Team:Heidelberg/Notebook/Sensing Group/Notebook/11thweek


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Monday, 10/13/2008

  • Miniprep of LuxS_mut_EcoRI and F1-YFP, F2-YFP
  • Digestion of Fusion-YFP with BamHI (NEBuffer3 + BSA)
    • Insert: 5937 bp and 1998 bp
    • No Insert: 7202 bp
  • Digestion of LuxS with EcoRI/NdeI (NEBuffer EcoRI)
    • Insert: 2773 bp and 1862 bp
    • 4635 bp
  • Fusion-PCR for LuxS standardization with Phusion
    • 30s @ 98°C || 10s @ 98°C | 30s @ 55°C | 1min @ 72°C || 5min @ 72°C | 4°C (30 cycles)

Tuesday, 10/14/2008

  • PCR Purification, Digestion of LuxS and pSB2K3 with EcoRI/PstI (EcoRI buffer + BSA). 1h @ 37°C
  • Fusion PCR for LuxS with Phusion and Annealing Temperature Gradient (50-60°C)
Fused LuxS parts for standardization seem positive
  • Transformation of Fusion-YFP into MG1655

Wednesday, 10/15/2008

  • PCR Purification of LuxS_standard and F1-GeneArt, F2-GeneArt
  • Digestion of LuxS and Fusion Constructs with EcoRI/PstI (NEBuffer EcoRI + BSA)
  • Digestion of LuxS with EcoRI/xbaI (NEBuffer2 + BSA) to check for restriction sites
  • Cloning into pSB2K3
  • Test Digestion of LuxS with EcoRI and xbaI to test if mutation was successful
  • Digestion of Fusion1-YFP and Fusion2-YFP constructs with BamHI
LuxS digestions with EcoRI and xbaI to test for successful mutagenesis
Digestion of LuxS to test for mutations and Fusion-YFP with BamHI constructs to confirm insert.

  • Inoculation of 50µl Fusion-YFP O/N culture in 5mL LB. Incubation for 2h @ 37°C. Induction of Fusion-YFP cells with 0.01% Arabinose for 3h. Visualization under microscope
  • Expression of Fusion-Receptor positive for both constructs. Even Localization to the membrane can be seen in some cells.
Fluorescence Image of Fusion1-YFP
Fluorescence Image of Fusion2-YFP

Thursday, 10/16/2008

  • Picked 16 LuxS colonies and Inoculation in LB-Kan
  • Inoculation of UU1250 cells containing Fusion constructs

Friday, 10/17/2008

  • Miniprep of LuxS and Fusion constructs
  • Digestion of LuxS with EcoRI/PstI (NEBuffer EcoRI + BSA) and Fusion with BamHI/PstI (NEBuffer 3 + BSA)
Digestion of LuxS with EcoRI/PstI. Positive clones show two bands
Sequencing @ GATC reveal that mutagenesis did not work. bcause restriction sites are still there.