IGEM:IMPERIAL/2007/Notebook/Protocols

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