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Team:Bielefeld-Germany/Results/Summary
From 2012.igem.org
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<h1>Laccases</h1> | <h1>Laccases</h1> | ||
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Zusammenfassung | Zusammenfassung | ||
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<h1>Immobilization</h1> | <h1>Immobilization</h1> | ||
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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] | ||
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<h1>Subtrate Analytics</h1> | <h1>Subtrate Analytics</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. Due to time reasons we could not measure the Analgesics and 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. For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click here] | 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. Due to time reasons we could not measure the Analgesics and 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. For more informations [https://2012.igem.org/Team:Bielefeld-Germany/Results/substrate click 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 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 and to characterize a GFP has been introduced as a C-terminal domain of the cellulose binding protein. After delays in cloning the constructs for both 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.<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 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 and to characterize a GFP has been introduced as a C-terminal domain of the cellulose binding protein. After delays in cloning the constructs for both 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.<html> | ||
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<h1>Shuttle vector</h1> | <h1>Shuttle vector</h1> | ||
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A shuttle vector for recombination into the yeast <i>P. pastoris</i> could be developed. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein in the media. This protein of interest could be cloned in frame with one restiction-ligate-cloning-step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy. [https://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.] | A shuttle vector for recombination into the yeast <i>P. pastoris</i> could be developed. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein in the media. This protein of interest could be cloned in frame with one restiction-ligate-cloning-step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy. [https://2012.igem.org/Team:Bielefeld-Germany/Results/vector Read more.] | ||
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<h1>Collaboration with UCL</h1> | <h1>Collaboration with UCL</h1> | ||
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[https://2012.igem.org/Team:Bielefeld-Germany/Results/london Read more.] | [https://2012.igem.org/Team:Bielefeld-Germany/Results/london Read more.] | ||
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Revision as of 00:53, 27 September 2012
Summary
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.| 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 | strain | promoter | name of protein | name given by the iGEM Team |
| <partinfo>K863000</partinfo> | Bacillus pumilus DSM 27 | T7 promoter | CotA | BPUL |
| <partinfo>K863005</partinfo> | E. coli BL21(DE3) | T7 promoter | CueO | ECOL |
| <partinfo>K863010</partinfo> | Thermus thermophilus HB27 | T7 promoter | tthL | TTHL |
| <partinfo>K863012</partinfo> | Thermus thermophilus HB27 | constitutive promoter (<partinfo>BBa_J23100</partinfo>) | tthL | TTHL |
| <partinfo>K863015</partinfo> | Xanthomonas campestris pv. campestris B100 | T7 | CopA | XCCL |
| <partinfo>K863020</partinfo> | Bacillus halodurans C-125 | T7 | Lbh1 | BHAL |
| <partinfo>K863022</partinfo> | Bacillus halodurans C-125 | constitutive promoter (<partinfo>BBa_J23100</partinfo>) | Lbh1 | BHAL |
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.
Datapage
==bla==
bal
Laccases
==bla== Zusammenfassung
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For Coli Read more.
For Pumi Read more.
For Thermo Read more.
For Halo Read more.
For Trametis Read more.
Subtrate Analytics
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. Due to time reasons we could not measure the Analgesics and 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. 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 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 and to characterize a GFP has been introduced as a C-terminal domain of the cellulose binding protein. After delays in cloning the constructs for both 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. Read more
Shuttle vector
A shuttle vector for recombination into the yeast P. pastoris could be developed. With this system it is possible to recombine a protein of interest with a N-terminal mating factor alpha 1 for secretion the protein in the media. This protein of interest could be cloned in frame with one restiction-ligate-cloning-step. The selection depends not on an antibiotic resistance like zeocine, but on a complementation of histidine auxotrophy. Read more.
Collaboration with UCL
The BioBrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 BBa_K729006] from the University College London was characterized by us. Therefore E. coli KRX containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] was cultivated and analysed by SDS-PAGE as well as tested with a laccase activity assay. [http://partsregistry.org/wiki/index.php?title=Part:BBa_K729006 BBa_K729006] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K7863005] showed a similar behaviour in oxidizing ABTS. Read more.
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