Team:Bielefeld-Germany/Labjournal/week11

Week 11 (07/09 - 07/15/12)

Monday July 9th

• Team Student Academy:
  • This week the Student Academy took place. Today we held a presentation about the background of our experiments and answered all the questions the pupils had. Furthermore two of us participated at the an unconstrained meeting between the instructors and the pupils in the evening.
• Team Site Directed Mutagenesis: Council with Katharina Thiedig, who did the Site Directed Mutagenesis for the last years iGEM-Team-Bielefeld, about how to use the “QuikChange Primer Design”-program and the SDM-protocol

Tuesday July 10th

• Team Site Directed Mutagenesis: Generated primer-sequences with Agilent Technologies “QuikChange Primer Design” for bpul, xccl, ecol and tthl and named the primers by their source-organism and place of mutation flanked on the left side by the original base and on the right by the mutated base.
  • ecol-g2307a
  • tthl-g2796a
  • bpul-a2883t
  • bpul-g2317t
  • xccl-g2247c
  • xccl-g3633c
• Team Cellulose Binding Domain: Searched NCBI for Cellulose, Chitin & Keratin binding motifs in accessible organisms; found two Chitin-binding-domains in Bacillus halodurans
• Team Student Academy:
  • The experiments were performed by one half of the pupils in groups of 2-3. Afterwards we held a presentation about the iGEM competition, the project of the last two teams from Bielefeld and about our project. In the evening we had a barbecue with the pupils and the iGEM team with a lot of interesting discussions.

• Team Fungal and Plant Laccases:
  • We got the sequences for five different fungal laccases from the requested plasmids from Monday 2nd and they wanted to send us plasmids which contain the cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus.
    • tvel5 from Trametes versicolor
    • tvel10 from Trametes versicolor
    • tvel13 from Trametes versicolor
    • tvel20 from Trametes versicolor
    • pcil35 from Pycnoporus cinnabarinus
  • We designed primer pairs with prefix and suffix overhanging ends for cloning in pSB1C3 and a primer pair for cloning in shuttle vector.
  • The first primer pairs were designed with standard prefix and suffix sequence and 20 bases complementary to the start and end of the ORF sequences.
  • Additional primer pairs were designed with AarI restriction site and Kozak consensus sequence before the first 20 bases from the start of the ORF (forward primers). The reverse primers were designed with the last 20 bases of the laccase genes and a terminator overhanging end and a AarI restriction site.

Wednesday July 11th

• Team Cellulose Binding Domain: Looked the sequence of Clostridium cellulovorans cellulose binding protein gene (cbp A) up and made a Clonemanager-file. We used a protein-BLAST of the translated protein-sequence to find the location of the cellulose binding domain within the protein: We found it should be from base 103 to base 378 of the open reading frame.
• Team Student Academy:
  • The experiments were performed by the second half of the pupils and the groups from yesterday analyzed their plates.
• Team Cloning of Bacterial Laccases:

• We made glycerin cultures of the finished BioBricks.

Thursday July 12th

Friday July 13th

• Team Student Academy:
  • The groups from Wednesday analyzed their plates. Together with the pupils we made a final analysis of the results, discussed about all problems and questions and helped with the preparation of a presentation, they had to hold in front of all instructors and participants.

• Team Cloning of Bacterial Laccases:

• We got new Streptomyces DNA from or supervisor C. Rueckert. He did an alignment with our S. griseus and S. lavendulae sequences against a local database for Streptomyces and identified S. rosechromogenes, S. tuebingen and S.goettingen. Those laccase genes showed simularity to our laccase genes. Since he had isolated chromosomal DNA we were able to work with them. We set the PCR with the three Streptomyces. On this PCR S. tuebingen DNA could be amplified, but it wasn't specific for that what we expected. No products could be amplified on the other two.

Saturday July 14th

Sunday July 15th

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