Team:Bielefeld-Germany/Labjournal/week20

From 2012.igem.org

(Difference between revisions)
(Week 20 (09/10 - 09/16/12))
(Friday September 14th)
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* '''Team Cellulose Binding Domain:'''
* '''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.
** 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.
 +
[[File:Bielefeld2012_Bild1_0914.jpg|400px|thumb|right|'''Figure 1: Fermentation from [https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week20#Thursday_September_13th 09/13]''' of ''E.coli'' KRX with [http://partsregistry.org/wiki/index.php?title=Part:BBa_K863000 BBa_K863000],
 +
6L in NFL22, [https://2012.igem.org/Team:Bielefeld-Germany/Protocols/Materials#Autoinduction_medium autoinduction medium], 37 °C, pO<sub>2</sub> 50 % for 12 hours.]]
* '''Team Cultivation & Purification: '''
* '''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.
** Cell disruption of fermentation 09/13 via high-pressure homogenizer and purification via Ni-NTA column. Made SDS-Pages of purificated fractions.

Revision as of 17:59, 24 September 2012


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:
Figure 1: Fermentation from 09/07 of E. coli KRX containing BBa_K863005, 6 L in NFL22, autoinduction medium, 37 °C, pO2 30 % for 12 hours
  • 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 Shuttle Vector:
    • Digest of shuttle shuttle vector with PvuII and HindIII as control. Agarose gel looks good.
  • 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, ligation and transformation in E. coli KRX cells.
  • Team Cellulose Binding Domain:
    • Assembly of <partinfo>BBa_K863104</partinfo> and <partinfo>BBa_K863114</partinfo>:
    • Restriction 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 Fungal and Plant Laccases:
    • Ligation of shuttle vector with tvel5 (digested with AarI).
  • 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 fluorescing, 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 <partinfo>BBa_K863022</partinfo> (09/09) behind a constitutive promoter.
    • SDS-Pages of the flask cultivation from 09/09 ( E. coli Rosetta Gami 2 containing BBa_K863012(09/09) and <partinfo>BBa_K863022</partinfo>)

Thursday September 13th

  • Team Fungal Laccases and Team Shuttle Vector:
    • Bad day for us. We realized that our primers we used for amplification of the laccase genes for cloning in the shuttle vector don't have overhanging ends which were needed for an optimal activity of the enzyme AarI. So we had to order new primers with some bases prior to the recognition sites. But after there is little time left until wiki freeze and the new primers need some time until they are here we started a last trial for cloning this 'wrong' PCR products in the shuttle vector. The idea was to phosphorylate and ligate the ends of the PCR product from tvel5 together. Then the problem with the missing bases at the end would be solved and AarI can digest the end of the PCR product. We digested the shuttle vector with AarI and dephosphorylated it. After ligation we transformed the approaches in E. coli KRX cells.
  • 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 fluorescend); Plated both positive and one additional fluorescend.
  • Team Cultivation & Purification:
    • Fermentation of E. coli KRX without 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 Fungal Laccases and Team Shuttle Vector:
    • Waiting for colonies from our transformation the day before.
  • Team Cellulose Binding Domain:
File:Bielefeld2012 Bild1 0914.jpg
Figure 1: Fermentation from 09/13 of E.coli KRX with BBa_K863000, 6L in NFL22, autoinduction medium, 37 °C, pO2 50 % for 12 hours.
  • 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 Fungal Laccases and Team Shuttle Vector:
    • We had some colonies on our LB + CM plates from our transformation of shuttle vector + tvel5. So we did Colony PCR to see if the gene was inserted. But sadly all colonies are negative.
  • 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 (pH 8.0)
      • If needed: Elution with 80% ethylen-glycol (EG) or 1/5 Pellet to 4/5 EG (100%) of the overall volume.
Figure 1: Fermentation from 09/13 of E.coli KRX with BBa_K863000, 6L in NFL22, autoinduction medium, 37 °C, pO2 50 % for 12 hours.
  • Team Cultivation & Purification:
    • Made SDS-Pages of fermentation from 09/13.
    • 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.
  • Team Activity Test:
    • Today we got the chance to work with Team Immobilization again. The samples they gave us were supernatants from different immobilization approaches. The team tested diverse bead concentrations with even higher amounts of beads then before. Check their labjournal for further information about the output of the measurements. Besides the supernatants we also received beads with immobilized laccases TEVL0 and ECOL. So finally we got to use the filter plates that helped us to carry out the activity measurements and then stop the reaction at a precice point of time via centrifugation. Team Immobilization gave us enough samples for 7 measurements after different reactions times. We chose to stop the reaction after 2, 4, 8, 16, 32 and 64 minutes, respectively, to check the behaviour of the immobilizied laccase in relation to the time. Check their labjournal for the very exciting results!

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.
55px Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg