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From 2011.igem.org

Production Protocols: These are the protocols for the cultivation and the downstream processing.

Contents 1 Cultivation 1.1 Expression of [http://expasy.org/sprot/hamap/CORGL.html Brevibacterium flavum] and [http://ijs.sgmjournals.org/cgi/content/abstract/54/3/779 Corynebacterium halotolerans] S-layer genes in E. coli 1.2 Expression of bisphenol A degrading BioBricks in E. coli 1.3 Bioreactor cultivations with E. coli KRX 2 Purification methods 2.1 Release of periplasmic protein fraction from E. coli by cold osmotic shock 2.2 Inclusion body clean-up 2.3 Ultra-/Diafiltration 2.4 Ion exchange chromatography (IEX) for S-layer proteins from Corynebacterium 2.5 Ni-NTA spin columns 2.6 His-tag affinity chromatography 3 Immobilization of S-layer proteins on silica beads

Cultivation

Expression of [http://expasy.org/sprot/hamap/CORGL.html Brevibacterium flavum] and [http://ijs.sgmjournals.org/cgi/content/abstract/54/3/779 Corynebacterium halotolerans] S-layer genes in E. coli

• Used BioBricks: <partinfo>K525131</partinfo>, <partinfo>K525133</partinfo>, <partinfo>K525231</partinfo>, <partinfo>K525232</partinfo>, <partinfo>K525233</partinfo>, <partinfo>K525234</partinfo>, <partinfo>K525304</partinfo>, <partinfo>K525305</partinfo>, <partinfo>K525306</partinfo>, <partinfo>K525406</partinfo>, <partinfo>K525323</partinfo>

• Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]

• Medium: LB medium supplemented with 20 mg L^-1 Chloramphenicol or autoinduction medium
  • Cultivations in LB-medium were supplemented with 0,1 % L-rhamnose as inducer, when the designated OD₆₀₀ was reached.
  • Autoinduction medium for expressing <partinfo>K525304</partinfo>, <partinfo>K525305</partinfo>, <partinfo>K525306</partinfo>, <partinfo>K525406</partinfo>, <partinfo>K525323</partinfo> was supplemented with 1 mM IPTG.

• 150 mL culture in 500 mL shaking flask with baffles (Schott) with silicon plugs

• Cultivation temperature: 37 °C at 120 rpm

Expression of bisphenol A degrading BioBricks in E. coli

• Used BioBricks: <partinfo>K525502</partinfo>, <partinfo>K525512</partinfo>, <partinfo>K525517</partinfo>, <partinfo>K525552</partinfo>

• Chassis: Promega's [http://www.promega.com/products/cloning-and-dna-markers/cloning-tools-and-competent-cells/bacterial-strains-and-competent-cells/single-step-_krx_-competent-cells/ E. coli KRX]

• Medium: LB medium supplemented with 100 mg L^-1 Ampicillin and 120 mg L^-1 bisphenol A
  • BPA is thermally stable -> you can autoclave it together with the medium

• 100 mL culture in 300 mL shaking flask without baffles (Schott) with silicon plugs

• Cultivation temperature: 24 °C, 30 °C or 37 °C, tempered with Infors AG AQUATRON at 120 rpm

• for characterizations: automatic sampling every three hours with Gilson fraction controller F2XX cooled (< 4 °C) with Julabo F10 water bath BURMA-SHAVE!!
  • the characterization experiment setup is shown on the picture on the right

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 DO.

• Bioreactor: [http://www.bioengineering-inc.com/standard-reactors.php?id=2.1 Bioengineering NLF22 7 L] with Bioengineering DCU

• Medium: HSG medium with 20 mg L^-1 chloramphenicol

• Culture volume: 4 L

• Starting OD₆₀₀: 0.2

• DO: 40 % airsaturation (controlled with stirrer cascade starting with 200 rpm)

• pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)

• Antifoam: BASF pluronic XXX

• Induction after 4 h cultivation time with 2 % rhamnose (in culture medium)

• Harvest after 13 h

Purification methods

Release of periplasmic protein fraction from E. coli by cold osmotic shock

Modified protocol from [http://www.jbc.org/content/240/9/3685.full.pdf+html?sid=4a90c176-0ec3-489f-8c82-4734274cebf5 Neu & Heppel, 1965].

• Centrifuge E. coli cell suspension for 5 min at 14000 g (4 °C) to collect the cells.
• Discard the entire supernatant.
• Resuspend the cells ice-cold cell fractionating buffer #1. The resulting volume should be 1/4 of the former suspension volume.
• Incubate for 20 min on ice. Ivert the suspension at regular intervals to counteract sedimentation.
• Centrifuge the cell suspension for 15 min at 14000 g (4 °C).
• Discard the entire supernatant.
• Resuspend the cells ice-cold cell fractionating buffer #2. The resulting volume should be 1/4 of the former suspension volume.
• Incubate for 10 min on ice under regular invertion.
• Centrifuge the cell suspension for 15 min at 14000 g (4 °C).
• Save the supernatant, which contains the periplasmatic proteins.
• If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter.

Inclusion body clean-up

• harvest the cells by centrifugation (30 min, 10,000 g, 4 °C)
• resuspend pellet and disrupt cells
• centrifuge lysate (60 min, >17,000 g, 4 °C)
• wash pellet at least two times with water to remove water-soluble proteins
• after washing the pellet: incubate the pellet in denaturation buffer for 60 min, 4 °C with vertical rotator
  • final concentration in denaturation buffer: 0.5 mg wet biomass per mL
• centrifuge (60 min, >17,000 g, 4 °C)
  • the higher the speed, the better the result
• collect supernatant and incubate the pellet again in denaturation buffer (60 min, 4 °C, vertical rotator)
• centrifuge (60 min, >17,000 g, 4 °C)
• collect supernatant and discard pellet

Ultra-/Diafiltration

• Arrange the filtration module as shown on the right side.

• Microfiltration (0.22 µm) of sample before ultrafiltration.

• For concentrating the sample just filter it until the desired volume is left in the feed reservoir. For diafiltration (e.g. buffer exchange, desalting) dilute the feed reservoir several times and filter continously.

• Used membranes: [http://www.millipore.com/catalogue/module/C7493 Milipore Pellicon XL 50] or XL 100 membranes
  • 50 or 100 kDa cut-off
  • 50 cm² filtration area
  • tangential flow filter
  • Hydrophilic polyvinylidene fluoride membrane

• Used pump: SciLog TANDEM 1081 peristaltic pump
  • flow rate during filtration: 40 mL min^-1

Ion exchange chromatography (IEX) for S-layer proteins from Corynebacterium

• used column: DEAE HiTrap 1 mL with GE Healthcare ÄKTA
• flow rate: 1 mL min^-1

• equilibrate column with 5 column volumes of binding buffer
• inject sample and wash column with 5 column volumes of binding buffer
• elute with 40 % binding / elution buffer mix
• elute S-layer with 60 % elution buffer
• elute remaining proteins with 100 % elution buffer

Ni-NTA spin columns

denaturating

non-denaturating

His-tag affinity chromatography

• Column: 1 mL HisTrap FF crude by [http://www.gehealthcare.com/ GE Healthcare]
• Harvest cells by centrifugation at 10000 g for 10 min at 4 °C
• Discard the supernatant and freeze bacterial pellet at -20 °C for at least 30 min
• Resuspend the pellet in 5 mL binding buffer for each gram of cell paste
• Wash column with 5 - 10 mL of deionized water
• Equilibrate column with 5 - 10 mL of binding buffer

denaturing

• For buffers see Table buffers for his-tag affinity chromatography
• Mechanical lysis:
  • Sonication on ice for approx. 5 min with Sonifier 450 by [http://www.gehealthcare.com/ Branson]
• Centrifuge at 10000 g for 30 min at 4 °C
• Load sample onto the column
• Wash with 10 ml binding buffer
• Elute with 5 mL of elution buffer with increasing imidazole concentrations
• Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction
• Re-equilibrate the column with binding buffer

non-denaturing

• For buffers see Table buffers for his-tag affinity chromatography
• Enzymatic lysis:
  • Add 0.2 mg L^-1 lysozyme, 3 Units Benzonase per mL of culture volume and 1 mM MgCl₂
  • Stir for 30 min at 4 °C
• Centrifuge at 10000 g for 30 min at 4 °C
• Load sample onto the column
• Wash with 10 ml binding buffer
• Elute with 5 mL of elution buffer with increasing imidazole concentrations
• Collect the eluate in 1 mL fractions, the purified protein is most likely in the second or third fraction
• Re-equilibrate the column with binding buffer

Immobilization of S-layer proteins on silica beads

• after purification: measure protein concentration

• dilute purified S-layer solution to 1 mg mL^-1 protein with ddH₂O

• suspend silicium dioxide beads in HBSS and mix it with the 1 mg mL^-1 S-layer solution
  • ratio of beads to protein can be varied

• incubate on vertical rotator at room temperature

• after incubation: centrifuge down the beads (1 min, > 15,000 g), wash them twice with ddH₂O and store them afterwards in ddH₂O at 4 °C in the dark