Labjournal

Week 12 (07/16 - 07/22/12)

Team A. thaliana laccase: Our plants had a great time during the last weeks in the climate chamber. So today it was time for them to donate their seeds for RNA isolation, cDNA synthesis and a PCR (check protocols). We ran an additional sample with actin primers as a positive control. However both samples did not show any bands. Maybe the high salt concentration in our sample was responsible or the laccase concentration in the 1:10 deluted cDNA was too low. We will do some washing and try again.

Team Cultivation/Purification: Second cultivation
Team Site Directed Mutagenesis: Made Clone Manager files for Trametes versicolor sequences

Team Site Directed Mutagenesis: Made Primer-Mixes. Got everything ready for Lab work.

Team Site Directed Mutagenesis: PCR and DpnI-restriktion of the SDM to mutate the B.pumilus-Laccase-Plasmid at 2883, the E.coli-Plasmid at 2307, the X.campestris at 2247 and T.thermophilus-Plasmid at 2796.
- Generated SDM-Primer for the Trametes versicolor Laccase 10 gene (2 mutations - one illegal restriktion site in the signaling-sequence will not be mutated).
Team Cellulose Binding Domain: Got info about Carbohydrate binding domain X2 (which is a more common domain in the organisms we handle than a Cellulose Binding Domain.)
- Nucleotid BLASTed BBa_K392014 (Cellulose-binding motif from C. josui Xyn10A gene) -> 1526bp to 2608bp of AB041993.1 (Clostridium josui xynA gene for xylanase A)
- Portein-BLASTed AB041993.1 and found got one cellulose binding site and one carbohydrate binding domain.:
  - pfam02018: CBM_4_9 (Carbohydrate binding domain) from AS 193 to AS 332 - corresponds to 1088 - 1507 in Gene and is not in the coding sequence of BBa_K392014
  - cd09619: CBM9_like_4 (DOMON-like type 9 carbohydrate binding module) from AS 716 to AS 887 - corresponds to 2657bp to 3172bp in the gene and is also not in coding sequence of BBa_K392014
  - Given sequence of BBa_K392014 (1589bp to 2590bp) does corresponds to the sequence of the Glycosyl hydrolase 1589bp to 2590bp and not one of the binding motifs

Team Modeling: We need a contact to a clarification plant to get information about clarification plant itself and perhaps to proof our cleaner with real probes. therefor we are calling Mr. Bülter form the clarification plant Schloß Holte (near Bielefeld) and he invited us to present our projekt.