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