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• Start
• Team
  • Members
  • Photos
  • Bielefeld University
  • Acknowledgements
  • Contact
  • 
• Project
  • Description
  • Achievements
  • Approach
  • Model
  • Database
  • Background
  • Labjournal
  • Protocols
  • Outlook
  • 
• Results
  • Summary
  • Datapage
  • Laccases
  • Immobilization
  • Substrate Analysis
  • Cellulose Binding Domain
  • Shuttle Vector
  • Collaboration
  • 
• Since Regionals
  • Summary
  • Labjournal
  • 
• Human Practice
  • Overview
  • Expert
  • Politics
  • Industry
  • Outreach
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Summary

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

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	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

We have also characterized the following parts

1. <partinfo>BBa_K863012</partinfo> - tthl laccase (from T. thermophilus) with constitutive promoter J23100, RBS and HIS tag:
2. <partinfo>BBa_K863022</partinfo> - bhal laccase (from Bacillus halodurans) with constitutive promoter J23100, RBS and HIS tag:

Laccases

Zusammenfassung

For Coli Read more. For Pumi Read more. For Thermo Read more. For Halo Read more. For Trametis Read more.

Immobilization

For immobilization results see here

Subtrate Analytics

For LC-MS, HPLC and Spectrofluorophotometer substrate analysis results see 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.