Team:Bielefeld-Germany/Labjournal/week21

Week 21 (09/17 - 09/23/12)

Monday September 17th

• Team Cultivation & Purification:
  • Cell disruption of fermentation of E. coli Rosetta-Gami 2 with BBa_K863012 via high-pressure homogenization and purification via Ni-NTA column.

Tuesday September 18th

• Team Cellulose Binding Domain:
  • Primer arrived:
  • Gradient-PCR with the CBDcex(T7)+GFP_His-plasmid and S3N10-CBDcex_RV and S3N10-GFP_FW primer-mix. the right product appeared only at 66,5°C; Lower: wrong product; Higher: no product
    • Following PCR at 66,5°C(50µL) had the wrong product (main product at 2 kbp); could be because of a different template concentration or not exact temperature.
    • Alternativly took the 20 µL-tube with right product and added 0,5 µL DpnI for over-night digestion.
  • Gradient-PCR with CBDcex(T7)+GFP_His-template and a CBDcex_Freiburg- + GFP_Freiburg_compl-primer-mix to amplify the whole fusion-protein and get rid of the His-tag.
  • Gradient-PCR with CBDclos(T7)+GFP_His-template and a CBDclos_Freiburg- + GFP_Freiburg_compl-primer-mix to amplify the whole fusion-protein and get rid of the His-tag.
• Team Substrate Anayltic: We analyze Ethin estradiol and estradiol degradation with the Spectrofluorophotometer. The measure showed that the degradation product are detectable but the results weren't clear. We decide to do this measurement again to have better results so a new degradation of the two substrates were set.

Wednesday September 19th

• Team Cellulose Binding Domain:
  • Stopped over-night-DpnI-digestion of S3N10-clos-GFP_his PCR-product and cleaned up in gel. Ligated the PCR-product with itself via Blunt-end-ligation and transformed it into KRX. Plated on Select-Agar.
  • Gradient-PCRs of the CBDcex(T7)+GFP_His-plasmid and a CBDclos(T7)+GFP_His-plasmid (unsequenced) with the CBDcex_Freiburg- and GFP_Freiburg-compl- and the CBDclos_Freiburg- and GFP_Freiburg_compl primers, respectively. The CBDcex-setup showed no correct product, but the CBDclos-setup had the right bands at temperatures from 57°C to 64°C; Merged the correct fractions and cleaned them up through the gel.
  • Digested the cleaned-up product with SpeI and XbaI and DpnI.
  • Ligated <partinfo>J61101</partinfo> and CBDclos_Freiburg with GFP_Freiburg and transformed it into KRX.

Thursday September 20th

• Team Cellulose Binding Domain:
  • No Colonies on the S3N10-CBDclos(T7)+GFP_His-transformations-dish with the blunt-end ligation; reason: forgotten to phosphorylate the PCR-product.
  • Two colonies on the CBDclos+GFP-dish with the constitutive J23100 + J61101 promoter. But none of them is glowing. And them shouldn't ... used the wrong selection-agar (since J61101 carries pSB1A2-backbone with AMP-resistence.
• Team Activity Tests:
  • Today we started another exciting measuremnt. Today was all about temperatures. Through our cooperation with the clarification plant we found out that the waster water here in Germany has a minimal temperature of 10°C throughout the year. We used this value together with our previous measurements at 25°C to complete the temperatur gradient of our measurements. ECOL
• Team Substrate Analytics:
  • Degradation measure from Ethin estradiol and Estradiol with the Spectrofluorophotometer. Degradation was from the 18th September

Friday September 21st

• Team Cellulose Binding Domain:
  • Transformated <partinfo>J61101</partinfo> with CBDclos+GFP again and plated on the right selection-agar this time!

Saturday September 22nd

• Team Cellulose Binding Domain:
  • Once again two colonies on the agar-dish: can not say if they have a green glow...
    • picked the colonies and them put into AMP-LB-Medium.
  • Also made a flask with 10 mL LM-Medium and Cells with the CBDcex(J61101)+GFP_His-plasmid

Sunday September 23rd

• Team Cellulose Binding Domain:
  • All cultures did not show any sign of GFP-glow under uv-light.
  • Gradient-PCR of <partinfo>BBa_K863104</partinfo> with S3N10_Cex_compl- and S3N10_GFP-primermix to generate a 13 amino acid-linker between the cellulose binding domain and the GFP.
  • Transformated CBDcex(T7)+GFP_His and CBDclos(T7)+GFP_His into BL21, to test if the problem is in the expression-system.

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