Protoplasts Formation, Regeneration and Transformation - Revision history

H Otani: /* Materials */

2020-03-24T21:55:58Z

Materials

← Older revision		Revision as of 21:55, 24 March 2020
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	*[[P buffer]]		*[[P buffer]]
	*10.3% sucrose		*10.3% sucrose
	*1 g polyethylene glycol 1000 (PEG)
	*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.		*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
	*Autoclaved filter tubes/syringes plugged with cotton wool		*Autoclaved filter tubes/syringes plugged with cotton wool
	*[[R5]] or [[R2YE]] plate

	==Instructions==		==Instructions==

H Otani: /* Instructions */

2020-03-24T21:53:41Z

Instructions

← Older revision		Revision as of 21:53, 24 March 2020
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	#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)		#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
	#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].		#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].
	~~#Add 3.85 ml P buffer to 1 g PEG sterilised.~~
	~~#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.~~
	~~#Plate it onto [[R5]] or [[R2YE]].~~
	~~#Incubate the plate at 298-30ºC overnight.~~
	~~#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.~~

	==Notes==		==Notes==

H Otani: /* Materials */

2020-03-24T21:52:54Z

Materials

← Older revision		Revision as of 21:52, 24 March 2020
Line 15:		Line 15:
	*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.		*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
	*Autoclaved filter tubes/syringes plugged with cotton wool		*Autoclaved filter tubes/syringes plugged with cotton wool
			*[[R5]] or [[R2YE]] plate

	==Instructions==		==Instructions==

H Otani: /* Instructions */

2020-03-24T21:52:04Z

Instructions

← Older revision		Revision as of 21:52, 24 March 2020
Line 28:		Line 28:
	#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)		#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
	#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].		#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].
			#Add 3.85 ml P buffer to 1 g PEG sterilised.
			#Add 200 µl PEG-P buffer mixture and a DNA solution to 50 µl of the protoplast suspension.
			#Plate it onto [[R5]] or [[R2YE]].
			#Incubate the plate at 298-30ºC overnight.
			#Overlay 1 ml sterilised water containing nalidixic acid and an appropriate antibiotic.

	==Notes==		==Notes==

H Otani: /* Materials */

2020-03-24T21:44:35Z

Materials

← Older revision		Revision as of 21:44, 24 March 2020
Line 12:		Line 12:
	*[[P buffer]]		*[[P buffer]]
	*10.3% sucrose		*10.3% sucrose
			*1 g polyethylene glycol 1000 (PEG)
	*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.		*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
	*Autoclaved filter tubes/syringes plugged with cotton wool		*Autoclaved filter tubes/syringes plugged with cotton wool

H Otani: /* Instructions */

2020-03-24T21:40:53Z

Instructions

← Older revision		Revision as of 21:40, 24 March 2020
Line 17:		Line 17:
	==Instructions==		==Instructions==

	#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)		#Add 25 ml [[YEME]] medium to a baffled flask. Glycine can be optionally added to the final concentration of 0.5%. Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
	#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant		#Pour culture broth into a screw cap tube and spin in centrifuge (e.g. 1000x g, 10 min). Discard supernatant
	#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant		#Re-suspend pellet in 15 ml 10.3% sucrose solution and spin down as above. Discard supernatant
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	#Filter protoplasts through cotton wool and transfer to a fresh tube.		#Filter protoplasts through cotton wool and transfer to a fresh tube.
	#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)		#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
	#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].		#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

	==Notes==		==Notes==

H Otani: /* Materials */

2020-03-24T21:37:49Z

Materials

← Older revision		Revision as of 21:37, 24 March 2020
Line 12:		Line 12:
	*[[P buffer]]		*[[P buffer]]
	*10.3% sucrose		*10.3% sucrose
	*Lysozyme solution (~~1mg~~/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.		*Lysozyme solution (1 mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
	*Autoclaved filter tubes/syringes plugged with cotton wool		*Autoclaved filter tubes/syringes plugged with cotton wool

LisAlgora at 11:13, 4 September 2019

2019-09-04T11:13:36Z

← Older revision		Revision as of 11:13, 4 September 2019
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	Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.		Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

	~~'''~~Materials~~:'''~~		==Materials==
	*''Streptomyces'' spores, fresh suspension (c. 10<sup>9</sup> spores/ml) or frozen stock in 20% glycerol		*''Streptomyces'' spores, fresh suspension (c. 10<sup>9</sup> spores/ml) or frozen stock in 20% glycerol
	*[[YEME]] medium		*[[YEME]] medium
Line 15:		Line 15:
	*Autoclaved filter tubes/syringes plugged with cotton wool		*Autoclaved filter tubes/syringes plugged with cotton wool

	~~'''~~Instructions~~:'''~~		==Instructions==

	#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)		#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
Line 28:		Line 28:
	#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].		#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

	~~'''~~Notes~~:'''~~		==Notes==
	*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving		*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
	*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)		*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
Line 36:		Line 36:
	*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.		*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

	=Further Manipulations of ~~Protoplats~~=		=Further Manipulations of Protoplasts=

	[[Protoplast Fusion]]		[[Protoplast Fusion]]
Line 43:		Line 43:

	[[Protoplast Transfection]]		[[Protoplast Transfection]]

			=Alternative Methods=

			=References=
			Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. and Hopwood, D.A. (2000) Practical Streptomyces Genetics: John Innes Foundation, Norwich Research Park, Colney, Norwich NR4 7UH, UK

LisAlgora at 10:37, 4 September 2019

2019-09-04T10:37:38Z

← Older revision		Revision as of 10:37, 4 September 2019
Line 33:		Line 33:
	*Avoid vortexing, which induces foaming and consequent lysis		*Avoid vortexing, which induces foaming and consequent lysis
	*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.		*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
	*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly''')		*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly'''')
	*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.		*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

LisAlgora at 10:37, 4 September 2019

2019-09-04T10:37:12Z

← Older revision		Revision as of 10:37, 4 September 2019
Line 7:		Line 7:
	Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.		Sometimes the age and/or physiological state of the mycelium is important. For example, the best transformation or transfection frequencies in ''S. lividans'' are obtained with protoplasts prepared from mycelium grown to "late exponential" phase (36-40h), whereas for transfection of ''S. parvulus'' protoplasts needed to come from much younger mycelium (c. 18h) The details may also depend on the particular genotype: many morphological mutants are particularly idiosyncratic.

	Materials:		'''Materials:'''
	*''Streptomyces'' spores, fresh suspension (c. 10<sup>9</sup> spores/ml) or frozen stock in 20% glycerol		*''Streptomyces'' spores, fresh suspension (c. 10<sup>9</sup> spores/ml) or frozen stock in 20% glycerol
	*[[YEME]] medium		*[[YEME]] medium
Line 13:		Line 13:
	*10.3% sucrose		*10.3% sucrose
	*Lysozyme solution (1mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.		*Lysozyme solution (1mg/ml in [[P buffer]], filter sterilised). For most purposes, lysozyme is dissolved in [[P buffer]]; use of [[L buffer]] gives more efficient transformation of ''S. lividans'' by plasmid DNA.
	*~~Filter~~ tubes plugged with cotton wool		*Autoclaved filter tubes/syringes plugged with cotton wool

	Instructions:		'''Instructions:'''

	#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)		#Add 25 ml [[YEME]] medium to a baffled flask, Add around 0.1 ml spore suspension and required growth factors. Incubate 36-40h at 30° in an orbital incubator shaker. (Cultures of ''S. lividans'' and ''S. coelicolor'' are ready for harvesting when they start to produce red pigment)
Line 23:		Line 23:
	#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.		#Resuspend mycelium in 4 ml lysozyme solution; incubate at 30°, 15-60 min. It is recommended to monitor protoplasts formation using the phase-contrast microscope, as lysozyme treatment times vary from strain to strain, e.g. 15 min is usually long enough for ''S. lividans'' and ''S. clavuligerus'', whereas ''S. coelicolor'' requires 60 min.
	#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.		#Draw in and out of a 5 ml pipette three times and incubate for a further 15 min.
	#Add 5 ml [[P buffer]]. Repeat step 6. This ~~help~~ to free protoplasts from the mycelium so that they will pass through the cotton wool.		#Add 5 ml [[P buffer]]. Repeat step 6. This helps to free protoplasts from the mycelium so that they will pass through the cotton wool.
	#Filter protoplasts through cotton wool and transfer to a fresh tube.		#Filter protoplasts through cotton wool and transfer to a fresh tube.
	#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)		#Sediment protoplasts gently by spinning in a centrifuge (e.g. 1000x g, 7 min)
	#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]]		#Discard supernatant and suspend protoplasts in 1 ml [[P buffer]].

			'''Notes:'''
			*Make sure all material (flasks, springs, etc.) is free from detergent. It is recommended to rinse the flasks 3 times in tap water followed by 3 times in distilled water prior to autoclaving
			*At any steps when pelleted protoplasts are to be re-suspended, re-suspend in the remaining drop of liquid by tapping the side of the tube repeatedly with a finger until protoplasts are dispersed to form a creamy suspension, then add the suspending [[P buffer]] (otherwise the protoplast pellet is difficult to disperse)
			*Avoid vortexing, which induces foaming and consequent lysis
			*To freeze the protoplasts for storage, place samples in small plastic tubes and place them in ice in a plastic beaker. Place the beaker at -80° overnight. Free the frozen protoplasts in their tubes from the ice and store at -80°.
			*To thaw, shake the frozen tube under running warm water (i.e. ''''freeze slowly, thaw quickly''')
			*To assess the proportion of non-protoplasted units in the suspension, samples can be diluted in parallel in [[P buffer]] and in dilute detergent (e.g. 0.01% SDS) and plated on regeneration plates. Any colonies arising after dilution in detergent are likely to have arisen from non-protoplasted units.

			=Further Manipulations of Protoplats=

			[[Protoplast Fusion]]

			[[Protoplast Transformation]]

			[[Protoplast Transfection]]