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