Tri-parental mating - Revision history

Alicia at 16:01, 11 August 2020

2020-08-11T16:01:18Z

← Older revision		Revision as of 16:01, 11 August 2020
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	===Tri-Parental Mating===		===Tri-Parental Mating===
	Tri-parental mating is useful for conjugating large plasmids, such as PAC clones (see [[PAC cloning]]), or those containing a kanamycin ~~resistane~~ gene as a selectable marker into ''Streptomyces''. In the standard [[ET Conjugation]] protocol a helper plasmid, [[pUZ8002]], is used in the ''Escherichia coli'' strain ET12567 (''E. coli'' ET12567) to transfer the desired vector to ''Streptomyces''. This [[pUZ8002]] helper plasmid contains a kanamycin resistance gene. This means that, if your desired plasmid contains a kanamycin resistance gene as marker (e.g. [[pRT802]]), it would be impossible to select for this vector when transforming ''E. coli'' ET12567 due to the kanamycin resistance gene already present on [[pUZ8002]].		Tri-parental mating is useful for conjugating large plasmids, such as PAC clones (see [[PAC cloning]]), or those containing a kanamycin resistance gene as a selectable marker into ''Streptomyces''. In the standard [[ET Conjugation]] protocol a helper plasmid, [[pUZ8002]], is used in the ''Escherichia coli'' strain ET12567 (''E. coli'' ET12567) to transfer the desired vector to ''Streptomyces''. This [[pUZ8002]] helper plasmid contains a kanamycin resistance gene. This means that, if your desired plasmid contains a kanamycin resistance gene as marker (e.g. [[pRT802]]), it would be impossible to select for this vector when transforming ''E. coli'' ET12567 due to the kanamycin resistance gene already present on [[pUZ8002]].

	To circumvent this issue, tri-parental mating is used. This transforms ''E. coli'' ET12567 with your vector of choice (donated from a parental ''E. coli'' strain) and a different helper plasmid, [[pR9604]], which contains an ampicillin/carbenicillin resistance gene as a selectable marker (donated from the parental strain ''E. coli'' TOP10 pR9604). The protocol then uses Amp/Carb to select for the helper plasmid, and kanamycin can be used to select for your desired vector. At the end an ''E. coli'' ET12567 strain is generated with an Carb/Amp selectable helper plasmid, and your kanamycin-marked vector of choice.		To circumvent this issue, tri-parental mating is used. This transforms ''E. coli'' ET12567 with your vector of choice (donated from a parental ''E. coli'' strain) and a different helper plasmid, [[pR9604]], which contains an ampicillin/carbenicillin resistance gene as a selectable marker (donated from the parental strain ''E. coli'' TOP10 pR9604). The protocol then uses Amp/Carb to select for the helper plasmid, and kanamycin can be used to select for your desired vector. At the end an ''E. coli'' ET12567 strain is generated with an Carb/Amp selectable helper plasmid, and your kanamycin-marked vector of choice.

Sam Prudence at 15:15, 29 August 2019

2019-08-29T15:15:49Z

← Older revision		Revision as of 15:15, 29 August 2019
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	Protocol adapted for ActinoBase by Samuel M.M. Prudence~~, PhD student at~~ the University of East Anglia.		Protocol adapted for ActinoBase by Samuel M.M. Prudence from the University of East Anglia.

Sam Prudence: /* Why Tri-Parental Mating? */

2019-08-09T14:47:56Z

Why Tri-Parental Mating?

← Older revision		Revision as of 14:47, 9 August 2019
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	*<em>[[Streptomyces venezuelae]]</em>		*<em>[[Streptomyces venezuelae]]</em>

	===~~Why~~ Tri-Parental Mating?===		===Tri-Parental Mating===
	Tri-parental mating is useful for conjugating large plasmids, such as PAC clones (see [[PAC cloning]]), or those containing a kanamycin resistane gene as a selectable marker into ''Streptomyces''. In the standard [[ET Conjugation]] protocol a helper plasmid, [[pUZ8002]], is used in the ''Escherichia coli'' strain ET12567 (''E. coli'' ET12567) to transfer the desired vector to ''Streptomyces''. This [[pUZ8002]] helper plasmid contains a kanamycin resistance gene. This means that, if your desired plasmid contains a kanamycin resistance gene as marker (e.g. [[pRT802]]), it would be impossible to select for this vector when transforming ''E. coli'' ET12567 due to the kanamycin resistance gene already present on [[pUZ8002]].		Tri-parental mating is useful for conjugating large plasmids, such as PAC clones (see [[PAC cloning]]), or those containing a kanamycin resistane gene as a selectable marker into ''Streptomyces''. In the standard [[ET Conjugation]] protocol a helper plasmid, [[pUZ8002]], is used in the ''Escherichia coli'' strain ET12567 (''E. coli'' ET12567) to transfer the desired vector to ''Streptomyces''. This [[pUZ8002]] helper plasmid contains a kanamycin resistance gene. This means that, if your desired plasmid contains a kanamycin resistance gene as marker (e.g. [[pRT802]]), it would be impossible to select for this vector when transforming ''E. coli'' ET12567 due to the kanamycin resistance gene already present on [[pUZ8002]].

Sam Prudence at 14:47, 9 August 2019

2019-08-09T14:47:34Z

← Older revision		Revision as of 14:47, 9 August 2019
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	===Protocol===		===Protocol===
	Strains needed:		Strains needed:
	# ''E. coli'' ~~TOPO10~~ donor strain containing the self‐transmissible helper plasmid pR9604 (CarbR/AmpR).		# ''E. coli'' TOP10 donor strain containing the self‐transmissible helper plasmid pR9604 (CarbR/AmpR).
	# ''E. coli'' donor strain containing your vector of choice		# ''E. coli'' donor strain containing your vector of choice
	# ''E. coli'' ET12567 recipient strain (ChlR)		# ''E. coli'' ET12567 recipient strain (ChlR)

Sam Prudence: /* Tri-parental Mating */

2019-08-07T13:03:41Z

Tri-parental Mating

← Older revision		Revision as of 13:03, 7 August 2019
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	=<strong>Tri-parental Mating</strong>=		=<strong>Tri-parental Mating</strong>=
	~~Protocol adapted for ActinoBase by Samuel M.M. Prudence, PhD student at the University of East Anglia.~~

	This protocol has been confirmed to work for the following organisms.		This protocol has been confirmed to work for the following organisms.
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	#Ampicillin and Carbenicillin can be used intergangably.		#Ampicillin and Carbenicillin can be used intergangably.
	#''E. coli'' ET12567 contains a chloramphenicol resistance gene integrated into the genome.		#''E. coli'' ET12567 contains a chloramphenicol resistance gene integrated into the genome.





			Protocol adapted for ActinoBase by Samuel M.M. Prudence, PhD student at the University of East Anglia.

Sam Prudence: /* Tri-parental Mating */

2019-08-06T19:07:32Z

Tri-parental Mating

← Older revision		Revision as of 19:07, 6 August 2019
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	This protocol has been confirmed to work for the following organisms.		This protocol has been confirmed to work for the following organisms.
	*<em>[[Streptomyces coelicolor]]</em>		*<em>[[Streptomyces coelicolor]]</em>
			*<em>[[Streptomyces formicae]]</em>
	*<em>[[Streptomyces venezuelae]]</em>		*<em>[[Streptomyces venezuelae]]</em>
	*<em>[[Streptomyces formicae]]</em>

	===Why Tri-Parental Mating?===		===Why Tri-Parental Mating?===

Sam Prudence at 19:07, 6 August 2019

2019-08-06T19:07:02Z

← Older revision		Revision as of 19:07, 6 August 2019
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	*<em>[[Streptomyces coelicolor]]</em>		*<em>[[Streptomyces coelicolor]]</em>
	*<em>[[Streptomyces venezuelae]]</em>		*<em>[[Streptomyces venezuelae]]</em>
			*<em>[[Streptomyces formicae]]</em>

	===Why Tri-Parental Mating?===		===Why Tri-Parental Mating?===

Sam Prudence at 18:31, 6 August 2019

2019-08-06T18:31:11Z

← Older revision		Revision as of 18:31, 6 August 2019
Line 3:		Line 3:

	This protocol has been confirmed to work for the following organisms.		This protocol has been confirmed to work for the following organisms.
	*''Streptomyces coelicolor''		*<em>[[Streptomyces coelicolor]]</em>
	*''Streptomyces venezuelae''		*<em>[[Streptomyces venezuelae]]</em>

	===Why Tri-Parental Mating?===		===Why Tri-Parental Mating?===

Sam Prudence: /* Tri-parental Mating */

2019-08-06T18:28:43Z

Tri-parental Mating

← Older revision		Revision as of 18:28, 6 August 2019
Line 4:		Line 4:
	This protocol has been confirmed to work for the following organisms.		This protocol has been confirmed to work for the following organisms.
	*''Streptomyces coelicolor''		*''Streptomyces coelicolor''
			*''Streptomyces venezuelae''

	===Why Tri-Parental Mating?===		===Why Tri-Parental Mating?===

Sam Prudence: /* Tri-parental Mating */

2019-08-06T18:26:53Z

Tri-parental Mating

← Older revision		Revision as of 18:26, 6 August 2019
Line 1:		Line 1:
	=<strong>Tri-parental Mating</strong>=		=<strong>Tri-parental Mating</strong>=
	Protocol adapted for ActinoBase by Samuel M.M. Prudence, PhD student at the University of East Anglia.		Protocol adapted for ActinoBase by Samuel M.M. Prudence, PhD student at the University of East Anglia.
	~~s.prudence@uea.ac.uk~~

	This protocol has been confirmed to work for the following organisms.		This protocol has been confirmed to work for the following organisms.