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Team:Bielefeld-Germany/Results/tvel5
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=== TVEL5 integrated in shuttle vector === | === TVEL5 integrated in shuttle vector === | ||
The laccase ''Trametes versicolor'' TVEL5 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863030 BBa_K863030] was cloned with classic restriction ligation steps into the shuttle vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863204 BBa_K863204]). This construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863207 BBa_K863207]) was linearized and integrated into the yeast genome of ''P. pastoris'' GS115. Positive Clones with single cross over were identified by PCR as shown in the following pictures. <<<<<<<BILDER PCR (2 pieces)>>>>>>>> | The laccase ''Trametes versicolor'' TVEL5 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863030 BBa_K863030] was cloned with classic restriction ligation steps into the shuttle vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863204 BBa_K863204]). This construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863207 BBa_K863207]) was linearized and integrated into the yeast genome of ''P. pastoris'' GS115. Positive Clones with single cross over were identified by PCR as shown in the following pictures. <<<<<<<BILDER PCR (2 pieces)>>>>>>>> | ||
| - | The clones were cultivated in minimal medium and induced with 1% (v/v) methanol twice a day for 2 days. The supernatant of the culture was examined to active laccases by testing the enzyme activity. | + | The clones were cultivated in minimal medium and induced with 1 % (v/v) methanol twice a day for 2 days. The supernatant of the culture was examined to active laccases by testing the enzyme activity. |
[[File:Bielefeld2012_TVEL5_Eppis.jpg|thumb|left|350px|Activity test of cultivation (culture 1, 2, 3) supernatant of [http://partsregistry.org/Part:BBa_K863207 BBa_K863207]. The control is the ''P. pastoris'' GS115.]] | [[File:Bielefeld2012_TVEL5_Eppis.jpg|thumb|left|350px|Activity test of cultivation (culture 1, 2, 3) supernatant of [http://partsregistry.org/Part:BBa_K863207 BBa_K863207]. The control is the ''P. pastoris'' GS115.]] | ||
Revision as of 01:16, 27 October 2012
Contents |
TVEL5 integrated in shuttle vector
The laccase Trametes versicolor TVEL5 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863030 BBa_K863030] was cloned with classic restriction ligation steps into the shuttle vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863204 BBa_K863204]). This construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K863207 BBa_K863207]) was linearized and integrated into the yeast genome of P. pastoris GS115. Positive Clones with single cross over were identified by PCR as shown in the following pictures. <<<<<<<BILDER PCR (2 pieces)>>>>>>>> The clones were cultivated in minimal medium and induced with 1 % (v/v) methanol twice a day for 2 days. The supernatant of the culture was examined to active laccases by testing the enzyme activity.
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