Team:Heidelberg/Notebook/Sensing Group/Notebook/8thweek


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Monday, 09/22/2008

  • test digestion of F1_LuxP_NdeI (F1 cloned in pDK48 with NdeI), F2_LuxP_NdeI, F1_LuxP_KpnI (F1 cloned in pDK48 with KpnI) and F2_LuxP_KpnI (F2 cloned in pDK48 with KpnI) with NcoI (NEBuffer 3)
Digestions of Fusion constructs with NcoI. Positive clones show two bands at 4308 and 2915 bp
  • Transformation of Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48) into MG1655 and HCB33 cells
  • Preparation of cultures for Maxipreps of Fusion constructs

Tuesday, 09/23/2008

  • Maxiprep of LuxP-Fusion constructs (F1(NdeI)_LuxP_pDK48, F1(KpnI)_LuxP_pDK48 and F2(NdeI)_LuxP_pDK48)
  • Cloning of Fusion_LuxP in pBAD33 with BamHI/PstI
  • digestions of Fusion constructs and pBAD33 as described in gel image descriptions
Digestion of Fusion constructs and pBAD33 with BamHI/PstI for subsequent cloning.
Digestion of Fusion constructs with NcoI and PstI to check for correct insert and Maxiprep digetions.

  • O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in TB-Kan
  • Transformation of Fusion-LuxP-pBAD33 constructs into DH5a

Wednesday, 09/24/2008

  • Glycerol-Stock of Fusion1-LuxP-pDK48 and Fusion2-LuxP-pDK48
  • O/N culture of Fusion-LuxP-pDK48 in HCB33 and MG1655 in LB
  • O/N culture of LuxS and GFP Plasmid from Marcus
  • Re-Sequencing of Fusion-1 and Fusion-2
Correct sequence for both Fusion constructs confirmed

Thursday, 09/25/2008

  • Miniprep of Fusion-LuxP-pDK48 constructs, LuxS, GFP Plasmid from Marcus
  • Competent Cells (Top-10, UU1250)
  • Picked colonies of Fusion-LuxP-pBAD33

Friday, 09/26/2008

  • Miniprep of Fusion-LuxP-pBAD33 constructs and Digestion with HindIII (NEBuffer2)
    • 5 µl DNA
    • 1µl NEBuffer 2
    • 0.5µl HindIII
    • 3.5µl H2O
  • Expected Fragments
    • Fusion1-LuxP-pBAD33: 7616bp and 769bp
    • Fusion2-LuxP-pBAD33: 7631bp and 769bp
    • pBAD33 only: 5360bp
Double digestion of Fusion constructs in pBAD33 with BamHI/PstI and single digestion with HindIII. Double digestion yielded positive results for all constructs.
  • Double Transformations of Fusion-LuxP-pBAD33 and pDK4 into UU1250 (2 Amp-CM plates) as well as LuxS and pDK6 into DH5a (Amp-Kan plate(s))
  • Transformation of pDK4 into DH5a (CM plate)
  • Preparation of 2x 3mL LB-CM-Amp, 2x 5mL TB-CM-Amp, 1x 3mL LB-Amp-Kan, 1x 5mL TB-Amp-Kan, 1x 5mL LB-CM

Sunday, 09/28/2008

  • Picked colony of each Fusion-LuxP-pBad/pDK4 UU1250 cells and inoculated in 5 mL TB and 5 mL LB medium
  • No colonies on LuxS/pDK6 DH5a cell plate. 5 mL liquid culture of LuxS DH5a cells.
  • No colonies on pDK4 cell plate. Waiting until tomorrow

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