"
Team:Bielefeld-Germany/Amsterdam/Results
From 2012.igem.org
JSchirmacher (Talk | contribs) |
|||
| Line 143: | Line 143: | ||
<p class="more"> | <p class="more"> | ||
</html> | </html> | ||
| - | The laccase TVEL5 from ''Trametes versicolor'' was successfully cloned via the ''Aar''I restriction sites in the shuttle vector <partinfo> | + | The laccase TVEL5 from ''Trametes versicolor'' was successfully cloned via the ''Aar''I restriction sites in the shuttle vector <partinfo>BBa_K863207</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. |
<html> | <html> | ||
</p> | </p> | ||
Revision as of 02:30, 27 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. Activity assays were done for all four laccases (ECOL, BPUL, BHAL and TTHL), especially in regard to optimal pH and temperature effects. Additionally a comparison of all four produced laccases concerning their activity has been done. 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. Estradiol and ethinyl estradiol was futher analyzed on LC-MS/MS
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
The laccase TVEL5 from Trametes versicolor was successfully cloned via the AarI restriction sites in the shuttle vector <partinfo>BBa_K863207</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.
| 55px |  |  |  |  |  |  |  |  | 
| 
|
