User:James Chappell/ Sandbox

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Contents

Characterization of Constitutive Expression

Aims:

  • To characterise the constitutive promoter and RBS combinations. This protocol is concerned with characterisation of the steady-state expression of GFPmut3b under the control of the 4 different promoter-RBS combinations. We will be measuring the fluorescence and O.D.600 of the cultures and normalizing our data to the units of rate of molecules of GFPmut3b synthesised per cell per min/sec,
  • In addition, from comparison of the rate of expression we should be able to determine the relative strength of the RBS.
  • With the data on the characterisation of the promoter-RBS combinations we will be able to make an informed decision on which we should use for our future test constructs.


  • The protocol below described what is needed for one combination of promoter and RBS, we will need to carry this out for all the combinations.


  • Normally we are only concerned with the steady state expression of an constitutive promoter. However, the conditions at which we measure this could change, for example what media the B.subtilis is grown in. In addition we may wish to see how the strength of expression changes during the growth of the B.subtilis. This could be achieved by measuring the change in fluorescence between the different growth phases.

Equipment

  • Fluorometer + PC
  • Pipette gun
  • Gilson p20,p200,p1000
  • Spectrometer
  • Shaking incubator

Reagents and Materials

  • 1 x 96 Fluorometer Plate (See through bottom)
  • Sticky plate lid
  • Curvettes
  • 4x10ml of Autoclaved LB media in a 100ml flask Containing suitable antibiotics


Protocol

Day 1

  1. Collect 10ml of LB media (containing suitable antibiotics) into a 100ml flask. Innoculate the media with a single colony from a B.subtilis plate and grow overnight at 37o.

Day 2

  1. Collect 3 x 100ml flasks containing 10ml of LB media (containing suitable antibiotics) and remove 1ml of the media and pipette into a blank curvette. Remove 1ml of the overnight culture and measure the O.D.600
  2. Dilute the overnight culture 1 in 10 in 3x20ml of fresh LB media containing suitable antibiotics . Mix thoroughly and remove 1ml of the culture and measure the O.D.600. Place into a shaking incubator and grow until reaches the stationary phase of growth, the time for this should be determined previously when we do the growth curve but always checked by removing 1ml of each culture and measuring the O.D.600 using LB media as a blank.
  3. Once the correct O.D600 has been reached then pipette 9x200μl of the B.subtilis into a 96 well plate following the plate schematic (in total want 3 repeats for each of the 3 cultures). In addition pipette 200μl of 3xLB media into the 96 well plate. Once the plate has been loaded then carefully place sticky tape onto the top of the plate.
  4. Place into the plate reader and open the protocol for characterisation of constitutive promoters and run protocol.
  5. This needs to be set up to measure the O.D.600 and fluorescence (488nm excitation filter and 525nm emission filter) every 10 minutes for an hour (What do we think of these data collection settings).
  6. Once data is collected dispose of the 96 well plate into the autoclaved rubbish bins (white).
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