Team:Bielefeld-Germany/Protocols/Production

From 2012.igem.org

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(Sonification)
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=Production=
=Production=
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Here all our methods according to cultivation and purification are listed.
Here all our methods according to cultivation and purification are listed.
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==Pre-Cultivation==
==Pre-Cultivation==
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===Precultivation of ''E.Coli'' KRX (with or without BioBrick)===
===Precultivation of ''E.Coli'' KRX (with or without BioBrick)===
* 50 mL [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#LB_medium LB]-medium, if nessassary with 20-60 mg L-1 chloramphenicol or with 100-300 mg L-1 ampicillin, in 300 mL shaking flask with baffles (Schott) with silicon plugs
* 50 mL [http://2012.igem.org/wiki/index.php?title=Team:Bielefeld-Germany/Protocols/Materials#LB_medium LB]-medium, if nessassary with 20-60 mg L-1 chloramphenicol or with 100-300 mg L-1 ampicillin, in 300 mL shaking flask with baffles (Schott) with silicon plugs
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* Shaking at 140 rpm
* Shaking at 140 rpm
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==Cultivation==
==Cultivation==
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===Expression of Laccase===  
===Expression of Laccase===  
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* Harvest after 12-13 h
* Harvest after 12-13 h
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==Cell Harvesting==
==Cell Harvesting==
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* Harvest cells by centrifugation at 10,000 g for 10 min at 4 °C
* Harvest cells by centrifugation at 10,000 g for 10 min at 4 °C
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* Resuspend the pellet in 5 mL [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#special_buffers_for_resuspending_the_cell_pellet special buffer] or [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography binding buffer] for each gramm of cell paste
* Resuspend the pellet in 5 mL [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#special_buffers_for_resuspending_the_cell_pellet special buffer] or [http://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Buffers_for_His-Tag_affinity_chromatography binding buffer] for each gramm of cell paste
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==Solubilization of inclusion bodies==
==Solubilization of inclusion bodies==
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* centrifugation of the celllysate at 40,000 g for 30 minutes
* centrifugation of the celllysate at 40,000 g for 30 minutes
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* resuspend the pellet in SDS running buffer
* resuspend the pellet in SDS running buffer
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=Cell disruption strategies=
=Cell disruption strategies=
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==B-PER lysis (chemical lysis)==
==B-PER lysis (chemical lysis)==
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The lysate is ready for a following purification step.
The lysate is ready for a following purification step.
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==Mechanical lysis==
==Mechanical lysis==
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The method of choice to disrupt the cells depends on the amount of biomass.
The method of choice to disrupt the cells depends on the amount of biomass.
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===Mechanical lysis of the (shaking flask) cultivation===
===Mechanical lysis of the (shaking flask) cultivation===
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====Sonication====
====Sonication====
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**fill the precellyse tubes with a sample volume between 1 mL up to 1,5 mL (for 2 mL tubes)
**fill the precellyse tubes with a sample volume between 1 mL up to 1,5 mL (for 2 mL tubes)
**homogenize the samples for 3 cycles (6500 rpm for 35 sec. ), to make sure not to heat the cells to much, the sample were stored for 5 min in ice between 2 cycles.
**homogenize the samples for 3 cycles (6500 rpm for 35 sec. ), to make sure not to heat the cells to much, the sample were stored for 5 min in ice between 2 cycles.
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===Mechanical lysis of the (bio-reactor) cultivation===
===Mechanical lysis of the (bio-reactor) cultivation===
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====Cell disruption with a high-pressure homogenizer====
====Cell disruption with a high-pressure homogenizer====
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* disruption of the cells by 3 cycles with cooling phases between the cycles, pressure = 1200 bar, make sure not to heat the cells too much
* disruption of the cells by 3 cycles with cooling phases between the cycles, pressure = 1200 bar, make sure not to heat the cells too much
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=Purification=
=Purification=
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==His-tag affinity chromatography==
==His-tag affinity chromatography==

Revision as of 00:05, 27 September 2012


Contents

Production

Here all our methods according to cultivation and purification are listed.

Pre-Cultivation

Precultivation of E.Coli KRX (with or without BioBrick)

  • 50 mL LB-medium, if nessassary with 20-60 mg L-1 chloramphenicol or with 100-300 mg L-1 ampicillin, in 300 mL shaking flask with baffles (Schott) with silicon plugs
  • 1 mL Glycerinculture or 1 colony
  • Cultivation temperature: 37 °C
  • Shaking at 140 rpm

Precultivation of E.Coli Rosetta Gami 2 (with or without BioBrick)

  • 50 mL LB-medium with 60 mg L-1 chloramphenicol and with 300 mg L-1 ampicillin, in 300 mL shaking flask with baffles (Schott) with silicon plugs
  • 1 mL Glycerinculture or 1 colony
  • Cultivation temperature: 37 °C
  • Shaking at 140 rpm

Precultivation of Pichia Pastoris GS115 (complex-medium)

  • 50 mL YPD-medium in 300 mL shaking flask with baffles (Schott) with silicon plugs
  • 1 mL Glycerinculture or 1 colony
  • Cultivation temperature: 30 °C
  • Shaking at 140 rpm

Precultivation of Pichia Pastoris GS115 (minimal-medium)

  • 50 mL YNB-medium in 300 mL shaking flask with baffles (Schott) with silicon plugs
  • 1 mL Glycerinculture or 1 colony
  • Cultivation temperature: 30 °C
  • Shaking at 140 rpm


Cultivation

Expression of Laccase

  • Chassis: Promega's E.Coli KRX
  • Medium: Autoinduction-Medium supplemented with Chloramphenicol (final concentration 60 μg mL-1)

Cultivation with E. coli KRX in shaking flask(with baffles):

  • 200 mL culture in 1000 mL shaking flask with baffles (Schott) with silicon plugs
  • Cultivation temperature: 37 °C
  • Autoinduction-medium with 20-60 mg L-1 chloramphenicol and if nessassary with 100-300 mg L-1 ampicillin
  • Shaking at 140 rpm
  • for characterizations: automatic sampling every 30 min

Bioreactor cultivations with E. coli KRX

To obtain higher amounts and concentration of proteins we cultivated and expressed in a bioreactor. It is possible to cultivate several liters and to control temperature, pH and pO_2.

  • Bioreactor: Braun Biostat B Bioreactor (3L), Infors Labfors Bioreactor (3L), Bioengineering NLF22 Bioreactor (7 L),
  • Autoinduction-medium with 60 mg L-1 chloramphenicol
  • Culture volume: 3,0-6,0 L
  • Starting OD600: 0.1 - 0.2
  • Airflow: 5 NL/min
  • pO2-Control: 30 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
    • pO2=100% calibration with 300rpm
  • pH: 7.0 (controlled with 2M phosphoric acid and 2 M NaOH)
  • Antifoam: BASF pluronic PE-8100
  • Harvest after 12-13 h


Cell Harvesting

  • Harvest cells by centrifugation at 10,000 g for 10 min at 4 °C
  • if the purification should start the next day store the cell pellet at 4°C !(the laccase must not be frozen!)
  • Resuspend the pellet in 5 mL special buffer or binding buffer for each gramm of cell paste


Solubilization of inclusion bodies

  • centrifugation of the celllysate at 40,000 g for 30 minutes
  • resuspend the pellet of the lysate in 1 mL 6M Urea solution, incubation for 1 hour
  • centrifugation for 10 minutes at 10,000 rpm
  • resuspend the pellet in SDS running buffer


Cell disruption strategies

B-PER lysis (chemical lysis)

B-Per bacterial Protein Extraction Reagnt was used for a cell disruption screening according to the following protocol of Thermo Scientific.

  • add 4 mL B-Per Reagent per gram of cell pellet
  • resuspend the cell pellet by pipetting the suspension up and down until it is homogenous.
  • incubate the solution for 10- 15 min at room temperature
  • centrifuge lysate at 15.000g for 10 min
  • decant the supernatant in a clean tube

The lysate is ready for a following purification step.

enzymatical lysis with lysozym

The lysis with lysozym was used for a cell disruption screening. The following protocol was utilized:

  • resuspend the cell pellet in 600 µL of lysozym-solution per gramm
  • incubate the solution for 1 h at 4°C
  • centrifuge the lysate toseperate soluble proteins from insoluble proteins and cell debris.

combination of chemical and enzymatical lysis

B-Per bacterial Protein Extraction Reagnt was used for a cell disruption screening according to the following protocol of Thermo Scientific.

  • add 4 mL B-Per Reagent per gram of cell pellet
  • add 2µL of lysozym-solution(50 mg mL -1) and 2 µL DNaseI (2500 U mL -1) per mL B-Per Reagent. (For laccases:Do not use EDTA!)
  • resuspend the cell pellet by pipetting the suspension up and down until it is homogenous.
  • incubate the solution for 10- 15 min at room temperature
  • centrifuge lysate at 15.000g for 10 min
  • decant the supernatant in a clean tube

The lysate is ready for a following purification step.


Mechanical lysis

The method of choice to disrupt the cells depends on the amount of biomass.


Mechanical lysis of the (shaking flask) cultivation

Sonication

  • Sonication of the re-suspended pellet on ice
    • cycle number depends on the volume of the resuspended cells (e.g. 3 mL means 3 cycles)
    • one cycle means sonification treatment for 1,5 min with Sonifier 450 by Branson, max. 50 %, cooled on ice, make sure not to heat the cells too much

Precellyse 24 homogenization

  • homogenization with the Precellyse 24
    • fill the precellyse tubes with a sample volume between 1 mL up to 1,5 mL (for 2 mL tubes)
    • homogenize the samples for 3 cycles (6500 rpm for 35 sec. ), to make sure not to heat the cells to much, the sample were stored for 5 min in ice between 2 cycles.

Mechanical lysis of the (bio-reactor) cultivation

Cell disruption with a high-pressure homogenizer

  • high-pressure homogenisation with a Rannie Homogenizer:
  • disruption of the cells by 3 cycles with cooling phases between the cycles, pressure = 1200 bar, make sure not to heat the cells too much


Purification

His-tag affinity chromatography


Used Chromatographycolumns:
left 1 mL HisTrap FF crude by GE Healthcare
middle 15 mL HisTrap FF crude by GE Healthcare
right TALON His-Tag Purification Resin by Clonetech
  • For buffers see here

Syringe method

  • Column: 1 mL HisTrap FF crude by GE Healthcare
  • Equilibrate with binding buffer(10mL)
  • Load sample onto column(max. 6 mL)
  • Wash with 10 mL binding buffer
  • Elute with 5 mL of elution buffer
  • Collect the eluate in 1 mL fractions, the purified protein is most likely in the first or second fraction
  • Re-equilibrate the column with binding buffer

ÄKTA method

  • Columns:
  1. 15 mL HisTrap FF crude by GE Healthcare
  2. 50 mL TALON-Histag-Purification Resin by Clonetech

Column preparation

  • If Column is not loaded with Ni-ions /Cobalt-ions:
    • Wash column with 5 - 8 Columnvolumes (CV) of deionized water
    • Load column with metal-ions(4 CV)
      • For HisTrap FF crude: 1,4% NiSO4-Solution
      • For TALON-Histag-Purification Resin: CoCl2-Solution

Chromatography protocol for the Äkta-system

Äkta Prime by GE Healthcare
  • Wash column with 10 CV of deionized water
  • Equilibrate column with 10 CV of binding buffer
  • Load column with supernatant of the lysed cells (Collect the Flow through for SDS-PAGE analysis)
  • Wash Column with 10 CV of binding buffer (Collect the Flow through for SDS-PAGE analysis)
  • Elute Protein with an increasing elutionbuffer ratio (gradient 0%-100%, length 200mL)
  • Collect the eluate in 10 mL fractions
  • Elute remaining proteins with 100% Elutionbuffer (4 CV)



An typical chromatogram of purified laccases is illustrated in the following grafic:

A typical chromatogram of the purification procedure of laccases. the different steps of the purification are illustrated: 1.) Equlibration, 2.) Sample Loading, 3.) Wasch, 4.)Elution, 5.) Regeneration




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