IGEM:IMPERIAL/2007/Notebook/Protocols

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Protocols for CBD and Biofilm applications

  • Construct all DNA assemblies.
  • Test out construction of vesicles using mineral oil method.
  • In vivo and in vitro testing of simple DNA constructs with the promoters pLux, pTet, pT7 and pcI; followed by a reporter GFP
    • 3 repeats to be done for each test
    • Positive control: Purified GFP in E.coli, and then in the cell extract
    • Negative control: Cell extract without any DNA insert or GFP
  • Fluorescence is measured for every 15 min until the levels of GFP reaches a steady state
  • The best promoter sequence is determined and implicated into the two applications.
    • Definition of best promoter:
      • Fastest expression of GFP
      • Highest detection of fluorescence
  • When Dsred Express gene sequence arrives, clone and express it (Temperature: 25°C
  • Draw up a calibration curve of DSred Express.
  • Positive control: Dsred Express in cell extract
  • Negative control: Cell extract only


Problems

  • The commercial cell extracts come in 50µl, while the wells in the fluorometer plates can hold up to 200µl.
  • Water evaporates when fluorescent readings are taken.


Protocol specific for CBD

  • Test for temperature range and Life span of system
  • Temperature range: 4°C- 60°C.
    • Temperature Intervals: 4°C 15°C 25°C 37°C 45°C 60°C
    • Readings are taken at every 15 min intervals
    • 3 readings are taken for each sample at a particular temperature
  • Subject system to temperature changes and measure effect on fluorescence levels
  • Type of gradients: Steep/ Gentle/ Pulse
  • Temperature increment: from 4°C to 25°C
  • Apply the temperature increment only after the GFP levels have reached steady state
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