28 August 2007

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 [http://www.openwetware.org/wiki/IGEM:IMPERIAL/2007/Notebook/General_Protocols#S30.2FS12_Cell_Extract#Miniprep| Miniprep] in the general protocols page

• [Vector DNA] = 137.9 ng/μl
• [Insert DNA] = 987.1 ng/μl

1. Set up digestion to cut out insert and vector
   • DNA (5 μl insert/ 15 μl vector)
   • Nde 0.75 μl
   • BamH1 0.75 μl
   • 10X BSA 2 μl
   • 10X BamH1 Buffer 2 μl
   • ddH20 to make up total volume of 20 μl
2. Digestion left at 37°C for 1 hour

Vesicles

Preparations

Two suspensions were prepared for the next day:

1. 125μl of POPC in 50ml of dodecane
2. 125μl of DOPC and 125μl of Span-80 in 50ml of Mineral Oil