Team:Bielefeld-Germany/Amsterdam/Results

From 2012.igem.org

(Difference between revisions)
Line 85: Line 85:
<p class="more">
<p class="more">
</html>
</html>
-
'''Using commercially acquired laccases from ''Trametes versicolor'' (named TVEL0) as a standard, it was possible to optimize an immobilization method of the purified laccases from ''E. coli'' BL21 (DE3) (named ECOL) and ''Bacillus pumilus''  (named BPUL) on CPC-silica beads. Both laccases were successfully bound to the beads and showed activity. Whereas ECOL showed the highest binding capacity, immobilized BPUL showed higher activity.'''
+
Coming soon!
<p class="more">
<p class="more">
For immobilization results see [https://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
For immobilization results see [https://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
Line 101: Line 101:
<p class="more">
<p class="more">
</html>
</html>
-
We tried to degrade our substrates with the TVEL0 (positive control) and our self-produced laccases. The HPLC results showed that the hormones are degradable with our laccases. Polycyclic aromatic hydrocarbons (PAHs) desintegrate themselves in the Briton buffer. The LC/MS measurements of anthracene for example, show a baseline, which can be decreased by adding laccases. This means that all of the tested Laccases are probably  able to degrade this substrate.  Due to the lack of time we could neither measure the analgesics nor lindane which was also one of our Substrates to test. but we have not had the opportunity. The spectrofluorophotometer data showed also that ethinyl estradiol and estradiol are degraded after laccase treatment.
+
Coming soon!
<p class="more">
<p class="more">
For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here]
For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here]
Line 120: Line 120:
<p class="more">
<p class="more">
</html>
</html>
-
A cheap alternative purification method combined with a powerful immobilization tool could be the solution to prevail over other more expensive water cleaning methods like oxidization with ozone or using tons of activated carbon which just capture microcontaminates, but does not dismantle them. A promising solution to this could be cellulose binding domains (CBDs). Cellulose is ubiquitous and sustainable. Following this idea fusion-protein-constructs with cellulose binding domains have been made. To characterize a GFP has been introduced as a C or N-terminal domain of the cellulose binding protein. After delays in cloning the constructs for two fusion proteins with a T7-promoter could be finished, but did not express the protein in ''E. coli'' KRX and BL21. An alternative construct with a constitutive promoter could also be finished, but gave the same results. Changing the order of CBD and GFP was carried out, but was hampered by a base deletion in the GFP gene causing a frame shift and could not be redone in time.
+
Coming soon!
<p class="more">
<p class="more">
<html>
<html>

Revision as of 13:06, 26 October 2012

Results since Regionals

Summary

coming soon

Laccases

The iGEM Team successfully produced four active bacterial laccases:

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. The laccase of TVEL0 fromTrametes versicolor was produced and tested for activity on oaxidation of ABTS.

Immobilization

Coming soon!

For immobilization results see here

Subtrate Analysis

Coming soon!

For more informations click here

Cellulose binding domain

Coming soon!

Read more

Shuttle vector

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. Read more.


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

About our Wiki

Fifteen students from different fields of study from the Bielefeld University aspired to complete a project called TOXIC COMPOUNDS IN NATURAL WATER - A CASE FOR LACCASE. In his Wiki we document our achievements and our approach.

Contact Us

Universität Bielefeld
Universitätsstraße 25
D-33615 Bielefeld

info(at)igem-bielefeld.de

Navigate our Wiki

Start

Team

Project

Results

Since Regionals

Human Practice

Safety

Judging

Partner

Retrieved from "http://2012.igem.org/Team:Bielefeld-Germany/Amsterdam/Results"