Team:Bielefeld-Germany/Labjournal/week10

Week 10 (07/02 - 07/08/12)

Monday July 2nd

Team Fungal Laccases: Sending a request for plasmids containing cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus from the Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis at Ernst-Moritz-Arndt-University in Greifswald.

Tuesday July 3rd

• Team Site Directed Mutagenesis: Decided how to insert silent mutations to get rid of the restriction-sites with an eye on the codon-usage, using the Kazusa “Codon Usage Database” (http://www.kazusa.or.jp/codon/)
  • ecol’s illegal NgoMIV at 2307 changing ggG to ggA (Glycin)
  • tthl’s illegal PstI at 2796 caG to caA (Glutamine)
  • bpul’s illegal XbaI at 2883 changing ctA to ctT (Leucin)
  • bpul’s mutation at 2317 changing Gag (Glutamat) to Aag (Lysin)
  • xccl’s illegal PstI at 2247 changing ctG to ctC (Valine)
  • xccl’s illegal PstI at 3633 changing ctG to ctC (Valine)

Wednesday July 4th

• Team Student Academy: Final meeting with all instructors of the student academy to talk about all details.

• Team Bacterial Laccases: After the failed attempts to isolate the laccase genes from Streptomyces lavendulae and Streptomyces griseus we assumed that maybe the sequences from this laccases and the laccase sequences we designed the primers for were to different to get a product (look here for more details). So we used the sequences of the laccase genes we tried to isolate from the Streptomycetes strains and blasted them against an intern database from our university. The results showed that we could use genomic DNA from Streptomyces sp. tuebingen, S.roseochromogenes and Streptomyces sp. goettingen, from which we can get already isolated genomic DNA. Therefore we designed primers for the isolation of the predicted laccase genes from these three strains.

Thursday July 5th

• Team shuttle vector: Some fragments for Gibson Assembly with overlapping DNA sequences were amplified via Phusion-PCR. The fragment 5'AOX1 was amplified with the primer pair pSB1C3-5aox1-f and pSB1C3-5aox1-r. The fragment MF-alpha1 was amplified with the primer pair 5aox1-mfalpha1-f and 5aox1-mfalpha1-r. The fragment 3'AOX1 was amplified with the primer pair his4-3aox1-f and his4-3aox1-r. The fragment pSB1C3 was amplified with the primer pair 3aox1-pSB1C3-f and 3aox1-pSB1C3-r.

Friday July 6th

Saturday July 7th

Sunday July 8th

Sunday

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