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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

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.


How our system works

Data for our favorite new parts

  1. BBa_K863000 - bpul (laccase from Bacillus pumilus) with T7 promoter, RBS and HIS tag:
  2. BBa_K863005 - ecol (laccase from E. coli) with T7 promoter, RBS and HIS tag:

Data for pre-existing parts

We have also characterized the following parts

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


Subtrate Analytics

Cellulose binding domain

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.

Collaboration with UCL

The BioBrick BBa_K729002 from the University College London was characterized by us. Therefore E. coli KRX containing BBa_K729002 and E. coli KRX as negative control were cultivated in shaking flasks and a growth kinetic was determined. The harvested cells were lysed via sonification and the supernatant was purified from substances with a low molecular weight. After purification it was analyzed by SDS-PAGE, activity assay as well as MALDI-TOF. For a comparison E. coli KRX containing BBa_K7863005 was cultivated and also analysed by SDS-PAGE and activity assay. BBa_K729002 and BBa_K7863005 showed a similar behaviour in oxidizing ABTS.

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