Team:Bielefeld-Germany/Labjournal/week13

Week 13 (07/23 - 07/29/12)

• From 07/23 - 07/25/12, some of our team members participated at the CAS conference in Munich.

Monday July 23rd

• Team Bacterial Laccase: The transformation from the 21st July showed no results.
• Team Fungal Laccases: Successful PCRs of laccase gene Tv5 from Trametes versicolor with plasmid DNA from Uni Greifswald as template.
• Team Site Directed Mutagenesis: Transfered 4 colonies per petri dish to an new one for plasmid isolation.

Tuesday July 24th

• Team Activity Tests: Today was the moment of truth. We received the first recombinant laccases from the cultivation team. We started with testing the supernatant from the cultivated cells to check whether they secrete laccase. We added 0,1 mM ABTS in each well that was filled with supernatant from Escherichia coli, Bacillus pumilus, Bacillus halodurans C-125, Xanthomonas campestris pv. campestris B100 and E. coli KRX (negative control - cells without plasmid) and measured as usual. There was no change in the OD so that we assume that no secretion takes place. Afterwards we tested potential laccase activity by filling each well with laccase and buffer (check protocols to see which buffer we used) and added 0,1 mM ABTS. Unfortunately no activity was seen. We will try again soon.
• Team Site Directed Mutagenesis: Plasmidisolation of B.pumilus 2883(I-IV); X.campestris 2247(I-IV); T.thermophilus 2796(I-IV) and E.coli 2307 (III).

Wednesday July 25th

• Team Site Directed Mutagenesis: Test restriction of the Plasmids revealed that B.pumilus III;IV; E.coli III;T.thermophilus.I;II seem to be mutated correctly but X.campestris is untouched (no Band at 3636).
  • SDM-PCR with B.pumilus III(III)+IV(IV) as template using the SDM-primermix to mutate bp 2317
  • SDM-PCR with X.campestris with the 2247-primermixand another one with the primermix to mutate 3633
  • Prepared test restriction-positives for Sequencing

Thursday July 26th

• Team Modeling and Team Sponsoring: meeting Mr. ??? from the clarification plant of Schloß Holte and finding a new cooperation partner. He want to give us the information we need to equate our model and design our cleaner. On top he wants to ask if the clarification plant can sponsor our project.
• Team Site Directed Mutagenesis: Went on with the PCR products and transformed them into XL1 Blue
• Team Fungal Laccases: We did PCR on Trametes versicolor laccase Tv5 and Pycnoporus cinnabarinus Pc35 with Tv_lac10.P.FW / Tv_lac10.S.RV and Pc_lac35.P.FW / Pc_lac35.S.RV primers. Additionally we want the laccases with overhanging ends for cloning in our shuttle vector for expression in Pichia pastoris. Therefore we used Pc_lac35_FW_oS / Pc_lac35_RV and Tv_lac5_FW_oS / Tv_lac5_RV primer pairs.
• Team Cultivation & Purification:
  • Glycerine cultures were made of all given BioBricks, to use them for Precultures.
  • Found out, that it could be important to add CuCl2 to the medium, because it is important for the active center of laccases. Another idea could be the incubation before measuring the activity.

Friday July 27th

• Team Site Directed Mutagenesis: picked colonies and plated them for plasmidisolation.
• Team Cellulose Binding Domain: After a chat with teammates made the decision to only use Cellolose binding domains and not carbohydrate binding domains to keep it comparable. This means we won't use the binding domains of Bacillus halodurans.
  • Redesigned Primers for CBDclos - added the bases TA in between RBS and ATG since the RBS can be made stronger by adding more adenines in the sequence upstream of the RBS
  • Made similar primers for CBDcex (the CBD of the Cellulomonas fimi exoglucanase we got for the fermentationgroup)
  • Checked the Osaka_2010 page about their BBa_K392014 BioBrick which should be a binding motif but is the Glycosyl hydrolase domain. I guess them messed it up...
• Team Fungal Laccases: The PCR on Tv5 laccase was positive after the first trial for both primer pairs. For the Tv35 laccase the PCR didn’t work.
• Team Cultivation & Purification:
  • We cultivated E.coli KRX with laccases from E.coli, B.halorudans, X.campestris and B.pumilus as well as without plasmid. -->Settings: 300mL flasks without baffles, final volume: 60mL, autoinduction medium with added 20µg/mL chloramphenicol in case of the first three laccases,with 100µg/mL ampicilline in case of B.pumilus, and without antibiotics for E.coli KRX without plasmid. Furthermore 0,25mM CuCl2 was added. Cultivation took place at 30°C.
  • We made glycerine cultures of E.coli KRX with correct plasmid for B.pumilus laccase.

Saturday July 28th

• Team Activity Tests: See, we are back already! Today we received different samples from Escherichia coli, Bacillus pumilus, Bacillus halodurans C-125, Thermus thermophilus, Xanthomonas campestris pv. campestris B100 and E. coli KRX (negative control - cells without plasmid). The cells were all cultivated at 30°C and supplied with copper. We tested each one of them but none of them showed laccase activity.
• Team Fungal Laccases: Purification of Trametes versicolor Tv5 laccase.
• Team Cultivation & Purification:
  • Cells of cultivation 07/28 were disrupted via sonification and given to the activity test team.

Sunday July 29th

Sunday

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