Team:Bielefeld-Germany/Labjournal/week17

Week 17 (08/20 - 08/26/12)

Monday August 20th

• Team Site Directed Mutagenesis:
  • Plasmid-isolation and test-digestion (with NotI) of the three tvel10-colonies showed two plasmids with the right insert-size and one wrong.
• Team Cellulose Binding Domain:

• CBD: Colony-PCR of 24 Assembly-colonies - all colonies only have CBDclos. Assembly didn't work at all...

• Team Bacterial Laccases:

• Digestion of pSB1C3 plasmid backbone, tthl_HIS and bpul_HIS for doing the assembly with the new t7 promoter and J23110 again.
• We did PCRs on bpul_HIS, bhal_HIS and ecol_HIS again.

Tuesday August 21st

• Team Cellulose Binding Domain: Restriction of pSB1C3+CBDclos with EcoRI+AgeI-HF in Buffer 4 + BSA to test the restriction enzymes - works
• Team Activity Tests: I know, all of you are expecting us to report about some laccase activity now, but today is going to be different. Today we started with a special task: a q-PCR. As written in one of our last labjournal entries we have discussed a lot and searched for reasons why our laccases are not active. We decided that one possible step to find the mistake is to analyze the transcript. The level of mRNA will show us if our plasmid is expressed at all or if there might be something wrong with it. Team Cultivation cultivated small samples of E.coli KRX with plasmids of laccases from the following organisms:Escherichia coli, Bacillus pumilus, Bacillus halodurans C-125, Xanthomonas campestris pv. campestris B100 and Thermus thermophilus. As controls a sample with a [http://partsregistry.org/wiki/index.php/Part:BBa_K525710 ligase a] plasmid (to ensure the induction works) and E. coli KRX (without any plasmid) were used. So our first step today was freezing some cell pellets for a RNA isolation and ordering primers. We will go on with our new mission as soon as the primers arrive.

• Team Bacterial Laccases:
  • We designed primers for q-PCR for Team Activity Tests (details, look above). The PCRs with this primer pairs should result in about 200 bases long fragments.
  • We realized that the primer pairs we anneal for the promoter parts had about 2000 ng/µl if diluted 1:10 from originally 100 pmol. Maybe our used amounts on promoter for the assemblys was a LITTLE to high. So now we try a dilution of 1:1000 with about 2 ng/µl.
  • PCRs of different laccases were purificated and digested for suffix insertion.
  • Ligation of the laccase genes ecol, ecol_HIS and bpul and bpul_HIS, the 1:000 diluted (0,1 pm/µl) promoters (pT7 and P110) in pSB1C3 vector.
  • Ligation of the digested ecol_pSB1C3 and ecol_HIS_pSB1C3 plasmids with the 1:000 diluted (0,1 pm/µl) promoters (pT7 and P110).
  • Cleanup of the PCRs from day before and digest with XbaI and PstI for the assembly with promoter and pSB1C3.
• Team Fungal Laccases:
  • We designed primers for the laccase BBa_K500002 for cloning in P. pastoris shuttle vector.

Wednesday August 22nd

• Street Science:

• Went to Dr. Joe Max Risse today and asked him about the mircoorganisms of the Fermentationgroup. He recommended Euglena gracilis, since it is without risk, big, colorful, and very healthy under the microscop.
• I asked for the bacteria they have and he told me I could check if their Kocuria rosea is a wild type.
• In the DMSZ catalog there are three strains of Kocuria rosea and all have been assessed to be of riskgroup 1, which is the safest group of bacteria and means it is unlikely that it will infect humans.
• I also asked for Penicillium chrysogenum but the one they use is a GVO.

• Team Site Directed Mutagenesis:

• Ordered new primers for Xantomonas Campestris SDM, since there is still no positive colony and even gradient-PCR gave the wrong product

• Team Cellulose Binding Domain:

• Test-restriction of all isolated CBDcex- and the GFP_Freiburg-plasmids with NotI - no positive result

Started new and made a gradient PCR for GFP-Freiburg and CBDcex (50-70°C)

• Team Cultivation & Purification:
  • Made the SDS-Pages of the cultivation on 08/15.
• Team Bacterial Laccases: Colony PCRs: some prefix insertions of promoters in pSB1C3 with ecol and ecol_HIS showed positive bands, so we picked the colonies for plasmid isolation. The problem with the prefix insertion of the promoters in pSB1C3 with the ecol gene is that we can’t easily prove that the promoters were ligated in the backbone because with control restriction there would be an about 50 bp longer fragment which is difficult to see even in 3 % agarose gel. For this reason we designed FW primers (J23103_K_FW and J23110/117_K_FW and T7_K_FW) for our colony PCR, which can just bind, if the new promoter sequence is included in the plasmid. Until all colonies from the promoter J23110 ligation were red, we had some white colonies for the pT7 promoter in pSB1C3 backbone. So we picked a positive colony and plated it for plasmid isolation.

Thursday August 23rd

• Team Bacterial Laccases: Plasmid isolation and sending plasmids for sequencing.
• Team Fungal Laccases: We did PCRs on tvel35 plasmid with primer pairs Pc_lac35.P.FW / Pc_lac35.S.RV for trying ligation in pSB1C3 again and with Pc_lac35_FW_oS / Pc_lac35_RV.

Friday August 24th

Saturday August 25th

STREET SCIENCE Visit of Danish guys till 08/27

Sunday August 26th

• Team Cultivation & Purification:
  • Made precultures of E.coli KRX without plasmid and with plasmids containing laccases from B.halodurans, B.pumilus, E.coli, T.thermophilus or X.campestris. As postive control we used [http://partsregistry.org/Part:BBa_K525710 BBa_K525710 (Ligase A)].

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