Team:Bielefeld-Germany/Labjournal/week20

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Contents

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:
  • Team Cultivation & Purification:
    • Cell disruption via high-pressure homogenizer and purification via Ni-NTA column were performed for the following samples: 3 L fermentation of E. coli KRX with BBa_K863000 (09/09), 6 L 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

  • Team Fungal and Plant 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>:
    • 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:

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 Cellulose Binding Domain:
    • There are only few colonies on all selection-agar-dishes, but none is obviously green fluoresensing, even with UV-light emission could not be stimulated.
    • Plated colonies of CBDcex_Freiburg and CBDclos_Freiburg to see if they are red or not.
    • Colony-PCR of CBDcex_Freiburg and CBDclos_Freiburg showed eight positive colonies to the <partinfo>K863104</partinfo>-insert. Plated the positive colonies for plasmid-isolation.
  • 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

  • Team Cellulose Binding Domain:
    • Designed a lot of Primers to cope with the expression problem. E.g. inserting a long S3N10-Linker via blunt-end-cloning between the CBD and the GFP, also primers to get rid of the His-tag on the GFP and a last set to easily change the order of CBD and GFP (via assembly but with no linker in between).
    • Plasmid-isolation of <partinfo>BBa_K863104</partinfo>-transformation clones
    • Colony-PCR of CBDcex_Freiburg and CBDclos_Freiburg colonies. All CBDclos_Freiburg-colonies are positive and half of the CBDcex_Freiburg-colonies.
    • Colony-PCR of CBDclos_F.+GFP with no positive result
    • Colony-PCR of const.GFP_His: 2 positive (one fluoreszend); Plated both positive and one additional fluorescend.
  • Team Cultivation & Purification:
    • Fermentation of E. coli KRX withoud plasmid (fermenter: Infors) and with BBa_K863000 (fermenter: Braun Biostat)
      • Settings: fermenter: Infors/Braun Biostat, final volume: 3 L, autoinduction medium, 60 µg/mL chloramphenicol added, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 2 NL/m, durance: 12 h.
    • Made preculture of E. coli KRX with BBa_K863103 (CBD-GFP-His).
    • Made preculture of P. pastoris GS115
    • Fermentation of E. coli KRX with BBa_K863000.
      • Settings: fermenter: Bioengineering NFL22(7 L), final volume: 6 L, autoinduction medium with 60 µg/mL chloramphenicol added, 37 °C, stirrer increased 2 % if the pO2 got below 30 %, airflow: 5 NL/m, 12 hours.

Friday September 14th

  • Team Cellulose Binding Domain:
  • Team Cultivation & Purification:
    • Cell disruption of fermentation 09/13 via high-pressure homogenizer and purification via Ni-NTA column. Made SDS-Pages of purificated fractions.
    • Repeat the preculture of P. pastoris GS115, because of using the wrong media.
    • Made preculure of E. coli Rosetta Gami 2 with BBa_K863012.

Saturday September 15th

  • Team Cellulose Binding Domain:
    • KRX culture of CBDcex(T7)-GFP_His seems to have a green glow.
    • CBDclos(T7) digested with SpeI and AgeI and deposphorylated.
    • GFP_His-PCR-product (gel-clean) digested with SpeI and NgoMIV.
    • Ligated CBDclos(T7) with GFP_His and plated on select-Agar.
    • Restriction-analysis showed that all const.GFP_His-plasmids are correct, as are the three CBDclos and two of the CBDcex
    • Collected 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% ethylen-glycol (EG) or 1/5 Pellet to 4/5 EG (100%) of the overall volume.
  • Team Cultivation & Purification:
    • Made competent P. pastoris GS115 cells.
    • Fermentation of E. coli KRX with BBa_K863012
      • Settings: fermenter: Bioengineering NFL22 (7 L), final volume: 6 L, LB-medium with 60 µg/mL chloramphenicol and 300 µg/mL ampicillin added, 37 °C, stirrer increased 2 % if the pO2 got below 30 %, airflow: 5 NL/m. Problem: stirrer cascade did not work at the beginning.
      • Harvest and centrifugation of cultivation & fermentations 09/14. Store pellet at 4 °C.

Sunday September 16th

  • Team Cultivation & Purification:
    • Harvest and centrifugation of fermentation of E. coli KRX with BBa_K863012. Store pellet at 4 °C.
    • Cell disruption via sonification and purification of cultivation of E. coli KRX with BBa_K863103 via Ni-NTA column.
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