Team:Bielefeld-Germany/Production

From 2012.igem.org

Revision as of 21:20, 22 June 2012 by KevinJarosch (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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

Contents

Cultivation

Expression of Laccase genes in E. coli

  • 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 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 Laccase genes in P. Pastoris

  • Chassis: Pichia Pastoris
  • Medium:
  • 200 mL culture in 1000 mL shaking flask with baffles (Schott) with silicon plugs
  • Cultivation temperature: 37 °C at 120 rpm


Purification methods

Enzymatic cell lysis with lysozyme

  • After cultivation biomass was collected by centrifugation at 5,000 g at 4 °C for 20 min.
  • 1 g of biomass (wet weight) was suspended in 10 mL of enzyme buffer containing 0.1 % Triton X-100, 2 µL benzonase (250 U/µL) and 40 µL of lysozyme (100 mg mL-1)
  • Incubation for 30 min at 4 °C
  • reaction mixture was centrifuged for 30 - 90 min at 15,000 g at 4 °C


His-tag affinity chromatography

  • Column: 1 mL HisTrap FF crude by [http://www.gehealthcare.com/ GE Healthcare]
  • Harvest cells by centrifugation at 10,000 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
  • For buffers see table buffers for his-tag affinity chromatography
  • Mechanical lysis:
    • Sonification on ice for 6 - 10 min with Sonifier 450 by [http://www.gehealthcare.com/ Branson], max. 20 W, cooled on ice
  • Incubate lysate 1 h at 4 °C shaking or rotating for solving inclusion bodies
  • Centrifuge at 15,000 g for 30 min at 4 °C
  • Filter sample (sterile filter or 300 kDa cut-off)


  • ÄKTA method
    • Equilibrate with 20 column volumes binding buffer, 0.5 mL min-1
    • Load sample onto column
    • Wash with binding buffer until UV signal is constant
    • Elute with 50 mM imidazol
    • Elute remaining proteins with 500 mM imidazol