Team:Bielefeld-Germany/Amsterdam/Results

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                 Results
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                 Results since Regionals
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<h1>Summary</h1>
<h1>Summary</h1>
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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.
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All BioBricks of the iGEM Team Bielefeld were screened to identify the best conditions for protein expression. The first trials were made by shaking flask cultivations with different parameters. These parameters were various shaking flask designs, different temperatures, different concentrations of chloramphenicol, various induction strategies, several cultivation times and some cultivations in absence or presence of CuCl<sub>2</sub>. To detect the produced laccases different analysis methods were performed like SDS-PAGE analysis as well as MALDI-TOF. The iGEM Team successfully produced four active bacterial laccases and accomplished to purify two of these (the ''Escherichia coli''-laccase (ECOL) and the ''Bacillus pumilis''-laccase (BPUL)). 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 Ethenyl estradiol and Estradiol. At this moment the self-designed Shuttle-vector for the production of eukaryotic laccases in yeast is ready to go and is waiting for its application. A cheap alternative purification and immobilization method via a cellulose binding tag is also close at hand. During our research we cultivated the following BioBricks and produced several laccase. To simplify the presentation of our results we named the produced laccase like the following system.
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{| class="wikitable"
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!colspan="5"|Produced and generated BioBricks with the source strain of the DNA-sequence, promoter, protein name and the names given by the iGEM Team Bielefeld
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|BioBrick code
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|strain
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|promoter
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|name  of protein
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|name given by the iGEM Team
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|<partinfo>K863000</partinfo>
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|''Bacillus pumilus'' DSM 27
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|T7 promoter
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|align="center"|CotA
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|align="center"|'''BPUL'''
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|<partinfo>K863005</partinfo>
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|''E. coli'' BL21(DE3)
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| T7 promoter
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|align="center"|CueO
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|align="center"|'''ECOL'''
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| <partinfo>K863010</partinfo>
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|''Thermus thermophilus'' HB27
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| T7 promoter
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|align="center"|tthL
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|align="center"|'''TTHL'''
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| <partinfo>K863012</partinfo>
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|''Thermus thermophilus'' HB27
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| constitutive promoter  (<partinfo>BBa_J23100</partinfo>)
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|align="center"|tthL
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|align="center"|'''TTHL'''
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| <partinfo>K863015</partinfo>
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| ''Xanthomonas campestris pv. campestris'' B100
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|T7
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|align="center"|CopA
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|align="center"|'''XCCL'''
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|<partinfo>K863020</partinfo>
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|''Bacillus halodurans'' C-125
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|T7
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|align="center"|Lbh1
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|align="center"|'''BHAL'''
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|<partinfo>K863022</partinfo>
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|''Bacillus halodurans''  C-125
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| constitutive promoter  (<partinfo>BBa_J23100</partinfo>)
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|align="center"|Lbh1
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|align="center"|'''BHAL'''
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<img src="https://static.igem.org/mediawiki/2012/9/99/Bielefeld-Germany_Lacman.jpg" />
<h1>Laccases</h1>
<h1>Laccases</h1>
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The iGEM Team successfully produced four active bacterial laccases (click for the results):
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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]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/tvel5 ''Trametes versicolor'' laccase TVEL5]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/comparison Comparison of the different laccases]
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:* [https://2012.igem.org/Team:Bielefeld-Germany/Results/trametis Purchased positive control ''Trametes versicolor'' laccase TVEL0]
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Two of these (ECOL and BPUL) we 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.
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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''.  
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The laccase of [https://2012.igem.org/Team:Bielefeld-Germany/Results/trametis ''Trametes versicolor''] is still waiting to be produced.
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<h1>Immobilization</h1>
<h1>Immobilization</h1>
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'''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.'''
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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
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For immobilization results see [https://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
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:* [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5 ''Bacillus halodurans'' C-125 ] (named BHAL) and from
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:*[http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzreso ''Thermus thermophilus'' HB27] (named TTHL).
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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]
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<img src="https://static.igem.org/mediawiki/2012/f/fa/Bielefeld2012-Estradiol-MS-measurement.JPG" />
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<h1>Subtrate Analysis</h1>
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<h1>Substrate Analysis</h1>
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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.
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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
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For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here]
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For more information about the new results [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate#Degradation_of_estrogens click here].
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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].
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<h1>Cellulose binding domain</h1>
<h1>Cellulose binding domain</h1>
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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.
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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.
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<a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Read more</a>
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<a href="https://2012.igem.org/Team:Bielefeld-Germany/Results/cbc#Since_Amsterdam">Read '''the Boston file'''</a>
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<h1>Shuttle vector</h1>
<h1>Shuttle vector</h1>
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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.
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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.
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[https://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.]
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[https://2012.igem.org/Team:Bielefeld-Germany/Results/vector#Since_Regionals:_TVEL5_integrated_in_shuttle_vector For more information read here.]
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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.

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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.

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