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Team:Bielefeld-Germany/Outlook
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JSchirmacher (Talk | contribs) (→Shuttle vector) |
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== Shuttle vector == | == Shuttle vector == | ||
| - | The [https://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#11 shuttle vector <partinfo> | + | The [https://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#11 shuttle vector <partinfo>K863204</partinfos> (pECPP11JS)] could be constructed. The following step is to do site directed mutagenesis to eliminate the illegal ''Xba''I restriction site in ''his4'' gene. At next the genotype of the ''P. pastoris'' mutants have to be characterized as Mut<sup>+</sup> or Mut<sup>s</sup> transformants via PCR with 5AOX-Genotype-FW and TT-Genotype-RV primer. Afterwards the GFP::pECPP11JS construct have to sequenced and transformed into ''P. pastoris'' cells. The same procedure have to be repeated with the laccases from ''Trametes versiculor'' TVEL5 and from ''Pycnoporus cinnabarinus'' PCIL35. |
After successfully site-directed integration of the shuttle vector in ''P. pastoris'' GS115 and genotype characterization, a methanol-induced cultivation is following. With an ion exchange chromatography the laccases can be purified and detected with a SDS-PAGE and MALDI-TOF-MS. | After successfully site-directed integration of the shuttle vector in ''P. pastoris'' GS115 and genotype characterization, a methanol-induced cultivation is following. With an ion exchange chromatography the laccases can be purified and detected with a SDS-PAGE and MALDI-TOF-MS. | ||
Revision as of 13:01, 26 September 2012
Shuttle vector
The shuttle vector <partinfo>K863204</partinfos> (pECPP11JS) could be constructed. The following step is to do site directed mutagenesis to eliminate the illegal XbaI restriction site in his4 gene. At next the genotype of the P. pastoris mutants have to be characterized as Mut+ or Muts transformants via PCR with 5AOX-Genotype-FW and TT-Genotype-RV primer. Afterwards the GFP::pECPP11JS construct have to sequenced and transformed into P. pastoris cells. The same procedure have to be repeated with the laccases from Trametes versiculor TVEL5 and from Pycnoporus cinnabarinus PCIL35.
After successfully site-directed integration of the shuttle vector in P. pastoris GS115 and genotype characterization, a methanol-induced cultivation is following. With an ion exchange chromatography the laccases can be purified and detected with a SDS-PAGE and MALDI-TOF-MS.
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