Team:Bielefeld-Germany/Results/Summary

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<li><a href="#3"><strong>Laccases</strong></a></li>
<li><a href="#3"><strong>Laccases</strong></a></li>
<li><a href="#4"><strong>Immobilization</strong></a></li>
<li><a href="#4"><strong>Immobilization</strong></a></li>
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<li><a href="#5"><strong>Substrate Analytics</strong></a></li>
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<li><a href="#5"><strong>Substrate Analysis</strong></a></li>
<li><a href="#6"><strong>CBD</strong></a></li>
<li><a href="#6"><strong>CBD</strong></a></li>
<li><a href="#7"><strong>Shuttle vector</strong></a></li>
<li><a href="#7"><strong>Shuttle vector</strong></a></li>
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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.
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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 succeeded to purify four of them. Besides the successfully scale-up fermentation, these 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 all of our produced laccases are able to degrade estradiol and the two laccases TTHL and BPUL are able to degrade ethinyl-estradiol in combination with a mediator. At this moment, the self-designed shuttle-vector for the production of eukaryotic laccases in yeast is ready to go. This vector was tested to integrate by courtesy of homologous recombination genes of eukaryotic laccases into Pichia Pastoris and produce them in an active form. First experiments show a successful production of one laccase of ''Trametes versicolor''. 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 corresponding system.
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All functional 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.  To detect the produced laccases different analysis methods were performed like SDS-PAGE analysis as well as MALDI-TOF.
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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 purify 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.
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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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|align="center"|Lbh1
|align="center"|Lbh1
|align="center"|'''BHAL'''
|align="center"|'''BHAL'''
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| <partinfo>K863030</partinfo>
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|''Trametes versicolor ''
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| AOX1 promoter
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|align="center"|TVL5
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|align="center"|'''TVEL5'''
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iGEM Team Bielefeld is developing a biological filter using immobilized laccases, enzymes able to radicalize and break down a broad range of aromatic substances. For the production of laccases from different bacteria, fungi and plants, two expression systems are used: Escherichia coli and the yeast Pichia pastoris. Immobilization is carried out either by using CPC-silica beads or by fusing the enzymes to cellulose binding domains. The concept could be extended to other toxic pollutants in drinking and wastewater, as well as to industrial applications in paper and textile industries or even for bioremediation of contaminated soil.  
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iGEM Team Bielefeld is developing a biological filter using immobilized laccases, enzymes able to radicalize and break down a broad range of aromatic substances. For the production of laccases from different bacteria, fungi and plants, two expression systems are used: ''Escherichia coli'' and the yeast ''Pichia pastoris''. Immobilization is carried out either by using CPC-silica beads or by fusing the enzymes to cellulose binding domains. The concept could be extended to other toxic pollutants in drinking and wastewater, as well as to industrial applications in paper and textile industries or even for bioremediation of contaminated soil.  
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[http://2012.igem.org/Team:Bielefeld-Germany/Judging#2 Read more.]
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[http://2012.igem.org/Team:Bielefeld-Germany/Results/Datapage Read more.]
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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):
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/coli ''Escherichia coli'' laccase ECOL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/coli ''Escherichia coli'' laccase ECOL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/pumi''Bacillus pumilus'' laccase BPUL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/pumi''Bacillus pumilus'' laccase BPUL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/halo''Bacillus halodurans'' laccase BHAL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/halo''Bacillus halodurans'' laccase BHAL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/thermo ''Thermus thermophilus'' laccase TTHL]
:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/thermo ''Thermus thermophilus'' laccase TTHL]
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:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/tvel5 ''Trametes versicolor'' laccase TVEL5]
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:* [http://2012.igem.org/Team:Bielefeld-Germany/Results/comparison Comparison of the different laccases]
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:* [http://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 Ethenyl estradiol and Estradiol
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All bacterial laccases (ECOL, BHAL, TTHL and BPUL) we accomplished to purify. Besides the successfully scale-up fermentation these 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 they are able to degrade estradiol and ethinyl-estradiol.
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The laccase of [http://2012.igem.org/Team:Bielefeld-Germany/Results/trametis ''Trametes versicolor''] is still waiting to be produced.
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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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'''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
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For immobilization results see [http://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
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:* [http://openwetware.org/wiki/E._coli_genotypes#BL21.28DE3.29 ''E. coli'' BL21 (DE3)] (named ECOL)
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:* [http://www.dsmz.de/catalogues/details/culture/DSM-27.html ''Bacillus pumilus'' DSM 27 (ATCC7061)] (named BPUL)
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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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on CPC-silica beads. All four purified laccases were successfully immobilized, with ECOL and BPUL showing the highest binding ability to beads. Moreover, all four immobilized laccases showed activity. Whereas immobilized BPUL showed a relatively high activity, the results couldn't be compared to BHAL und TTHL due to the low concentration of the latters. ''' For immobilization results see [http://2012.igem.org/Team:Bielefeld-Germany/Results/immo here]
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<img src="http://2012.igem.org/wiki/images/a/a7/Bielefeld2012-estradiol-control-spectroflurophotometer.JPG" />
<img src="http://2012.igem.org/wiki/images/a/a7/Bielefeld2012-estradiol-control-spectroflurophotometer.JPG" />
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<h1>Subtrate Analytics</h1>
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<h1>Substrate Analysis</h1>
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We tried to degrade our substrates with the TVEL0 (positiv control) and our self-produced laccases. The HPLC results showed that the hormones are degradeble with our laccases. Polycyclic Aromatic Hydrocarbons (PAHs) desintegrate themselves in the Britton buffer. The LC/MS measurements of anthracene for example, show a baseline, which can be decreased by additing 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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To establish the methods for degradation analysis of different substrates TVEL0 was used as positive control.
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For more informations [http://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here]
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After that the four produced bacterial laccases were analyzed. The HPLC results showed that estradiol and ethinyl estradiol ( with addition of ABTS) are degradable with our laccases.To determine degradation products of estradiol and ethinyl estradiol after laccase treatment LCMS-MS analysis were done. For more informations [http://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here].
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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 micro-contaminates, 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-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. Future research will focus on the linker between CBDs and the reporter GFP and in changing the order of CBD and GFP.
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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 href="http://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Read more</a>
<a href="http://2012.igem.org/Team:Bielefeld-Germany/Results/cbc">Read more</a>
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A shuttle vector for site-directed recombination into the yeast <i>P. pastoris</i> 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 procaryotic expression system <i>E. coli</i>, because the protein needs glycosylation.
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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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[http://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.]
[http://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.]
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Latest revision as of 00:15, 3 December 2012

Results

Summary

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 CuCl2. 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 succeeded to purify four of them. Besides the successfully scale-up fermentation, these 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 all of our produced laccases are able to degrade estradiol and the two laccases TTHL and BPUL are able to degrade ethinyl-estradiol in combination with a mediator. At this moment, the self-designed shuttle-vector for the production of eukaryotic laccases in yeast is ready to go. This vector was tested to integrate by courtesy of homologous recombination genes of eukaryotic laccases into Pichia Pastoris and produce them in an active form. First experiments show a successful production of one laccase of Trametes versicolor. 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 corresponding system.

Produced and generated BioBricks with the source strain of the DNA-sequence, promoter, protein name and the names given by the iGEM Team Bielefeld
BioBrick code strain promoter name of protein name given by the iGEM Team
BBa_K863000 Bacillus pumilus DSM 27 T7 promoter CotA BPUL
BBa_K863005 E. coli BL21(DE3) T7 promoter CueO ECOL
BBa_K863010 Thermus thermophilus HB27 T7 promoter tthL TTHL
BBa_K863012 Thermus thermophilus HB27 constitutive promoter (BBa_J23100) tthL TTHL
BBa_K863015 Xanthomonas campestris pv. campestris B100 T7 CopA XCCL
BBa_K863020 Bacillus halodurans C-125 T7 Lbh1 BHAL
BBa_K863022 Bacillus halodurans C-125 constitutive promoter (BBa_J23100) Lbh1 BHAL
BBa_K863030 Trametes versicolor AOX1 promoter TVL5 TVEL5

Datapage

iGEM Team Bielefeld is developing a biological filter using immobilized laccases, enzymes able to radicalize and break down a broad range of aromatic substances. For the production of laccases from different bacteria, fungi and plants, two expression systems are used: Escherichia coli and the yeast Pichia pastoris. Immobilization is carried out either by using CPC-silica beads or by fusing the enzymes to cellulose binding domains. The concept could be extended to other toxic pollutants in drinking and wastewater, as well as to industrial applications in paper and textile industries or even for bioremediation of contaminated soil.

Read more.

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) we accomplished to purify. Besides the successfully scale-up fermentation these 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 they are able to degrade estradiol and ethinyl-estradiol.

Immobilization

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

on CPC-silica beads. All four purified laccases were successfully immobilized, with ECOL and BPUL showing the highest binding ability to beads. Moreover, all four immobilized laccases showed activity. Whereas immobilized BPUL showed a relatively high activity, the results couldn't be compared to BHAL und TTHL due to the low concentration of the latters. For immobilization results see here

Substrate Analysis

To establish the methods for degradation analysis of different substrates TVEL0 was used as positive control. After that the four produced bacterial laccases were analyzed. The HPLC results showed that estradiol and ethinyl estradiol ( with addition of ABTS) are degradable with our laccases.To determine degradation products of estradiol and ethinyl estradiol after laccase treatment LCMS-MS analysis were done. For more informations click here.

Cellulose binding domain

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.

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.

Collaboration with UCL

The BioBrick BBa_K729006 from the University College London was characterized by us. Therefore E. coli KRX containing BBa_K729006 and E. coli KRX as a negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via sonication and substances with a low molecular weight were seperated out of the supernatant. After purification the sample was analyzed by SDS-PAGE and MALDI-TOF. For a comparison E. coli KRX containing BBa_K7863005 was cultivated and analysed by SDS-PAGE as well as tested with a laccase activity assay. BBa_K729006 and BBa_K7863005 showed a similar behaviour in oxidizing ABTS. Read more.


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