Team:Bielefeld-Germany/Labjournal/week6
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- | '''Team Bacterial Laccases:''' | + | '''Team Bacterial Laccases:''' We plated colonies for plasmid isolations on new plates and made a control restriction with ''Not''I. The electrophoretic separation showed gel bands in the right hight for the Tth-plasmid and the plasmid with B. halodurans laccase. |
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===Saturday June 9th=== | ===Saturday June 9th=== |
Revision as of 17:58, 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.
- Team Bacterial Laccases: Digest of T. thermophilus laccase and B. halodurans laccase PCR products and ligation in pSB1C3 backbone. After that we transformed the plasmids in competent E. coli KRX cells.
Thursday June 7th
- Team Modeling: becoming acquainted with matlab while reading the manual
- Team Bacterial Laccases: Colony PCRs of the transformed colonies from yesterday's transformation showed some positive PCR bands.
Friday June 8th
Team Bacterial Laccases: We plated colonies for plasmid isolations on new plates and made a control restriction with NotI. The electrophoretic separation showed gel bands in the right hight for the Tth-plasmid and the plasmid with B. halodurans laccase.
Saturday June 9th
Sunday June 10th
Sunday
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