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

Promoter pLux Protocol

For the followling protcol is for preparation of a culture and one set of 8 inducer concentration ranges. We wish to carry out 4 repeats of concentration and so carry out this protocol x4 in parrelel.

Protocol

Preparing the Over night Cultures

• Inoculate 5ml of M9 minimal media containing 50 μg/mL Ampicillin with 10μl of stored culture containing pLac test construct.
• Incubate at 37°C for overnight in a shaker.
  • Allows selected growth of E.coli with vector

Day 1

• Incubate M9 Amp Media 37^o
• 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:

volume culture = (0.1/O.D.600)*10ml

• 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 37^oC 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 IPTG.(PROCESS)

2 hours Incubation

• 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:

volume culture = (0.1/O.D.600)*15ml

• 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 0.2M AHL (Just need a high concentration to saturate promoter)

• Place into the fluorometer and set time intervals to relevant settings to give 50 time points.

Results

• Each concentration of inducer has four repeats each of which is composed of 50 time interval measurements.