negative strain)

===Isolation of phages from environmental samples===

It is often useful to isolate bacteriophage from environmental samples, to study bacteriophage diversity or to isolate new phages that can be used as genetic tools (i.e. for generalized transduction with a specific ''Streptomyces'' host strain or for isolating new phage recombinases to develop useful plasmids.)

This protocol allows for the enrichment of bacteriophage that are able to infect a specific ''Streptomyces'' indicator strain (see note 1). Note that any ''Streptomyces'' phage unable to infect the indicator strain, will not be enriched, so this protocol will not offer a comprehensive survey of all ''Streptomyces'' phage present in the environmental sample.

===Protocol===

Strains needed:
#suitable ''Streptomyces'' indicator strain(s)

Materials needed:
#Soil or environmental sample from which you wish to isolate bacteriophage
#Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
Enrichment -
#Mix 5 g of soil with 10 ml of DNB in a sterile universal bottle.
#Add 500 µl of a dense spore preparation (1 x 10 9)from a Streptomyces indicator strain of your choice - see note 1.
#Let soil/spore mixture stand at room temperature for 24 h with occasional agitation: this will enrich the phage population that can plaque on your ''Streptomyces'' indicator strain.
#After 24 hours, centrifuge the sample on a bench top centrifuge (3000 rpm) for 10 minutes to remove the particulate matter.
#Decant the enriched DNB and filter through a 0.45 µm syringe filter.
#The resulting filtrate can be used in the standard [[Streptomyces Bacteriophage plaque assay]], using the same ''Streptomyces'' indicator strain, to isolate new phage.

===Notes===
#Often host range is a key parameter in phage isolation, so it is worth looking at a number of strains. ''Streptomyces lividans'' TK24 has been used extensively in the past.
#New phage should be purified using the [[Isolation of phages from single plaques]] protocol.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Isolation of phages from single plaques

2020-05-05T12:17:09Z

Paulhoskisson:

=Isolation of phages from single plaques=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces coelicolor]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces lividans]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces venezuelae]] with ΦSV1

===Isolation of phages from single plaques===

It is often very useful to isolate phage from single plaques (this is the equivalent of working with single colonies of bacteria). At an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event (with all phage progeny being, in theory at least, genetically identical to the "parent" phage.)

Thus, when isolating novel phage from the environment, it is essential to go through at least one round of isolation from a single plaque.

===Protocol===

Materials needed:
#Plates containing the bacteriophage grown on a permissive host strain (see either [[Streptomyces Bacteriophage plaque assay]] or [[Isolation of phages from environmental samples]])
#Liquid [[DNB]] medium
#0.45µm syringe filter
#Sterile yellow pipette tips, Bijoux bottles, etc.

Instructions:
#The bacteriophage of choice should be grown on a permissive host strain, following either the enrichment procedure or bacteriophage plaque assay protocol.
#Identify the plaque(s) you wish to isolate. Ideally, these should be well-separated from any other plaques.
#Using a ‘yellow pipette tip’ pick the plaque from the SNA overlay and place it into 1 mL of DNB broth in a Bijoux bottle.
#Shake at room temperature for 2 hrs.
#Filter sterilise using a 0.45 µm syringe filter, and store at 4°C.
#This phage lysate can be used to generate high-titre preparations and for host range studies.

===Notes===
#The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).
#It is not necessary to add chloroform to the phage lysates.
#Plaques can be very variable in size (0.1-0.8 cm), shape and turbidity - this may also change from the initial isolation plate through to stable populations of the phage. Turbid plaques are characteristic of temperate phages, clear plaques of lytic phages. It is not uncommon to see clear plaques that are turbid at the edges.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

High titre preparation of phage

2020-05-05T12:12:52Z

Paulhoskisson:

=High titre preparation of ''Streptomyces'' phage=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces coelicolor]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces lividans]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces venezuelae]] with ΦSV1

===High titre preparation of phage===

High titre preparation of phage (phage lysates) can be used for any experiment requiring specific bacteriophages (e.g. for phage typing of strains, generalized transduction or any other experiments).

A single plate (from a [[Streptomyces Bacteriophage plaque assay]]) generates several millilitres of a high titre (~108 pfu/mL) phage lysate.

===Protocol===

Materials needed:
# Plates containing the bacteriophage grown on a permissive host strain (see [[Streptomyces bacteriophage plaque assay]])
# Liquid [[DNB]] medium
#0.45µm syringe filter

Instructions:
#Take plates from the [[Streptomyces Bacteriophage plaque assay]]
#Take the dilution(s) that show almost confluent lysis (You should still be able to distinguish the shapes of individual plaques, but the plate should display as much lysis as possible - to harvest the maximum number of phage. Often it pays to look at the edges of the plates where it looks like there is no growth of the indicator strain – these often display confluent lysis).
#Flood the plates exhibiting almost confluent lysis with 5 ml of Difco Nutrient broth (DNB), and leave at room temperature for 2 hrs. Plates can be gently rocked to increase the phage titre.
#Pour the broth off the plate into a Bijoux bottle (or other suitable container).
#Filter the DNB through a 0.45 µm syringe filter. The resulting filtrate is your high-titre phage preparation and should be around 108 pfu/mL.
#This phage preparation (phage lysate) should be stored at 4°C.

===Notes===
#The phage lysates remain viable for long periods when stored at 4°C (I have recovered phage from filtrates ~10 years old!).
#It is not necessary to add chloroform to the phage lysates.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Streptomyces Bacteriophage plaque assay

2020-05-05T12:09:19Z

Paulhoskisson: /* Protocol */

=Streptomyces bacteriophage plaque assay=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces coelicolor]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces lividans]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces venezuelae]] with ΦSV1

===Bacteriophage plaque assay===

The bacteriophage plaque assay can be used to calculate (titer) the number of plaque-forming units (pfu) present in a sample, i.e. a phage lysate or an environmental sample suspected to contain bacteriophage.

It is also useful for the preparation of phage lysates (see the protocol, [[High titre preparation of phage]]) which can be used e.g. for generalized transduction or other experiments.

It can also be used to isolate phage from single plaques (the equivalent of working with single colonies of bacteria), as at an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event. (See the protocol, [[Isolation of phages from single plaques]].)

===Protocol===
Strains needed:
# ''Streptomyces'' strain sensitive to the bacteriophage
# suitable phage lysate or sample suspected to contain phage particles

Materials needed:
# Solid [[DNA]] medium (approx. 8 plates/phage being assayed)
# Molten [[SNA]] medium (approx 20-30 mL/phage being assayed)
# Liquid [[DNB]] medium
#1M MgSO4 solution (sterile)
#0.8 M CaCl2 solution (sterile)
#Sterile Eppendorf tubes, pipet tips, etc.

[[File:PhiC31 serial dilution.jpg|thumb|'''Example of a ''Streptomyces'' bacteriophage plaque assay.''' A plaque assay was performed using decimal serial dilutions of ΦC31 and a ''pglW-'' strain of ''S. coelicolor''. The control (left) contains spores only, followed by 10-1-10-6 dilutions of a ΦC31 phage lysate]]

Instructions:
#Pour agar plates with Difco Nutrient agar ([[DNA]]) and allow to set and dry.
##For ΦC31, the DNA plates should contain 10 mM MgSO4 and 8 mM CaCl2 (to 100 mL of medium, add 1ml of 1M MgSO4 and 1ml of 0.8M CaCl2) - see note 1.
#Melt Difco soft nutrient agar ([[SNA]]) and store in 50°C water bath. Work carefully with the molten SNA: if it is too hot, it will kill some of the ''Streptomyces'' spores; if it is too cold, it will solidify.
#Dilute phage suspension (10-fold serial dilution) using Difco nutrient broth ([[DNB]]): 100-108 should cover most preparations of phage from high-titre lysates through to enriched environmental samples).
#Pipette 0.1 ml of each phage sample onto the centre of an agar plate.
#Add spores to the SNA to give a slightly turbid suspension (roughly around 0.1 mL of 1 x 108 spore stock per 10 mL of SNA) – it is often useful to do this in universal bottles as it makes pouring on to plates easier.
#Add 2-3ml of the SNA/spore mixture to the surface of the plate and swirl immediately to cover the whole plate. Leave to set for 10-15 minutes.
#Incubate the plates overnight at the required temperature (usually 30°C).
#The following morning, inspect the plates for plaques. Plaques can be visible within 10-12 hours, be careful not to allow the plates to overgrow as some lytic phage plaques may appear turbid and temperate phage (turbid plaques) ma be lost to overgrowth.
#Proceed to the [[High titre preparation of phage]] or [[Isolation of phages from single plaques]] protocol.

===Notes===
#Each phage with have an optimal divalent cation requirement for propagation – this is generally in the range of 0-10 mM MgSO4 and 8-25 mM CaCl2 but will need to be optimised for each phage. For isolation of novel phages, the standard ΦC31 parameters are suitable for isolation.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Streptomyces Bacteriophage plaque assay

2020-05-05T12:08:28Z

Paulhoskisson:

=Streptomyces bacteriophage plaque assay=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces coelicolor]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces lividans]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces venezuelae]] with ΦSV1

===Bacteriophage plaque assay===

The bacteriophage plaque assay can be used to calculate (titer) the number of plaque-forming units (pfu) present in a sample, i.e. a phage lysate or an environmental sample suspected to contain bacteriophage.

It is also useful for the preparation of phage lysates (see the protocol, [[High titre preparation of phage]]) which can be used e.g. for generalized transduction or other experiments.

It can also be used to isolate phage from single plaques (the equivalent of working with single colonies of bacteria), as at an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event. (See the protocol, [[Isolation of phages from single plaques]].)

===Protocol===
Strains needed:
# ''Streptomyces'' strain sensitive to the bacteriophage
# suitable phage lysate or sample suspected to contain phage particles

Materials needed:
# Solid [[DNA]] medium (approx. 8 plates/phage being assayed)
# Molten [[SNA]] medium (approx 20-30 mL/phage being assayed)
# Liquid [[DNB]] medium
#1M MgSO4 solution (sterile)
#0.8 M CaCl2 solution (sterile)
#Sterile Eppendorf tubes, pipet tips, etc.

[[File:PhiC31 serial dilution.jpg|thumb|'''Example of a ''Streptomyces'' bacteriophage plaque assay.''' A plaque assay was performed using decimal serial dilutions of ΦC31 and a ''pglW-'' strain of ''S. coelicolor''. The control (left) contains spores only, followed by 10-1-10-6 dilutions of a ΦC31 phage lysate]]

Instructions:
#Pour agar plates with Difco Nutrient agar ([[DNA]]) and allow to set and dry.
##For ΦC31, the DNA plates should contain 10 mM MgSO4 and 8 mM CaCl2 (to 100 mL of medium, add 1ml of 1M MgSO4 and 1ml of 0.8M CaCl2) - see note 1.
#Melt Difco soft nutrient agar ([[SNA]]) and store in 50°C water bath. Work carefully with the molten SNA: if it is too hot, it will kill some of the ''Streptomyces'' spores; if it is too cold, it will solidify.
#Dilute phage suspension (10-fold serial dilution) using Difco nutrient broth ([[DNB]]): 100-108 should cover most preparations of phage from high-titre lysates through to enriched environmental samples).
#Pipette 0.1 ml of each phage sample onto the centre of an agar plate.
#Add spores to the SNA to give a slightly turbid suspension (roughly around 0.1 mL of 1 x 108 spore stock per 10 mL of SNA)– it is often useful to do this in universal bottles as it makes pouring on to plates easier.
#Add 2-3ml of the SNA/spore mixture to the surface of the plate and swirl immediately to cover the whole plate. Leave to set for 10-15 minutes.
#Incubate the plates overnight at the required temperature (usually 30°C).
#The following morning, inspect the plates for plaques. Plaques can be visible within 10-12 hours, be careful not to allow the plates to overgrow as some lytic phage plaques may appear turbid and temperate phage (turbid plaques) ma be lost to overgrowth.
#Proceed to the [[High titre preparation of phage]] or [[Isolation of phages from single plaques]] protocol.

===Notes===
#Each phage with have an optimal divalent cation requirement for propagation – this is generally in the range of 0-10 mM MgSO4 and 8-25 mM CaCl2 but will need to be optimised for each phage. For isolation of novel phages, the standard ΦC31 parameters are suitable for isolation.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Streptomyces Bacteriophage plaque assay

2020-05-05T12:07:11Z

Paulhoskisson: /* Streptomyces bacteriophage plaque assay */

=Streptomyces bacteriophage plaque assay=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces coelicolor]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces lividans]] with ΦC31, ΦBT1, ΦHau3, ΦR4
*[[Streptomyces venezuelae]] with ΦSV1

===Bacteriophage plaque assay===

The bacteriophage plaque assay can be used to calculate (titer) the number of plaque-forming units (pfu) present in a sample, i.e. a phage lysate or an environmental sample suspected to contain bacteriophage.

It is also useful for the preparation of phage lysates (see the protocol, [[High titre preparation of phage]]) which can be used e.g. for generalized transduction or other experiments.

It can also be used to isolate phage from single plaques (the equivalent of working with single colonies of bacteria), as at an appropriate multiplicity of infection (MOI), each plaque will represent a single infection event. (See the protocol, [[Isolation of phages from single plaques]].)

===Protocol===
Strains needed:
# ''Streptomyces'' strain sensitive to the bacteriophage
# suitable phage lysate or sample suspected to contain phage particles

Materials needed:
# Solid [[DNA]] medium (approx. 8 plates/phage being assayed)
# Molten [[SNA]] medium (approx 20-30 mL/phage being assayed)
# Liquid [[DNB]] medium
#1M MgSO4 solution (sterile)
#0.8 M CaCl2 solution (sterile)
#Sterile Eppendorf tubes, pipet tips, etc.

[[File:PhiC31 serial dilution.jpg|thumb|'''Example of a ''Streptomyces'' bacteriophage plaque assay.''' A plaque assay was performed using decimal serial dilutions of ΦC31 and a ''pglW-'' strain of ''S. coelicolor''. The control (left) contains spores only, followed by 10-1-10-6 dilutions of a ΦC31 phage lysate]]

Instructions:
#Pour agar plates with Difco Nutrient agar ([[DNA]]) and allow to set and dry.
##For ΦC31, the DNA plates should contain 10 mM MgSO4 and 8 mM CaCl2 (to 100 mL of medium, add 1ml of 1M MgSO4 and 1ml of 0.8M CaCl2) - see note 1.
#Melt Difco soft nutrient agar ([[SNA]]) and store in 50°C water bath. Work carefully with the molten SNA: if it is too hot, it will kill some of the ''Streptomyces'' spores; if it is too cold, it will solidify.
#Dilute phage suspension (10-fold serial dilution) using Difco nutrient broth ([[DNB]]): 100-108 should cover most preparations of phage from high-titre lysates through to enriched environmental samples).
#Pipette 0.1 ml of each phage sample onto the centre of an agar plate.
#Add spores to the SNA to give a slightly turbid suspension (roughly around 0.1 mL of 1 x 10e8 spore stock per 10 mL of SNA)– it is often useful to do this in universal bottles as it makes pouring on to plates easier.
#Add 2-3ml of the SNA/spore mixture to the surface of the plate and swirl immediately to cover the whole plate. Leave to set for 10-15 minutes.
#Incubate the plates overnight at the required temperature (usually 30°C).
#The following morning, inspect the plates for plaques. Plaques can be visible within 10-12 hours, be careful not to allow the plates to overgrow as some lytic phage plaques may appear turbid and temperate phage (turbid plaques) ma be lost to overgrowth.
#Proceed to the [[High titre preparation of phage]] or [[Isolation of phages from single plaques]] protocol.

===Notes===
#Each phage with have an optimal divalent cation requirement for propagation – this is generally in the range of 0-10 mM MgSO4 and 8-25 mM CaCl2 but will need to be optimised for each phage. For isolation of novel phages, the standard ΦC31 parameters are suitable for isolation.

Protocol adapted for ActinoBase by Paul Hoskisson from the University of Strathclyde.

Streptomyces Bacteriophages

2020-05-05T07:08:31Z

Paulhoskisson: /* Introduction */

Streptomyces bacteriophages

Prof Paul A Hoskisson, University of Strathclyde.

=Introduction=

Bacteriophages (or phages) are amongst the most abundant biological entities on the planet and exhibit enormous diversity[1]. There has been a huge amount of interest in ''Streptomyces'' phages over many years as they have been a source of many valuable tools for molecular biology and genetic manipulation. These include the widely used integrating vector systems[2] based on phage integrases, such as [[pSET152]], [[pMS82]] and their derivatives, along with newer integrase systems[3–5], mutagenesis[6] and the older, seldom-used nowadays, phage cloning vectors[7,8]. ''Streptomyces'' bacteriophages have also been extensively studied from the perspective of their own biology and diversity[1,9,10], and also in ''Streptomyces'' from the perspective of bacteriophage resistance[11,12].

''Streptomyces'' phages are readily isolated from a range of soil and environmental samples on soft agar overnight (see protocol for [[Isolation of phages from environmental samples]]), with isolation of novel phage from single phages easily achieved. Success can be increased through enrichment procedures (see protocol 'Isolation of phages from environmental samples'), with the isolation of lytic and temperate phages being relatively straightforward.

Phages can also readily be induced from lysogens (prophages) using the standard methods of UV-induction, mitomycin C-induction and from spontaneous induction events following growth in liquid medium, which provides free phage for plaque formation on suitable indicator strains (not always straightforward as endogenous phage often have narrow host ranges and the original strain is likely immune to a phage it carries).

The model Streptomyces phage (our equivalent of the model coliphage, λ) is the temperate phage [[ΦC31]], with clear plaque (lytic) mutants (cΔ25) available and often used in the laboratory. The R4-like group of phages appear to be the most abundant Streptomyces phages globally[1]. Beyond the Streptomycetes there have been limited investigations of phage within the Actinobacteria - the exception being the extensive work on Mycobacteriophages[10,13,14] and Corynephages[15].

=Protocols=
[[Streptomyces Bacteriophage plaque assay]]

[[High titre preparation of phage]]

[[Isolation of phages from single plaques]]

[[Isolation of phages from environmental samples]]

=References=
1. Smith, M. C. M. et al. Evolutionary relationships among actinophages and a putative adaptation for growth in ''Streptomyces'' spp. J Bacteriol 195, 4924–35 (2013).

2. Baltz, R. H. ''Streptomyces'' temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms). J Ind Microbiol Biot 39, 661–72 (2011).

3. Bierman, M. et al. Plasmid cloning vectors for the conjugal transfer of DNA from ''Escherichia coli'' to ''Streptomyces'' spp. Gene 116, 43–49 (1992).

4. Gregory, M., Till, R. & Smith, M. Integration Site for ''Streptomyces'' Phage BT1 and Development of Site-Specific Integrating Vectors. Journal of Bacteriology 185, (2003).

5. Fogg, P. C. M., Haley, J. A., Stark, W. M. & Smith, M. C. M. Genome Integration and Excision by a New ''Streptomyces'' Bacteriophage, ϕJoe. Appl Environ Microb 83, e02767-16 (2017).

6. Khodakaramian, G. Expression of Cre recombinase during transient phage infection permits efficient marker removal in ''Streptomyces''. Nucleic Acids Research 34, (2006).

7. Foor, F. & Morin, N. Construction of a shuttle vector consisting of the ''Escherichia coli'' plasmid pACYC177 inserted into the ''Streptomyces cattleya'' phage TG1. Gene 94, 109–113 (1990).

8. Zhou, X., Deng, Z., Hopwood, D. A. & Kieser, T. Characterization of phi HAU3, a broad-host-range temperate ''streptomyces'' phage, and development of phasmids. J Bacteriol 176, 2096–2099 (1994).

9. Kieser, T. et al. Practical Streptomyces Genetics. (2000).

10. Hendrix, R. W., Smith, M. C., Burns, R. N., Ford, M. E. & Hatfull, G. F. Evolutionary relationships among diverse bacteriophages and prophages: all the world’s a phage. Proceedings of the National Academy of Sciences of the United States of America 96, 2192–7 (1999).

11. Hoskisson, P. A. & Smith, M. C. Hypervariation and phase variation in the bacteriophage ‘resistome’. Current Opinion in Microbiology 10, (2007).

12. Hoskisson, P. A., Sumby, P. & Smith, M. C. M. The phage growth limitation system in ''Streptomyces coelicolor'' A(3)2 is a toxin/antitoxin system, comprising enzymes with DNA methyltransferase, protein kinase and ATPase activity. Virology (2015) doi:10.1016/j.virol.2014.12.036.

13. Kessel, J. C. van & Hatfull, G. F. Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets. Molecular Microbiology 67, (2008).

14. Hatfull, G. F. Molecular Genetics of ''Mycobacteria.'' 81–119 (2014) doi:10.1128/9781555818845.ch5.

15. Sangal, V. & Hoskisson, P. A. Corynephages: Infections of the Infectors. In: ''Corynebacterium diphtheriae'' and Related Toxigenic Species. pp67–81; Springer; Edited Burkovski, A. (2013). doi:10.1007/978-94-007-7624-1_4.