Team:Sydney Australia/Contribution

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Characterisation

Basic Part Characterisation - Thermal Stability of sfGFP

To qualify for a Bronze medal for Characterisation, we decided to improve the characterisation of Superfolder GFP (BBa_I746916) through production of melt curve to quantify its thermal stability. We also hope to improve the characterisation of sfGFP and our new part, VVD_MET130I (BBa_K3140010) by comparison of their thermal stability.

The results of the assay can be found in the Registry pages of these basic parts.

Protocol for Measuring Thermal Stability of a Fluorescent Protein

This protocol allows you to quantify the thermal stability of fluorescent proteins by qPCR. You will need to know the excitation and emission spectra of the protein of interest before you start so if you are using a new or mutated variant first measure the excitation and emission using a fluorimeter.

  1. Prepare overnight 50ml cultures of an appropriate chassis E. coli transformed with a vector containing the fluorescent protein you wish to study.
  2. Pellet cells and resuspend in 1mL of Zietkiewicz buffer (40mM Tris-HCl, pH 7.8; 50mM NaCl: 20mM KCl; 20mM MgCl2; 5mM b-mercapto-ethanol; 10% glycerol).
  3. Transfer 1mL of cells to a bead beating tube and beat 3 times for 30 seconds placing on ice for 2 minutes between each beating.
  4. Centrifuge tubes at 15,000 rpm for two minutes and remove supernatant into fresh tubes.
  5. In a 96 well qPCR plate perform a serial dilution of the resuspended cells by first adding 50mL of Zietkiewicz buffer to 6 wells. In the first well add 50mL of the cell solution and mix by pipetting up and down before transferring 50mL from this well into the next. Discard 50mL from the 6thwell.
  6. Set a Real Time PCR machine with a FAM filter to measure excitation and emission at appropriate wavelengths for your protein of interest at an initial temperature of 60°C for one minute before increasing by 0.3 degrees per second up to 95°C.