IGEM:IMPERIAL/2009/M3/Assays/F2620/GFPmaturation

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Contents

GFP maturation rate

  • Could be used as modelling parameter, instead of doing experiments, although the maturation rate could vary with temperature.


Equipment

  • Spectrophotometer
  • Fluorimeter
  • 96 well plates


Reagents

  • M9 medium


Protocol

Inoculation

1. Prepare two 5 ml cultures of M9 minimal medium5 supplemented with 0.2% casamino acids and 1mM Thiamine Hydrochloride (supplemented M9 medium) and antibiotic (kanamycin, 20 µg/ml).


2. Inoculate each test tube with single colonies (~2mm ø) of a cell with GFP expressed and a control cell, both from a freshly streaked plate


3. Grow cultures in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.

Growing up cells

4. Cultures were diluted 1:1000 into 5.5 ml of pre-warmed fresh medium and grown to an OD600 of 0.15 under the same conditions as before (this growth took 4.5 hrs on average).

5. Twelve 200 μl aliquots of each culture were transferred into a flat-bottom 96 well plate.

6. Three wells were each filled with 200 μl of medium to measure the absorbance background. Inducer was added.

7. The plate was incubated in a Wallac Victor3 multi-well fluorimeter at 37°C and assayed with an automatically repeating protocol of fluorescence measurements, absorbance measurements, and shaking (all as above). Time between repeated measurements was 54 sec.

8. After 20 min, spectinomycin (Sigma-Aldrich) was added to a final concentration of 500μM to three wells containing 3OC6HSL and to three wells not containing 3OC6HSL for each culture.

9. The plate was again incubated in the multi-well fluorimeter and assayed as before.

10. Relative GFP accumulation was plotted as a function of time.

Modelling parameter

9. We measured an average time constant for GFP maturation of 6.3 min with a 95% confidence interval over three replicates of ±0.4 min. The corresponding average GFP maturation rate is 1.8E-3 sec-1.

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