Labjournal

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Week 2 (05/07 - 05/13/12)

weekly seminar:

• Found our first sponsors: [http://corporate.evonik.com/en/Pages/default.aspx Evonik], [http://www.biocircle.com/en-ca/ BioCircle] and [http://www.merckgroup.com/en/index.html Merck], now treaties have to be created and signed
• Julia is working on the database
• Decision to organize waver sell to fill up our petty cash
• Gabi and Isabel are designing a poster for the waver sell
• For our human practices we wanted to find a sociology student, willing to think about bioethics, but failed
• Our video is nearly done, is cutted and only needs be underlain with music

• Team Modeling: Looking for suitable Software and enzymkinetics to model the degradation of our substrates with the different laccases. Finding the Michaelis-Menten kinetics and matlab.

Monday May 7th

• Team Student Academy: First transformation of [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522] and plating on selective agar.
  • Electroporation setup: U= 2,5 kV C= 25 µF and R= 400 Ω

• Result: We got little colonies. There weren’t any green colonies and only some pale red fluorescent colonies.

Team Bacterial Laccases:

• More PCRs of laccase genes CopA from Xanthomonas campestris pv. campestris B100 and CueO from E. coli BL21(DE3) with the isolated genomic DNA as template and Xcc_LAC_FW_T7 / Xcc_LAC_RV_HIS and E.coli_LAC_FW_T7 / E.coli_LAC_RV_HIS primer pairs.
• Since we wanted to characterize laccases from different bacteria we had to order the bacterial strains which weren't available at the University Bielefeld from [http://www.dsmz.de/|DSMZ]. Below is a list of the ordered strains and the laccases we want to isolate from this strains.

• [http://www.ncbi.nlm.nih.gov/protein/46197298 Laccase] from [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Thermus thermophilus HB27]

• [http://www.ncbi.nlm.nih.gov/protein/10174701 BH2082] from [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Bacillus halodurans C-125]

• We ordered [http://www.dsmz.de/catalogues/details/culture/DSM-40069.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|S. lavendulae sp. lavendulae ATCC 14158]. Originally we wanted the strain Streptomyces lavendulae REN-7 but this strain isn't available at DSMZ. So we now hope that the laccase gene [http://www.ncbi.nlm.nih.gov/nuccore/23491745 STSL] from Streptomyces lavendulae REN-7 is similar to that from S. lavendulae sp. lavendulae ATCC 14158 because there's no DNA sequence for the laccase from this strain available.

• We wanted the laccase [http://www.ncbi.nlm.nih.gov/protein/182434812 EpoA] from Streptomyces griseus IFO 13350. This strain was not available so we ordered [http://www.dsmz.de/catalogues/details/culture/DSM-40236.html?tx_dsmzresources_pi5%5BreturnPid%5D=304| Streptomyces griseus ATCC 10137]. Unfortunately for this strain are no blast results after blasting the [http://www.ncbi.nlm.nih.gov/protein/182434812 laccase] from Streptomyces griseus IFO 13350 against database. So we decided to make primers for the laccase sequence from Streptomyces griseus IFO 13350 in the hope that the sequences are similar enough to get a PCR product.

Tuesday May 8th

• Team Student Academy: Repetition of the transformation didn’t change the result. We made a liquid culture of [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], but it did not fluoresce. Searching for mistakes and alternatives. Maybe competent cells are not that good and in case of RFP the lacI sensitivity could be the problem.
• Team Bacterial Laccases: After some empty agarose gels we finally isolated the laccase gene CotA from Bacillus pumilus ATCC7061 as PCR product with the desired overhangig end. As template we used the plasmid we have got from the Swiss working group.

Wednesday May 9th

Thursday May 10th

• Team Student Academy Testing the competent cells by transformation of pUC19. The transformation did not work that good, so that we produced new ones.

• Team Bacterial Laccases PCR on Thermus thermophilus genomic DNA. First we dissolved some of the lyophilized powder in water and for opening the cells we boiled them for a few minutes. The primers we used were T.thermo_LAC_FW_T7 and T.thermo_LAC_RV_HIS to get the laccase with the same overhangs described in Monday April 30th. Finally with additional DMSO and GC-buffer we had a product of the GC-rich laccase.

Friday May 11th