IGEM:IMPERIAL/2007/Projects/Hrp System/Systems/Hrp Device 1/TestingValidation/Preliminary

Promoter Protocol
Preparing the Over night Cultures Day 1 volume culture = (0.1/ O.D.600 )*10ml
 * Inoculate 5ml of M9 minimal media containing 50 μg/mL Ampicillin with 10μl of stored culture containing pBad test construct.
 * Incubate at 37°C for overnight in a shaker.
 * Allows selected growth of E.coli with vector
 * Incubate M9 Amp Media 37o
 * Remove the overnight culture from incubator and measure Optical Density at 600nm (O.D.600) of a 4ml sample.  Click here for technique on spectrometer.
 * Calculate the volume of culture needed to give a O.D.600 in a 10ml volume, use simple calculate:
 * With the calculated volume of culture add pre-warmed M9 media to make total volume of 10ml
 * Return the 10ml culture to the incubator and incubate at 37oC for 2 hours.
 * The purpose of inoculating a new media is because over night E.coli will go into the stationary phase, but by inoculating a new medium we are returned cells to the exponential phase
 * Whilst the cultures are incubating prepare the standard dilutions of arabinose.(PROCESS)

2 hours Incubation volume culture = (0.1/ O.D.600 )*15ml
 * Remove culture and measure O.D.600.
 * Calculate the volume of culture needed to give a O.D.600 in a 15ml volume, use simple calculate:
 * With the calculated volume of culture add pre-warmed M9 media to make total volume of 15ml
 * Spin the 15ml culture in a centrifuge for 10 mins at 300 to 2000g minutes(check). Cells will form a pellet in the bottom of the tube.
 * Carefully remove the liquid in the tube (supernatant) to leave just the pellet.
 * Re-suspend the cells in 15ml of prewarmed M9 Amp and gentle vortex.
 * This is to supply fresh media, ensures the cultures remain in the exponential phase

Fluorometer Plate Reader
 * Retrieve the Plate
 * Remove 200μL of the culture from the 15ml into 10 wells.
 * Pipette samples into a 96 well plate
 * Add 2μL of pre-prepared AHL solutions to relevant well (Follow schematic)
 * In addition want the following control wells:
 * 1) 200μl of growth medium.
 * 2) Cultures without vector.
 * 3) A 200μL culture with 2μL of 0.2mM AHL
 * Place into the fluorometer and set time intervals to relevant settings to give 50 time points.

Results

 * From this preliminary experiment we will be able to further adapted the test construct protocol to ensure that our data range will be sufficient.
 * For example, if our graph has a sharp transition from low output to high output we will want to increase the data points in this transition.
 * The parameters of the protocol we may wish to adjust will be concentration range and the time intervals of measurements.