IGEM:IMPERIAL/2007/Notebook/2007-8-28

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7

In vitro Testing of pTet-LuxR-pLux-GFP at room temperature
Testing of the construct was done at a low concentration of AHL 0.1nM to emulate a real biofilm. This experiment tests whether the system is sensitive enough to respond to the low amounts of AHL. Protocol can be found here under Phase 1-In vitro testing on the experimental design page. Results: Very low levels of fluorescence was observed.

Construction of pT7-GFP

 * 1) Grew cells with plasmids containing pT7-GFP and cells with vector plasmids overnight
 * 2) Spun down cells and minipreped

Protocols can be found at Miniprep in the general protocols page


 * [Vector DNA] = 137.9 ng/&mu;l
 * [Insert DNA] = 987.1 ng/&mu;l


 * 1) Set up digestion to cut out insert and vector
 * 2) *DNA (5 &mu;l insert/ 15 &mu;l vector)
 * 3) *Nde 0.75 &mu;l
 * 4) *BamH1 0.75 &mu;l
 * 5) *10X BSA 2 &mu;l
 * 6) *10X BamH1 Buffer 2 &mu;l
 * 7) *ddH20 to make up total volume of 20 &mu;l
 * 8) Digestion left at 37°C for 1 hour

Vesicles
Preparations

Two suspensions were prepared for the next day:
 * 1) 125&mu;l of POPC in 50ml of dodecane
 * 2) 125&mu;l of DOPC and 125&mu;l of Span-80 in 50ml of Mineral Oil