Team:Bielefeld-Germany/Labjournal/week6

From 2012.igem.org


Contents

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

weekly seminar

  • As part of the sponsoring with Merck we will present our project in Darmstadt. Kevin, Nadine and Gabi travel to Darmstadt on 21th of june.

The team will gather on 14th, 15th and 18th of june to practice the presentation.

  • Participants at the CAS conference for Synthetic Biology: Hakan, Derya, Miriam, Julia S., Gabi, Malak, Timo, Robert, Isabel, Nils.
  • Robert is responsible for ordering the following BioBricks from the Partsregistry:

BBa_K500000
BBa_K500001
BBa_K500002
BBa_K500003
BBa_K392014

  • The treaty with our sponsor BioCircle is signed.
  • All teams presented their lab results.

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
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.

  • 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 BBa_J04450 or pMTE cp46 His and 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. 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 BBa_K863015. 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

Promega logo 180x109.gif Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg