Team:Bielefeld-Germany/Labjournal/week6

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Contents

Labjournal

Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21


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 laccase:
    • 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