Team:Bielefeld-Germany/Labjournal/week6

Week 6 (06/04 - 06/10/12)

Monday June 4th

• Team Cloning of Bacterial Laccases:

• 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

• • PCR program

Cycle between step 2 and 4 35 times.

• Team Fungal and Plant Laccases:
  • 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.

Tuesday June 5th

• Team Student Academy:
  • Transformation of a plasmid mixture of either pMTE cp46 His and [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] or pMTE cp46 His and [http://partsregistry.org/Part:BBa_J23100 BBa_J23100]. We plated both on LB agar without antibiotics and with Kanamycin. The first one was also plated on LB agar with ampicillin and the second on LB agar with chloramphenicol. Result: Works as expected. [http://partsregistry.org/Part:BBa_J23100 BBa_J23100] has a more intense fluorescence and was chosen for the experiment.

• Team Cloning of Bacterial Laccases:
  • The sequencing results for isolated plasmids xccl(T7)_His, bpul(T7)_His and ecol(T7)_His came. The results showed that only the xccl(T7)_His was ok – our first finished biobrick *yeha*. We proudly name it <partinfo>BBa_K863015</partinfo>. The sequence of 'ecol(T7)_His showed that there are missing 4 bases in the promoter region and the bpul(T7)_His sequence showed a mutation which leads to another amino acid in protein sequence.
  • Again we did PCRs on 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,this time with enough material for a restriction.

Activity measurements of the bought laccase from T.versiolor analyzed through the OD₄₂₀ of oxidized ABTS in sodium acetate buffer at different pHs depending on time. Values are calculated by taking the average and standard deviation out of 4 measurements (n=4).

• 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 sodium acetate buffer at pH 1, 3, 7 an 9. Result: We agree with the literature that pH 5 seems 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 lower temperaturers. Stay tuned.

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 Cloning of Bacterial Laccases: Digest of tthl(T7)_His and bhal(T7)_His PCR products and ligation in pSB1C3 backbone. After that we transformed the plasmids in competent E. coli KRX cells.

Thursday June 7th

• Team Student Academy:
  • Repeating of Transformation of 06/05 to verify the function. It is reproducible :)

• Team Cloning of Bacterial Laccases: Colony PCRs of the transformed colonies from yesterday's transformation showed some positive PCR bands.

• Team Modeling: becoming acquainted with matlab while reading the manual

Friday June 8th

• Team Cloning of 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 height for the Tthl(T7)_His and with bhal(T7)_His.

Saturday June 9th

Sunday June 10th

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