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

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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/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
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