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 [http://partsregistry.org/Part:BBa_K863102 CBDcex(T7)] is completely right!
  • Nanodropping plasmids of isolated [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His] showed that the cells did not have a plasmid at all (selection-agar did not work)
  • Gradient-PCR with [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] as template and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg]-primers did work just fine (best temperature 61,7°C); Digestion with XbaI and AgeI.
  • Gel-Clean-up of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863121 GFP_His]
  • Digestion of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] with XbaI+AgeI.
  • Digestion of [http://partsregistry.org/Part:BBa_K863121 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] (09/09), 6L fermentation of E.coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 BBa_K863005] (09/07) and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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 [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] with [http://partsregistry.org/Part:BBa_K863121 GFP_His] and <partinfo>J61101</partinfo> and plated the ligation on AMP-selection-agar (because of the pSB1A2-backbone).
  • Restriktion of [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] and [http://partsregistry.org/Part:BBa_K863101 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863005 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 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 [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] to see if they are red or not.
  • Colony-PCR of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863111 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 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 S2N10 Linker between the CBD and the GFP, also getting rid of the His-tag on the GFP to easily change the order of CBD and GFP.
  • Plasmid-isolation of <partinfo>BBa_K863104</partinfo>-transformation clones
  • Colony-PCR of [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg] and [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] colonies. All [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg]-colonies are positive and half of the [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg]-colonies.
  • Colony-PCR of CBDclos_F.+GFP with no positive result
  • Colony-PCR of [http://partsregistry.org/Part:BBa_K863122 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863103 BBa_K863103] (CBD-GFP-His).
  • Made preculture of P. pastoris GS115
  • Fermentation of E. coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 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 NF/m, 12 hours.

Friday September 14th

• Team Cellulose Binding Domain:
  • Isolated three glowing dishes of KRX with the [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]-plasmid, three with the [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg]-plasmid and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg]-plasmid.
• 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012].

Saturday September 15th

• Team Cellulose Binding Domain:
  • KRX culture of [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)-GFP_His] seems to have a green glow.
    • Isolation of four pellets of [http://partsregistry.org/Part:BBa_K863103 CBDcex(T7)-GFP_His] for us and SDU Denmark.
  • [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] digested with SpeI and AgeI and deposphorylated.
  • [http://partsregistry.org/Part:BBa_K863121 GFP_His]-PCR-product (gel-clean) digested with SpeI and NgoMIV.
  • Ligated [http://partsregistry.org/Part:BBa_K863112 CBDclos(T7)] with [http://partsregistry.org/Part:BBa_K863121 GFP_His] and plated on select-Agar.
  • Restriction-analysis showed that all [http://partsregistry.org/Part:BBa_K863122 const.GFP_His]-plasmids are correct, as are the three [http://partsregistry.org/Part:BBa_K863111 CBDclos] and two of the [http://partsregistry.org/Part:BBa_K863101 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.

Sunday September 16th

• Team Cellulose Binding Domain:
  • Colony-PCR of 15 colonies from the [http://partsregistry.org/Part:BBa_K863113 CBDclos(T7)+GFP_His] transformation-plate (biotaq - Armin Nestat's recipe) with the result of a lot of positive clones.
  • Prepared the sequencing of three [http://partsregistry.org/Part:BBa_K863122 const.GFP_His], three [http://partsregistry.org/Part:BBa_K863111 CBDclos_Freiburg] and [http://partsregistry.org/Part:BBa_K863101 CBDcex_Freiburg]
  • Lysis of [http://partsregistry.org/Part:BBa_K863113 CBDcex(T7)+GFP_His] isn't glowing anymore, or never was.

• Team Cultivation & Purification:
  • Harvest and centrifugation of fermentation of E. coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863012 BBa_K863012]. Store pellet at 4 °C.
  • Purification of cultivation of E. coli KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863103 BBa_K863103] via Ni-NTA column.

55px