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Team:Bielefeld-Germany/Amsterdam/Results
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All bacterial laccases (ECOL, BHAL, TTHL and BPUL) were successfully scaled-up to a working volume of 12 L. Additional a optimized medium was chosen in the hope to enhance the protein amount. Especially the two laccases BHAL and TTHL could be produced in sufficient quantities to realize their characterization of the optimal activity conditions regarding pH, temperature, buffer solutions and organic solvent resistance. In addition an active eukaryotic laccases of ''Trametes versicolor'' was produced with a self-designed Shuttle-vector in yeast ''Pichia Pastoris''. | All bacterial laccases (ECOL, BHAL, TTHL and BPUL) were successfully scaled-up to a working volume of 12 L. Additional a optimized medium was chosen in the hope to enhance the protein amount. Especially the two laccases BHAL and TTHL could be produced in sufficient quantities to realize their characterization of the optimal activity conditions regarding pH, temperature, buffer solutions and organic solvent resistance. In addition an active eukaryotic laccases of ''Trametes versicolor'' was produced with a self-designed Shuttle-vector in yeast ''Pichia Pastoris''. | ||
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Revision as of 23:55, 26 October 2012
Summary
coming soon
Laccases
The iGEM Team successfully produced four active bacterial laccases and an eukaryotic laccase (click for the results):
All bacterial laccases (ECOL, BHAL, TTHL and BPUL) were successfully scaled-up to a working volume of 12 L. Additional a optimized medium was chosen in the hope to enhance the protein amount. Especially the two laccases BHAL and TTHL could be produced in sufficient quantities to realize their characterization of the optimal activity conditions regarding pH, temperature, buffer solutions and organic solvent resistance. In addition an active eukaryotic laccases of Trametes versicolor was produced with a self-designed Shuttle-vector in yeast Pichia Pastoris.
Substrate Analysis
After the regional jamboree Team Activity Test, Team Immobilization and Team Substrate Analysis got laccases from the same pool from the Cultivation Team. With these laccases the degradation experiments of estradiol and ethinyl estradiol were repeated for the laccases BPUL and ECOL. The new laccases BHAL and TTHL were characterized for estradiol and ethinyl estradiol degradation, too. For more information about the new results click here. Possible resulting degradation products after treatment of estradiol and ethinyl estradiol with TVEL0 were further analyzed via LCMS-MS. The results are shown here.
Cellulose binding domain
A lot of efforts were made, to change the order of the fusion proteins, to change the promoter and the RBS and to insert a different linker between the cellulose binding domain and the reporter GFP, but to the last day of lab work no green glowing colony to work with and execute the binding assay could be generated.
Shuttle vector
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The laccase TVEL5 from Trametes versicolor was successfully cloned via the AarI restriction sites in the shuttle vector <partinfo>BBa_K863205</partinfo>. We could show that the gene of interest is integrating in yeast genome over site directed recombination and the produced protein of interest is secreted in the cultivation medium and detectable there. With this results we could show that the shuttle vector is working as expected and therefor can be used for the production of any protein of interest. Read more.
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