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

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)

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

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

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.