Team:Bielefeld-Germany/Labjournal/week11

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

Monday July 9th

• Ordering primers for isolation of fungal laccases from plasmids, we got from the Ernst-Moritz-Arndt-University in Greifswald. The plasmids contain the cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus.

• lac5 from Trametes versicolor
• lac10 from Trametes versicolor
• lac13 from Trametes versicolor
• lac20 from Trametes versicolor
• lac35 from Pycnoporus cinnabarinus

• The first primerpairs were designed with standard prefix and suffix sequence and 20 bases complementary to the start and end of the ORF sequences.

• Additional primerpairs 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 desigend with the last 20 bases of the lacase genes and a terminator overhaning end and a AarI restriction site.

Problem: We forgot to delete the signal peptid sequences, which are present in the fungal laccases. Solution: Ordering the forward primers again with the first 20 bases after the signal peptides.

• 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.

Tuesday July 10th

• Team Site Directed Mutagenesis: Generated the first Site-Directed-Mutagenesis primer for Bacillus pumilus (two sites), Xanthomonas Campestris (two sites), Escherichia coli (one site)und Thermus thermophilus (one site).
• 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 Bacterial Laccases: We got the sequences for five different fungal laccases from the requested plasmids on Monday 2nd and they wanted to send us plasmids with the laccase ORFs on it. The laccases are Tv5, Tv10, Tv13 and Tv20 from Trametes versicolor and Pc35 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.

Wednesday July 11th

• Team Cellulose Binding Domain: Looked up the sequence of Clostridium cellulovorans cellulose binding protein gene (cbp A) http://www.ncbi.nlm.nih.gov/nuccore/M73817 made a Clonemanagerfile and protein-BLASTed the sequence to find out the domain of the Cellulose-Binding-Domain: 103bp-378bp
• Team Student Academy: The experiments were performed by the second half of the pupils and the groups from yesterday analyzed their plates.
• Team 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 Bacterial Laccase: 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. tue and S.goe. Thoose Laccase genes showed simularity to our Laccase genes. Since he had isolated chromosomol DNA we were able to work with them. We set the PCR with the three Streptomyces. On this PCR S. tue 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

Sunday

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