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Summary

Since the European Jamboree in Amsterdam all four laccases (ECOL, BPUL, TTHL, BHAL) were produced in a 12 L scale and purified to get a high amount of the enzyme for a further characterization. They were analyzed and compared concerning their activity at different pH and temperature effects. Furthermore they were all screened regarding to their ability of estradiol and ethinyl estradiol degradation. The immobilization of the four laccases using CPC-beads was optimized and the activity tested. All of them were active. Additionally a shuttle vector for Pichia pastoris was constructed, which works as expected. The eukaryotic laccase TVEL5 was successfully cloned into the vector, produced and secreted. Finally lots of efforts were made to construct a protein fused to a cellulose binding domain, but until now, no working fusion protein could be produced.

Laccases

The iGEM Team successfully produced four active bacterial laccases and an eukaryotic laccase (click for the results):

• Escherichia coli laccase ECOL
• Bacillus pumilus laccase BPUL
• Bacillus halodurans laccase BHAL
• Thermus thermophilus laccase TTHL
• Trametes versicolor laccase TVEL5
• Comparison of the different laccases
• Purchased positive control Trametes versicolor laccase TVEL0

All bacterial laccases (ECOL, BPUL, BHAL and TTHL) 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.

Immobilization

The immobilization method on CPC-beads was further optimized. It has been proved that an incubation time of 6 hours is actually enough for immobilization. Furthermore, two additional laccases were successfully immobilized: from

• [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 Bacillus halodurans C-125 ] (named BHAL) and from
• [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzreso Thermus thermophilus HB27] (named TTHL).

Moreover, activity tests were carried out on all four immobilized laccases (ECOL, BPUL, BHAL, and TTHL). All of them showed activity. For immobilization results see here

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. <p class="more"> Read '''the Boston file'''

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.

For more information read here.