Team:Bielefeld-Germany/Amsterdam/Results

From 2012.igem.org

(Difference between revisions)
 
(69 intermediate revisions not shown)
Line 34: Line 34:
<!-- tab panes -->
<!-- tab panes -->
-
<div id="panesresult">
+
<div id="panesresult2">
<div id="anzeige">
<div id="anzeige">
 +
<img src="https://static.igem.org/mediawiki/2012/0/0b/Bielefeld2012_Amsterdamgruppe.jpg" />
 +
 +
<h1>Summary</h1>
<h1>Summary</h1>
  </html>
  </html>
-
coming soon
+
<p class="more">
 +
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.
<html>
<html>
-
+
</p>
Line 53: Line 57:
<div id="anzeige">
<div id="anzeige">
-
<img src="https://static.igem.org/mediawiki/2012/6/6c/Bielefeld2012_Plan.jpg" />
+
<img src="https://static.igem.org/mediawiki/2012/9/99/Bielefeld-Germany_Lacman.jpg" />
<h1>Laccases</h1>
<h1>Laccases</h1>
<p class="more">
<p class="more">
</html>
</html>
-
The iGEM Team successfully produced four active bacterial laccases:
+
The iGEM Team successfully produced four active bacterial laccases and an eukaryotic laccase (click for the results):
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/coli ''Escherichia coli'' laccase ECOL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/coli ''Escherichia coli'' laccase ECOL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/pumi''Bacillus pumilus'' laccase BPUL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/pumi''Bacillus pumilus'' laccase BPUL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/halo''Bacillus halodurans'' laccase BHAL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/halo''Bacillus halodurans'' laccase BHAL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/thermo ''Thermus thermophilus'' laccase TTHL]
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/thermo ''Thermus thermophilus'' laccase TTHL]
 +
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/tvel5 ''Trametes versicolor'' laccase TVEL5]
 +
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/comparison Comparison of the different laccases]
 +
:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/trametis Purchased positive control ''Trametes versicolor'' laccase TVEL0]
<html>
<html>
 +
</p>
<p class="more">
<p class="more">
</html>
</html>
-
All of the bacterial laccases were accomplished to purify. Besides the successfully scale-up fermentation these two laccases could be purified in a high amount to characterize the optimal activity conditions regarding  pH, temperature, buffer solutions  and organic solvent resistance. Furthermore the iGEM Team Bielefeld demonstrated that the produced laccases can be immobilized maintaining their activity and the degradation capacity was screened for several micro-contaminants. These tests indicate that the ECOL and BPUL are able to degrade ethinyl estradiol and estradiol.
+
All bacterial laccases ([https://2012.igem.org/Team:Bielefeld-Germany/Results/coli#Since_Regionals:_12.C2.A0L_Fermentation_E._coli_KRX_with_BBa_K863005 ECOL], [https://2012.igem.org/Team:Bielefeld-Germany/Results/pumi#Since_Regionals:_12.C2.A0L_Fermentation_E..C2.A0coli_KRX_with_BBa_K863000 BPUL], [https://2012.igem.org/Team:Bielefeld-Germany/Results/halo#Since_Regionals:_12L_Fermentation_of_E._coli_Rosetta-Gami_2_with_BBa_K863022 BHAL] and [https://2012.igem.org/Team:Bielefeld-Germany/Results/thermo#Since_Regionals:_12_L_Fermentation_of_E._coli_Rosetta_Gami_2_with_BBa_K863012 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 ([https://2012.igem.org/Team:Bielefeld-Germany/Results/coli#Initial_activity_tests_of_purified_fractions ECOL], [https://2012.igem.org/Team:Bielefeld-Germany/Results/pumi#Since_Regionals:_Initial_activity_tests_of_purified_fractions BPUL], [https://2012.igem.org/Team:Bielefeld-Germany/Results/halo#Since_Regionals:_Initial_activity_tests_of_purified_fractions BHAL] and [https://2012.igem.org/Team:Bielefeld-Germany/Results/thermo#Since_Regionals:_Initial_activity_tests_of_purified_fractions TTHL]), especially in regard to optimal pH and temperature effects. Additionally a [https://2012.igem.org/Team:Bielefeld-Germany/Results/comparison 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''.  
-
The laccase of [https://2012.igem.org/Team:Bielefeld-Germany/Results/tvel0 TVEL0 from''Trametes versicolor''] was produced and tested for activity on oaxidation of ABTS.
+
<html>
<html>
</p>
</p>
-
</p>
+
 
</div>
</div>
Line 79: Line 86:
<div id="anzeige">
<div id="anzeige">
-
<img src="https://static.igem.org/mediawiki/2012/2/25/Bielefeld2012_Immo.jpeg" />
+
<img src="https://static.igem.org/mediawiki/2012/7/79/Bielefeld2012_Immosum2.jpg" />
<h1>Immobilization</h1>
<h1>Immobilization</h1>
Line 85: Line 92:
<p class="more">
<p class="more">
</html>
</html>
-
Coming soon!
+
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
-
<p class="more">
+
<html>
-
For immobilization results see [https://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
+
</p>
</p>
 +
</html>
 +
:* [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).
 +
<html>
 +
<p class="more">
 +
</html>
 +
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 [https://2012.igem.org/Team:Bielefeld-Germany/Results/immo#Since_Regionals:_Activity_tests_of_immobilized_ECOL.2C_BPUL.2C_BHAL_and_TTHL here]
<html>
<html>
</p>
</p>
Line 96: Line 109:
<div id="anzeige">
<div id="anzeige">
-
<img src="https://static.igem.org/mediawiki/2012/a/a7/Bielefeld2012-estradiol-control-spectroflurophotometer.JPG" />
+
<img src="https://static.igem.org/mediawiki/2012/f/fa/Bielefeld2012-Estradiol-MS-measurement.JPG" />
-
<h1>Subtrate Analysis</h1>
+
<h1>Substrate Analysis</h1>
<p class="more">
<p class="more">
</html>
</html>
-
Coming soon!
+
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
-
<p class="more">
+
<html>
-
For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here]
+
</p>
</p>
 +
<p class="more">
 +
</html>
 +
For more information about the new results [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate#Degradation_of_estrogens 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 [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate#Further_analysis_.28after_Regionals_Amsterdam.29 here].
<html>
<html>
Line 114: Line 130:
<div id="anzeige">
<div id="anzeige">
-
<img src="https://static.igem.org/mediawiki/2012/6/6d/Bielefeld2012_GFP.jpg" />
+
<img src="https://static.igem.org/mediawiki/2012/b/b4/Bielefeld2012_no_GFP.jpg" />
<h1>Cellulose binding domain</h1>
<h1>Cellulose binding domain</h1>
Line 120: Line 136:
<p class="more">
<p class="more">
</html>
</html>
-
Coming soon!
+
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">
<p class="more">
<html>
<html>
-
<a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc#Since_Amsterdam">Read more</a>
+
<a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc#Since_Amsterdam">Read '''the Boston file'''</a>
<html>
<html>
</p>
</p>
Line 133: Line 149:
<div id="anzeige">
<div id="anzeige">
-
<img src="https://static.igem.org/mediawiki/2012/1/17/Bielefeld2012_PECPP11JS.JPG" />
+
<img src="https://static.igem.org/mediawiki/2012/a/a5/Bielefeld2012_TVEL5_Eppis.jpg" />
<h1>Shuttle vector</h1>
<h1>Shuttle vector</h1>
<p class="more">
<p class="more">
</html>
</html>
-
A shuttle vector for site-directed recombination into the yeast ''P. pastoris'' does not exist in the parts registry and could be developed by our team. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein into the media. This protein of interest could be cloned in frame with one restriction ligate cloning step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy. This system is for us important because some of our laccases can not be expressed in the prokaryotic expression system ''E. coli'', because the protein needs glycosylation.
+
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.
-
[https://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.]
+
<html>
</p>
</p>
 +
<p class="more">
 +
</html>
 +
[https://2012.igem.org/Team:Bielefeld-Germany/Results/vector#Since_Regionals:_TVEL5_integrated_in_shuttle_vector For more information read here.]
<html>
<html>
-
 
+
</p>
-
</p>
+
</div>
</div>
Line 156: Line 175:
-
$("#tab ul").tabs("#panesresult > div", {effect: 'fade', fadeOutSpeed: 400});
+
$("#tab ul").tabs("#panesresult2 > div", {effect: 'fade', fadeOutSpeed: 400});
});
});
</script>
</script>

Latest revision as of 03:40, 27 October 2012

Results since Regionals

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):

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.

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