Team:Bielefeld-Germany/Labjournal/week20

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

Monday September 10th

• 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.

• Team Cellulose Binding Domain:
  • Sequencing results arrived: One CBDcex(T7) is completely right!
  • Nanodropping plasmids of isolated CBDclos(T7)+GFP_His showed that the cells did not have a plasmid at all (selection-agar did not work)
  • Gradient-PCR with CBDclos(T7) as template and CBDclos_Freiburg-primers did work just fine (best temperature 61,7°C); Digestion with XbaI and AgeI.
  • Gel-Clean-up of CBDcex_Freiburg and GFP_His
  • Digestion of CBDcex_Freiburg with XbaI+AgeI.
  • Digestion of GFP_His with NgoMIV and PstI.

• 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 BBa_K863000 (09/09), 6L fermentation of E.coli KRX with BBa_K863005 (09/07) and BBa_K863000 (09/09).
  • We made SDS-Pages of purification of ECOL.

Tuesday September 11th

• Fungal Laccases:
  • PCR on tvel5 laccase for cloning in shuttle vector. Digest of shuttle vector and digest of tvel35 with AarI enzyme.

• Team Shuttle Vector:
  • Digest of shuttle shuttle vector with PvuII and HindIII as control. Agarose gel looks good.

• Team Cellulose Binding Domain:
  • Assembly of <partinfo>BBa_K863104</partinfo> and <partinfo>BBa_K863114</partinfo>:
    • Assembled CBDclos_Freiburg and CBDcex_Freiburg with GFP_His and <partinfo>J61101</partinfo> and plated the ligation on AMP-selection-agar (because of the pSB1A2-backbone).
  • Restriktion of CBDclos_Freiburg and CBDcex_Freiburg with EcoRI and PstI.
    • Ligation of CBDcex_Freiburg and CBDclos_Freiburg with the pSB1C3-backbone and transformated and plated on selection-agar

• Team Cultivation & Purification:
  • Made precultures of E.coli KRX with BBa_K863000 and BBa_K863005 as well as of E.coli KRX.

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.
• Team Cultivation & Purification
  • Cell disruption via sonification and purification via Ni-NTA column were performed for the following samples: 200 mL cultivation of E.coli Rosetta Gami 2 containing BBa_K863012(09/09) and laccase from B.halodurans (09/09) behind a constitutive promotor.
  • SDS-Pages of the flask cultivation from 09/09 ( E.coli Rosetta Gami 2 containing BBa_K863012(09/09) and laccase from B.halodurans)

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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