Team:Bielefeld-Germany/Labjournal/week20

Week 20 (09/10 - 09/16/12)

Monday September 10th

• Team Cultivation & Purification:
  • Cell disruption via high-pressure homogeniser and purification via Ni-NTA column were performed for the following samples: 3L fermentation of E.coli KRX with laccase from B.pumilus (09/09), 6L fermentation of E.coli KRX with laccase from E.coli (09/07) and B.pumilus (09/09).
  • We made SDS-Pages of purification of laccase from B.pumilus
• Team Site Directed Mutagenesis:
  • Digestion of the six tvel10-plasmids. Three were unmutated and other three hadn't lost the illegal SpeI-restriction-site, but their second fragment was of a smaller size.
  • plated three additional tvel-t243g-colonies.

Tuesday September 11th

• Team Shuttle Vector: Digest of shuttle shuttle vector with PvuII and HindIII as control. Agarose gel looks good.
• Fungal Laccases: PCR on tvel5 laccase for cloning in shuttle vector. Digest of shuttle vector and digest of tvel35 with AarI enzyme.

Wednesday September 12th

• Team Site Directed Mutagenesis:
  • Plasmid-isolation of the three tvel10-plasmids and digestion with SpeI showed two unmutated plasmids and one with the same wrong restriction-fragments as Monday. There must be a systematical error. pfu-PCR should be done again.

Thursday September 13th

Friday September 14th

• Team Cellulose Binding Domain:
  • Isolated three glowing colonies with the constitutive GFP_His, three with ClosF 36-38 and Cex 43, 45, 46

Saturday September 15th

• Team Cellulose Binding Domain:
  • KRX culture of CBDcex(T7)-GFP_His seems to have a green glow.
  • Isolation of four pellets of CBDcex(T7)-GFP_His for us and SDU Denmark.
  • CBDclos(T7) cut with SpeI+AgeI and deposphorylated.
  • GFP_Freiburg (PCR) Gel-clean cut with SpeI and NgoMIV.
  • Ligated and plated on select-Agar.
  • Restriktionanalysis showed that all GFP_His plasmids are correct, as are the three CBDclos and two of the CBDcex
  • Collect data to make a protocol for a Cellulose binding assay:
    • Avicel: about 11,4 mg protein (CBD) binds to 1 g Avicel (0,14 mg/12,3 mg)
    • Duration of incubation for CBD to bind to Avicel: about 30 minutes
    • Washing and Lysis-buffer: 50mM Tris-HCl (pH8.0)
    • If needed: elution with 80% EG or 1/5 Pellet to 4/5 EG (100%) of the overall volume.

Sunday September 16th

• Team Cellulose Binding Domain:
  • Colony-PCR of 15 colonies from the CBDclos(t7)+GFP_His transformation-plate (biotaq - Armins recipe) with the result of the positive clones.
  • Prepared the sequencing of GFP_Freiburg 6-8 (1-3) CBDclos 1-3 (36-38) and CBDcex 1-2 (43, 45)
  • CBDcex(T7)+GFP_His isn't glowing anymore, or never was.

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