Team:Bielefeld-Germany/Labjournal/week2

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 xccl from Xanthomonas campestris pv. campestris B100 and ecol 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 screen and characterize laccases from different bacteria we had to order the bacterial strains which weren't available at Bielefeld University from [http://www.dsmz.de/|DSMZ]. Below is a list of the ordered strains and the laccases we want to isolate from these 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] (look [http://www.ncbi.nlm.nih.gov/pubmed/15999224 here] for a publication to this laccase)

• [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] (look [http://www.ncbi.nlm.nih.gov/pubmed/15293032 here] for a publication to this laccase)

• 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. [http://www.ncbi.nlm.nih.gov/pubmed/14586105 publication]

• We wanted the laccase [http://www.ncbi.nlm.nih.gov/protein/182434812 EpoA] from Streptomyces griseus IFO 13350 (for the publication look [http://jb.oxfordjournals.org/content/133/5/671.full.pdf here]. 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 bpul from Bacillus pumilus ATCC7061 as PCR product with the desired overhanging ends. As template we used the plasmid we got from the Swiss working group.

Wednesday May 9th

• Team Activity Test: From the information we collected during our literature research we created a protocol for our first experiments. We decided to check the activity via a photometer. The one we may use here at the Cebitec is a Tecan Microplate reader. Check protocols for further information. If oxidized by laccase, ABTS can me measured at 420 nm. Also we found out that sodium acetate buffer (100 mM / pH 5) would give an optimal environment to our enzyme. So let´s have a look at our protocol:

Initial laccase activity test:

100 mM sodium acetate buffer, pH 5.0

5 mM ABTS

8 U laccase

ad 200 µL deionized H₂0

Also we talked about further characterization after accomplishing the first experiments and confirming that the used concentrations are a good choice. We are planning to buy and characterize the laccase from T.versicolor (TVEL0), to have a comparison to our future recombinant laccases. That laccase we are going to analyze in sodium acetate buffers that are adjusted to pH 1, 3, 5, 7 and 9. Further we are going to analyze the effect of different temperatures on the enzymes activity. For that we will first do some more research on the temperatures of the waste water in clarification plants here in Germany. Also we found out that an addition of copper does enhance the laccases activity, so we are going to do some measurements with copper concentrations from 0.1 mM to 0.5 mM in each sample. This seems like some great experiments for the start, so next we are going to order what we need to do the measurements.

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
  • We got the ordered strains from [http://www.dsmz.de/ DSMZ]. So we did 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

• Team Activity Tests: For some pre test and characterization for our future laccase activity standard we ordered [http://www.sigmaaldrich.com/catalog/product/sigma/53739?lang=de&region=DE laccase] from Trametes versicolor. As well we had to order a substrate that the laccase could use to demonstrate its abilities. According to the literature [http://www.sigmaaldrich.com/catalog/product/sigma/a1888?lang=de&region=DE ABTS] is a well working substrate to characterize oxidizing enzym activity. So we ordered.

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