Conjugation using ET12567/pUZ8002 - Revision history

Sam Prudence at 22:17, 25 March 2020

2020-03-25T22:17:00Z

← Older revision		Revision as of 22:17, 25 March 2020
Line 18:		Line 18:
	#Dilute the overnight culture 1:100 in fresh [[LB]] + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.		#Dilute the overnight culture 1:100 in fresh [[LB]] + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.
	#Wash the cells twice with an equal volume of [[LB]] (to remove antibiotics) and resuspend in 0.1 volume of [[LB]]		#Wash the cells twice with an equal volume of [[LB]] (to remove antibiotics) and resuspend in 0.1 volume of [[LB]]
	#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.		#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth and germinate via heat shock at 50° for 10 minutes, then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
	#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and ~~thn~~ resuspend the pellet in the residual liquid.		#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and then resuspend the pellet in the residual liquid.
	#Plate out on [[SFM \| MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9. (Note: for <em>[[Streptomyces venezuelae]]</em> incubate for 16-20 h at room temperature to avoid overgrowth which may make antibiotic selectiom more challenging)		#Plate out on [[SFM \| MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9. (Note: for <em>[[Streptomyces venezuelae]]</em> incubate for 16-20 h at room temperature to avoid overgrowth which may make antibiotic selectiom more challenging)
	#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°		#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
	#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.		#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Sam Prudence: /* Intructions */

2019-11-07T18:20:37Z

Intructions

← Older revision		Revision as of 18:20, 7 November 2019
Line 12:		Line 12:
	*Acceptor ''Streptomyces'' strain		*Acceptor ''Streptomyces'' strain

	=~~Intructions~~=		=Instructions=

	#Transform ET12467/pUZ8002 with ''oriT''-containing vector, and select for the incoming plasmid only		#Transform ET12467/pUZ8002 with ''oriT''-containing vector, and select for the incoming plasmid only

Sam Prudence: /* Intructions */

2019-10-05T15:18:31Z

Intructions

← Older revision		Revision as of 15:18, 5 October 2019
Line 20:		Line 20:
	#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.		#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
	#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.		#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
	#Plate out on [[SFM \| MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.		#Plate out on [[SFM \| MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9. (Note: for <em>[[Streptomyces venezuelae]]</em> incubate for 16-20 h at room temperature to avoid overgrowth which may make antibiotic selectiom more challenging)
	#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°		#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
	#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.		#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Sam Prudence at 15:14, 5 October 2019

2019-10-05T15:14:27Z

← Older revision		Revision as of 15:14, 5 October 2019
Line 5:		Line 5:
	*[[LB]] broth		*[[LB]] broth
	*[[2X YT]]		*[[2X YT]]
	*[[SFM \| MS]] plates containing 10 mM MgCl<sub>2</sub>		*[[SFM \| MS]] (also known as SFM) plates containing 10 mM MgCl<sub>2</sub>
	*Antibiotics: nalidixic acid (25 mg/ml), chloramphenicol (25 mg/ml), kanamycin (25 mg/ml) and selection antibiotic. See [[Antibiotic stocks and working concentrations]]		*Antibiotics: nalidixic acid (25 mg/ml), chloramphenicol (25 mg/ml), kanamycin (25 mg/ml) and selection antibiotic. See [[Antibiotic stocks and working concentrations]]

DaveMark at 12:50, 1 October 2019

2019-10-01T12:50:03Z

← Older revision		Revision as of 12:50, 1 October 2019
Line 3:		Line 3:

	=Materials=		=Materials=
	*[[LB]]		*[[LB]] broth
	*[[2X YT]]		*[[2X YT]]
	*[[SFM \| MS]] plates containing 10 mM MgCl<sub>2</sub>		*[[SFM \| MS]] plates containing 10 mM MgCl<sub>2</sub>

LisAlgora at 11:14, 4 September 2019

2019-09-04T11:14:00Z

← Older revision		Revision as of 11:14, 4 September 2019
Line 1:		Line 1:
	=Strains=		Intergeneric transfer of plasmids from ''E. coli'' to ''Streptomyces'' strains was demonstrated after modifying the mobilisation system established by Simon ''et al.'' (1983) for Gram-negative bacteria. It works well also for non-replicative vectors and integrate into the chromosome either site-specifically at the φC31 or pSAM2 attachment sites, or via insert-directed homologous recombination.
	*ET12567/pUZ8002 containing vector		Vectors containing ''oriT'' require transfer functions to be supplied by the ''E. coli'' donor strain such as the methylation-deficient ET12567 (MacNeil ''et al.'', 1991) carrying the non-transmissible pUZ8002.
	*~~Donor "~~Streptomyces" strain
			=Materials=
			*[[LB]]
			*[[2X YT]]
			*[[SFM \| MS]] plates containing 10 mM MgCl<sub>2</sub>
			*Antibiotics: nalidixic acid (25 mg/ml), chloramphenicol (25 mg/ml), kanamycin (25 mg/ml) and selection antibiotic. See [[Antibiotic stocks and working concentrations]]

			===Strains===
			*''E. coli'' donor strain ET12567/pUZ8002 containing vector
			*Acceptor ''Streptomyces'' strain

	=Intructions=		=Intructions=

	#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only		#Transform ET12467/pUZ8002 with ''oriT''-containing vector, and select for the incoming plasmid only
	#Inoculate a colony in to 10 ml LB containing chloramphenicol, kanamycin and the antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.		#Inoculate a colony in to 10 ml [[LB]] containing chloramphenicol, kanamycin and the antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
	#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.		#Dilute the overnight culture 1:100 in fresh [[LB]] + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.
	#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB		#Wash the cells twice with an equal volume of [[LB]] (to remove antibiotics) and resuspend in 0.1 volume of [[LB]]
	#While washing the E. coli cells, for each conjugation add approximately ~~108~~ Streptomyces ~~spires~~ to 500 µl ~~2 x~~ YT broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.		#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
	#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.		#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
	#Plate out on MS agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.		#Plate out on [[SFM \| MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
	#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°		#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
	#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.		#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

LisAlgora at 10:49, 4 September 2019

2019-09-04T10:49:07Z

← Older revision		Revision as of 10:49, 4 September 2019
Line 1:		Line 1:
	=Strains=		=Strains=
	*ET12567/pUZ8002		*ET12567/pUZ8002 containing vector
	*Donor "Streptomyces" strain		*Donor "Streptomyces" strain

Line 6:		Line 6:

	#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only		#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
	#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and ~~Kanamycin~~ sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.		#Inoculate a colony in to 10 ml LB containing chloramphenicol, kanamycin and the antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
	#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an ~~OD600~~ of 0.4-0.6.		#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.
	#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB		#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
	#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.		#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.

LisAlgora at 10:46, 4 September 2019

2019-09-04T10:46:50Z

← Older revision		Revision as of 10:46, 4 September 2019
Line 9:		Line 9:
	#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.		#Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
	#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB		#Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
	#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at ~~50 oC~~ for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.		#While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spires to 500 µl 2 x YT broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
	#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.		#Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
	#Plate out on MS agar + 10 mM ~~MgCl2~~ and incubate at ~~30 oC~~ for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.		#Plate out on MS agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9.
	#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at ~~30 oC~~		#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
	#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.		#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

LisAlgora at 10:45, 4 September 2019

2019-09-04T10:45:08Z

← Older revision		Revision as of 10:45, 4 September 2019
Line 2:		Line 2:
	*ET12567/pUZ8002		*ET12567/pUZ8002
	*Donor "Streptomyces" strain		*Donor "Streptomyces" strain

			=Intructions=

	#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only		#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only

REMcHugh at 14:43, 30 August 2019

2019-08-30T14:43:14Z

← Older revision		Revision as of 14:43, 30 August 2019
Line 1:		Line 1:
			=Strains=
			*ET12567/pUZ8002
			*Donor "Streptomyces" strain

	#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only		#Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
	#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.		#Inoculate a colony in to 10 ml LB containing Cm, Km and antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and Kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.