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Team:Bielefeld-Germany/Results/Summary
From 2012.igem.org
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. 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. 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. 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 | ||||
|---|---|---|---|---|
| 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.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. The iGEM Team successfully produced four active bacterial laccases and accomplished to purify two of these (ECOL and 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.
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.
Laccases
The iGEM Team successfully produced four active bacterial laccases :
- Echerichia coli laccase laccase ECOL
- Bacillus Pumilus laccase BPUL
- Bacillus Halondurans laccase BHAL
- Thermus thermophiles laccase TTHL
Two of these (ECOL and BPUL) we accomplished to purify. 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 The laccase of Trametes vesicolor is still waiting to be produced.
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 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. For immobilization results see here
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. 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. 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 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 procaryotic expression system E. coli, because the protein needs glycosylation.
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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