Prbbbb:small scale expression v1

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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==
 +
 +
If not mentioned otherwise, the LB medium must contain the appropriate antibiotic
 +
(or both if the plasmid contains two resistances).
'''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
Line 18: Line 22:
'''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 + Antibiotic
# grow over night @ 37 °C
# grow over night @ 37 °C
Line 26: Line 30:
#* 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)  
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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)
Line 80: Line 84:
# 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 up 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)
 +
* 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)
 +
* 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 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==
 +
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]]
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 +
[[Category:In vitro]]
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 +
[[Category:In vivo]]
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[[Category:In silico]]
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[[Category:DNA]]
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[[Category:RNA]]
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[[Category:Protein]]
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 +
[[Category:Chemical]]
 +
 +
[[Category:Escherichia coli]]
 +
 +
[[Category:Yeast]]
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-->

Current revision

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Contents

Overview

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 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

If not mentioned otherwise, the LB medium must contain the appropriate antibiotic (or both if the plasmid contains two resistances).

day 1

  1. 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)
  2. transform expression construct into BL21DE3 cells using normal protocol
  3. plate transformation on LB + 1% Glucose + Antibiotic
    • grow over night @ 37°C but avoid overgrowing clones -- 13 or 14 h are best

day 2

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

day 3

  1. prepare glycerol stock of each clone -- use 10 % final glycerol concentration
    • 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
  2. inoculate 10 ml 2xTY + 1% Glucose + Antibiotic from over night culture at 1:100 dilution
  3. incubate shaking @ 37°C until OD600 reaches 0.5 (usually 2-3 h)
  4. induce expression: add IPTG to final concentration of 0.1 - 1 mM (0.5mM as a start)
  5. let induced cultures grow (A) 3 h @ 37 and (B) over night @ 20°C
  6. measure OD600 of 37°C cultures
  7. aliquot 10 ml into 5 x 2ml tubes
  8. pellet by centrifugation
  9. store pellet @ -20°C

day 4

  1. measure OD600 of 20°C cultures
  2. aliquot 10 ml into 5 x 2ml tubes
  3. pellet by centrifugation
  4. store pellet @ -20°C

Lysis and Analysis

Cell lysis by sonication

  1. resuspend pellet in 500 µl extraction buffer
    • extraction buffer: add 1 "complete mini" Protease inhibitor tablet to 10 ml PBS
  2. transfer into 1.5 ml Eppendorf tubes
  3. sonicate
    • miniprobe: apply 2 x 20s intermediate strength burst
    • ultrasound bath: 5 min @ intermediate strength
    • check solution and adapt length accordingly

or: Enzymatic cell lysis

  1. prepare BugBuster (Novagen) buffer (1ml 10x buffer + 9ml ddH20 + 1 tablet Complete, Mini (protease inhibitor, Roche))
  2. resuspend pellet in 300 µl BugBuster buffer
  3. shake 20 min @ RT
  4. centrifuge 1 min @ max. r.p.m. in table-top centrifuge to remove intact cells

Gel electrophoresis

  1. transfer supernatant
  2. centrifuge 30 - 40 min @ 20.000 g (@ 4°C, cold room)
    • supernatant = soluble fraction
    • glassy pellet = membrane bound and inclusion bodies
  3. soluble fraction:
    • prepare 2 samples (a) and (b) of 20µl each:
    • 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)
    • add 5µl 5x sample buffer with SDS
    • denature 5 min @ 95°C
  4. insoluble fraction:
    • resuspend in 50µl 5x sample buffer by repeated up/down pipetting
    • denature 5 min @ 95°C
    • prepare 2 samples (a) and (b) of about 5µl each:
    • normalize exact amount according to OD after induction
  1. load samples (a) on gel A for Commassi staining
  2. load samples (b) on gel B for Western blot

Modifications

scaling up 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)
  • 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)
  • 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 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

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

raik: share your experience!


References

Contact

or instead, discuss this protocol.

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