Prbbbb:small scale expression v1 - Revision history

Raik: /* Modifications */

Raik — Mon, 21 Sep 2009 17:21:41 GMT

Modifications

←Older revision		Revision as of 17:21, 21 September 2009
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	==Modifications==		==Modifications==

-	===scaling ~~down~~ to 24-deepwell plates===	+	===scaling up to 24-deepwell plates===

	24 deepwell plates can be used for culture volumes up to 4 ml.		24 deepwell plates can be used for culture volumes up to 4 ml.
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	still on plate:		still on plate:
	* pellet 10 min @ 1500 g; vigorously discard medium (but beware of soft/loose pellets)		* pellet 10 min @ 1500 g; vigorously discard medium (but beware of soft/loose pellets)
-	* ~~redisolve~~ pellet in 600 µl bugbuster by up-down-pipetting (Eppendorf shaker may work too)	+	* re-dissolve pellet in 600 µl bugbuster by up-down-pipetting (Eppendorf shaker may work too)
	* shake 20' - 30' min @ 250 r.p.m. (large incubator) or more (small shaker)		* shake 20' - 30' min @ 250 r.p.m. (large incubator) or more (small shaker)
	* centrifuge 5-10 min @ 1500 g in plate to remove cell debris		* centrifuge 5-10 min @ 1500 g in plate to remove cell debris
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	'''later steps'''		'''later steps'''
-	Supernatant and ~~resolubilized~~ pellet can be manipulated in 96 deepwell plates from here on. That means the content of four 24-well plates can then be processed on one 96 well plate.	+	Supernatant and re-stabilized pellet can be manipulated in 96 deepwell plates from here on. That means the content of four 24-well plates can then be processed on one 96 well plate.
		+
		+	===Reducing post-induction stress===
		+
		+	All these measures aim to slow down protein production and reduce the shock response of cells that are facing rapid induction.
		+
		+	* grow pre-induction cells at production temperature -- no temperature change before/after induction
		+	* lower IPTG concentration -- even less than 10 µM has been reported to work well
		+	* avoid yeast extract -- contains galactosides that induce the lac operon
		+
		+	'''generic protocol for difficult proteins?'''
		+	* 25C growth before and after induction
		+	* test production growth with 10 and 50 µM IPTG overnight
		+	* Use synthetic rich medium - for example, [[Neidhardt_EZ_Rich_Defined]]

	==Notes==		==Notes==

Raik: /* Culture Growth */

Raik — Mon, 21 Sep 2009 17:08:26 GMT

Culture Growth

←Older revision		Revision as of 17:08, 21 September 2009
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	'''day 2'''		'''day 2'''

-	# inoculate 3-4 clones from each transformation plate into 3 ml LB + 1% Glucose	+	# inoculate 3-4 clones from each transformation plate into 3 ml LB + 1% Glucose + Antibiotic
	# grow over night @ 37 °C		# grow over night @ 37 °C

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	#* backup for later as different clones of one construct can have varying expression strengths		#* backup for later as different clones of one construct can have varying expression strengths
	#* glycerol content is a bit lower than normal; decreases risk of plasmid loss		#* glycerol content is a bit lower than normal; decreases risk of plasmid loss
-	# inoculate 10 ml 2xTY + 1% Glucose from over night culture at 1:100 dilution	+	# inoculate 10 ml 2xTY + 1% Glucose + Antibiotic from over night culture at 1:100 dilution
	# incubate shaking @ 37°C until OD600 reaches 0.5 (usually 2-3 h)		# incubate shaking @ 37°C until OD600 reaches 0.5 (usually 2-3 h)
	# induce expression: add IPTG to final concentration of 0.1 - 1 mM (0.5mM as a start)		# induce expression: add IPTG to final concentration of 0.1 - 1 mM (0.5mM as a start)

Raik: /* Culture Growth */

Raik — Mon, 21 Sep 2009 17:07:23 GMT

Culture Growth

←Older revision		Revision as of 17:07, 21 September 2009
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	==Culture Growth==		==Culture Growth==
		+
		+	If not mentioned otherwise, the LB medium must contain the appropriate antibiotic
		+	(or both if the plasmid contains two resistances).

	'''day 1'''		'''day 1'''

Raik: /* Notes */

Raik — Wed, 26 Aug 2009 10:06:05 GMT

Notes

←Older revision		Revision as of 10:06, 26 August 2009
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	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

-	<font face="courier"><nowiki>raik:</nowiki></font>	+	<font face="courier"><nowiki>raik:</nowiki></font> share your experience!
-	share your experience!	+

	<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->		<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->

Raik: /* Overview */

Raik — Wed, 26 Aug 2009 10:05:40 GMT

Overview

←Older revision		Revision as of 10:05, 26 August 2009
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	This protocol describes how to express small protein amounts from 10 ml culture for expression screening.		This protocol describes how to express small protein amounts from 10 ml culture for expression screening.
-	The constructs are supposed to be under control of the T7 promoter. BL21 strains contain ~~an inducible~~ T7 polymerase.	+	The constructs are supposed to be under control of the T7 promoter. BL21 strains contain a genomic T7 polymerase that is under the control of the Lac repressor -- it is induced by IPTG and leaky expression is repressed by adding extra Glucose.

	==Culture Growth==		==Culture Growth==

Raik: add categories and Notes section

Raik — Wed, 26 Aug 2009 10:04:35 GMT

add categories and Notes section

←Older revision		Revision as of 10:04, 26 August 2009
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	'''later steps'''		'''later steps'''
	Supernatant and resolubilized pellet can be manipulated in 96 deepwell plates from here on. That means the content of four 24-well plates can then be processed on one 96 well plate.		Supernatant and resolubilized pellet can be manipulated in 96 deepwell plates from here on. That means the content of four 24-well plates can then be processed on one 96 well plate.
		+
		+	==Notes==
		+	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
		+
		+	<font face="courier"><nowiki>raik:</nowiki></font>
		+	share your experience!
		+
		+	<!--Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.-->
		+
		+	==References==
		+	<!-- If this protocol has papers or books associated with it, list those references here. See the [[OpenWetWare:Biblio]] page for more information. -->
		+	<!--
		+	<biblio>
		+	#Ptashne-Genetic-Switch isbn=0879697164
		+	</biblio>
		+	-->
		+
		+	==Contact==
		+	*[[Special:Emailuser/Raik\|Raik]]
		+
		+	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].
		+
		+	<!-- You can tag this protocol with various categories. See the [[Categories]] page for more information. -->
		+	[[Category:Protocol]]
		+	[[Category:Protein]]
		+	[[Category:In vitro]]
		+
		+	<!-- Move the relevant categories above this line to tag your protocol with the label
		+	[[Category:Protocol]]
		+
		+	[[Category:Needs attention]]
		+
		+	[[Category:In vitro]]
		+
		+	[[Category:In vivo]]
		+
		+	[[Category:In silico]]
		+
		+	[[Category:DNA]]
		+
		+	[[Category:RNA]]
		+
		+	[[Category:Protein]]
		+
		+	[[Category:Chemical]]
		+
		+	[[Category:Escherichia coli]]
		+
		+	[[Category:Yeast]]
		+	-->

Raik at 08:43, 26 August 2009

Raik — Wed, 26 Aug 2009 08:43:53 GMT

←Older revision		Revision as of 08:43, 26 August 2009
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	# resuspend pellet in 300 µl BugBuster buffer		# resuspend pellet in 300 µl BugBuster buffer
	# shake 20 min @ RT		# shake 20 min @ RT
		+	# centrifuge 1 min @ max. r.p.m. in table-top centrifuge to remove intact cells

	'''Gel electrophoresis'''		'''Gel electrophoresis'''

-	~~# centrifuge 1 min @ max. r.p.m. in table-top centrifuge to remove intact cells~~
	# transfer supernatant		# transfer supernatant
	# centrifuge 30 - 40 min @ 20.000 g (@ 4°C, cold room)		# centrifuge 30 - 40 min @ 20.000 g (@ 4°C, cold room)
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	# load samples (a) on gel A for Commassi staining		# load samples (a) on gel A for Commassi staining
	# load samples (b) on gel B for Western blot		# load samples (b) on gel B for Western blot
		+
		+	==Modifications==
		+
		+	===scaling down to 24-deepwell plates===
		+
		+	24 deepwell plates can be used for culture volumes up to 4 ml.
		+
		+	'''incubation'''
		+	* Incubate 24-deepwell plates at high shaking in aerated incubator (i.e. sticky bottom if available)
		+	* incubate starting cultures (3 ml) over night @ 37 C as described
		+	* then inoculate 4 ml production cultures with 40 µl starting culture (using multi-channel pipette if appropriate)
		+
		+	'''lysis'''
		+	still on plate:
		+	* pellet 10 min @ 1500 g; vigorously discard medium (but beware of soft/loose pellets)
		+	* redisolve pellet in 600 µl bugbuster by up-down-pipetting (Eppendorf shaker may work too)
		+	* shake 20' - 30' min @ 250 r.p.m. (large incubator) or more (small shaker)
		+	* centrifuge 5-10 min @ 1500 g in plate to remove cell debris
		+	* then transfer supernatant into 1.5 ml tubes for high speed cooled centrifugation
		+
		+	'''later steps'''
		+	Supernatant and resolubilized pellet can be manipulated in 96 deepwell plates from here on. That means the content of four 24-well plates can then be processed on one 96 well plate.

Raik: /* Culture Growth */

Raik — Wed, 26 Aug 2009 08:26:21 GMT

Culture Growth

←Older revision		Revision as of 08:26, 26 August 2009
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	'''day 1'''		'''day 1'''

-	# prepare LB+~~Amp (or~~ appropriate antibiotic) Agar plates with Glucose added to 1% final concentration	+	# prepare LB+appropriate antibiotic Agar plates with Glucose added to 1% final concentration
		+	#* (not Ampicillin alone, if possible -- Amp increases risk of plasmid loss)
	#* (prepare stock of sterile-filtered 20% Glucose)		#* (prepare stock of sterile-filtered 20% Glucose)
	# transform expression construct into BL21DE3 cells using normal protocol		# transform expression construct into BL21DE3 cells using normal protocol
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	'''day 2'''		'''day 2'''

-	# inoculate 4 clones from each transformation plate into 3 ml LB + 1% Glucose	+	# inoculate 3-4 clones from each transformation plate into 3 ml LB + 1% Glucose
	# grow over night @ 37 °C		# grow over night @ 37 °C

Raik at 17:34, 3 July 2009

Raik — Fri, 03 Jul 2009 17:34:52 GMT

←Older revision		Revision as of 17:34, 3 July 2009
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	'''or: Enzymatic cell lysis'''		'''or: Enzymatic cell lysis'''

-	# prepare ~~bug buster~~ buffer (~~add~~ 1 tablet ~~complete mini~~ protease inhibitor ~~to 10 ml buffer~~)	+	# prepare BugBuster (Novagen) buffer (1ml 10x buffer + 9ml ddH20 + 1 tablet Complete, Mini (protease inhibitor, Roche))
-	# resuspend pellet in 300 µl ~~bug buster~~ buffer	+	# resuspend pellet in 300 µl BugBuster buffer
	# shake 20 min @ RT		# shake 20 min @ RT

Raik at 17:14, 3 July 2009

Raik — Fri, 03 Jul 2009 17:14:03 GMT

←Older revision		Revision as of 17:14, 3 July 2009
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	# ''soluble fraction'':		# ''soluble fraction'':
	#* prepare 2 samples (a) and (b) of 20µl each:		#* prepare 2 samples (a) and (b) of 20µl each:
-	#* normalize samples to 20µl of culture with lowest OD after induction	+	#* normalize samples to 20µl of culture with lowest OD after induction (e.g. directly take 2 x 20µl from lowest ID supernatant but take less from the others and fill up to 20 µl)
-	(e.g. directly take 2 x 20µl from lowest ID supernatant but take less from the others and fill up to 20 µl)	+
	#* add 5µl 5x sample buffer with SDS		#* add 5µl 5x sample buffer with SDS
	#* denature 5 min @ 95°C		#* denature 5 min @ 95°C