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:
- 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
- Assembly of <partinfo>BBa_K863104</partinfo> and <partinfo>BBa_K863114</partinfo>:
- 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 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 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 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 NF/m, 12 hours.
- Fermentation of E. coli KRX withoud plasmid (fermenter: Infors) and with BBa_K863000 (fermenter: Braun Biostat)
Friday September 14th
- Team Cellulose Binding Domain:
- Isolated three glowing dishes of KRX with the const.GFP_His-plasmid, three with the CBDclos_Freiburg-plasmid and 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 BBa_K863012.
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) 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.
- KRX culture of CBDcex(T7)-GFP_His seems to have a green glow.
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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