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

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Promoter Protocol

Preparing the Over night Cultures

  • Inoculate 5ml of M9 minimal media containing 40 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


Day 1

  • 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:

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 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

  • 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 arabinose 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 1mM arabinose
  • Place into the fluorometer and set time intervals to relevant settings to give 50 time points.
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