Team:Bielefeld-Germany/Protocols/Production
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Contents |
Production
Here all our methods according to cultivation and purification are listed.
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
Purification
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