Team:Bielefeld-Germany/Outlook

From 2012.igem.org

(Difference between revisions)
(Shuttle vector)
(Shuttle vector)
Line 12: Line 12:
== Shuttle vector ==
== Shuttle vector ==
-
The [https://2012.igem.org/Team:Bielefeld-Germany/Results/Summary#11 shuttle vector <partinfo>BBa_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.
+
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

Outlook
This page will soon report about annoying substrates in our water that we would like to deal with during our project.

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.

Bielefeld2012 Under construction.png


55px Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg