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Team:Bielefeld-Germany/Labjournal/week6
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Revision as of 13:38, 13 September 2012
Contents |
Week 6 (06/04 - 06/10/12)
Monday June 4th
- Primerdesign for isolating a laccase from Arababidopsis thaliana cDNA. Since we want to express the laccase in E.coli we designed the primers like before for the bacterial laccases with T7 promotor and HIS-tag.
- Team bacterial laccase: The bacteria S. griseus and S. lavendulae has been delivered so we can start with PCRs. We set the first PCR with them as followed:
- PCR table
| Material | Volume |
|---|---|
| Buffer (10x Phusion) | 10µL |
| Phusion Polymerase | 0,5µL |
| dNTPs | 1µL |
| Primer Mix | 1µL |
| Template DNA | 1µL |
| DMSO | 1,5µL |
| Water | 35µL |
- PCR program
| Temperature | Time |
|---|---|
| 1) 98°C | 7 mins |
| 2) 98°C | 20 sec |
| 3) 55°C | 20 sec |
| 4) 72°C | 1 min |
| 5) 72°C | 3 min |
| 6) 12°C |
Cycle between step 2 and 4 35 times.
Tuesday June 5th
- Team Activity Tests: After testing the T. versicolor laccase under conditions that are optimal (pH 5, 25°C ) according to the literature we now started further characterization under different pHs. We analyzed the laccases behavior when working in 100 mM natrium acetate buffer at pH 1, 3, 7 an 9. Result: We agree with the literature that pH 5 and also pH 6 seem to make the laccase happy. Since not all waste waters (especially those here in Germany) are not as warm as 25°C we now wonder what our laccase might do when exposed to low temperature such as 4°C. Stay tuned.
- Team Bacterial Laccases:
- The sequencing results for isolated plasmids from Xanthomonas campestris CopA, B. pumilus CotA and E.coli CueO came. The results showed that only the sequence with CopA was ok – our first finished biobrick *yeha*. The sequence of E. coli showed that there are 4 bases in the promotor region missing and the CotA sequence showed a mutation which leads to another amino acid in protein sequence.
- Again we did PCRs from T. thermophilus laccase and B. halodurans laccase with B.halo_FW_T7 / B.halo_FW_HIS and T.thermo_LAC_FW_T7 / T.thermo_LAC_RV_HIS primers and purified the product but this time with enough material for a restriction.
Wednesday June 6th
- Team Wiki: Yay for Team Wiki´s first entry. Our first steps with the iGEM Bielefeld 2012 Wiki contain thinking about contents, layouts, programming and responsibilities. Our first rules are:
- we are programming static pages in HTML and all the other pages (those that will be updated by all team members) in wiki code.
- we created all pages and will fill them up with some nice and beautiful content constantly from now on.
- Our temporary banner contains our outstanding logo and a DNA but we will set up a new layout soon.
Thursday June 7th
- Team Modeling: becomeing acquainted with matlab while reading the manual
Friday June 8th
Saturday June 9th
Sunday June 10th
Sunday
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