<calendar> name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>
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
- Grew cells with plasmids containing pT7-GFP and cells with vector plasmids overnight
- Spun down cells and minipreped
Protocols can be found at Miniprep in the general protocols page
- [Vector DNA] = 137.9 ng/μl
- [Insert DNA] = 987.1 ng/μl
- 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
- Digestion left at 37°C for 1 hour
Two suspensions were prepared for the next day:
- 125μl of POPC in 50ml of dodecane
- 125μl of DOPC and 125μl of Span-80 in 50ml of Mineral Oil