Team:Bielefeld-Germany/Results/Summary

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==Cultivation and Purification of the different laccases==
 
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
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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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.
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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Revision as of 00:34, 27 September 2012

Results

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
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 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

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


bal

Laccases

Zusammenfassung

Immobilization

For immobilization results see here

Subtrate Analytics

We tried to degrade our Substrates with the TVEL0 and our self produced Laccases. The HPLC results showed that the hormons are degradeble with our Laccases. Polycyclic Aromatic Hydrocarbons (PAHs) distingrate themself in the Britton buffer but with Laccases there is so little Anthracene that the LC-MS could not detect it. This means that probably the Laccase is able to degradate it. 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 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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