IGEM:IMPERIAL/2009/M3/Assays/F2620/calib/fluor/expt1

Relating Fluorescence to extracellular GFP

Aim

The aim of this protocol is to produce a calibration curve of fluorescence vs molecules of GFPmut3b. This is to help us convert our fluorescence data in arbitrary units into the normalised units of rate of molecules of GFPmut3b synthesised per cell per min/sec.

Equipment

• Multi-well plate reader
• 96 well plate

Reagents and Materials

• Purified GFP- 100ul of 1mg/ml
• Lysozyme- 2mg (or other lysis solution)

Protocol

Things needed

• LB media with Strep
• Supplemented M9 Minimal Media (premade) with Strep
• 15ml falcon tubes

2) Inoculation of cells

• Inoculate single colonies of E. coli cells with no GFP into 15ml falcon tubes containing 5 ml of the pre-warmed (37°C) normal LB medium with kanamycin (20 ug/ml)

• Grow the cultures for O/N with spinning at 70 rpm.

• Place the culture in the cold room at noon time

Day 2 5PM:

• Dilute cells 1:50 in 10 ml of supplemented M9 media with 0.2% casamino acid.

• Grow the cultures for O/N with spinning at 37 degrees at 70 rpm.

Day 3 9AM:

Things needed

• Multi-well plate reader
• 96 well plate
• Lysozyme solution

Lysing cells

1. Collect the overnight culture from the incubator.

2. The OD is measured by transferring 200ul aliquots into a 96 well plate and measuring the absorbance at 600nm.

3. Return the cultures to the incubator and grow until an O.D.600 of 1.0 is reached in 10ml media. (returning to an O.D.600 of 1 because this gives a suitable cell density for cells in the exponential phase of growth were we are going to characterize the test constructs.)

4. Now prepare the lysozyme solution by adding 2mg of lysozymes to 1ml of ddH2O, mix thoroughly and store on ice. Add 200μl of this solution, to the 10ml culture drop by drop mixing gently in between drops.

5. Incubate at 37oC for 15 minutes with occasional mixing - Lysis should be indicated by a change in the viscosity of the culture.

6. Whilst the cells are lysing the GFPmut3b dilutions can be prepared. The stock solution is 1mg/ml (27.8kDa or 37μΜ), using this the following dilutions can be performed:

[1.] 32.4μl of stock GFPmut3b into 7.6μl o ddH2O = 30μM
[2.] 16μl of [1.] into 10μl of ddH2O = 24μM
[3.] 16μl of [2.] into 10μl of ddH2O = 18μM
[4.] 16μl of [3.] into 10μl of ddH2O = 12μM
[5.] 16μl of [4.] into 10μl of ddH2O = 6μM

7. Once the lysis is complete, we are ready to carry out the dilutions of GFPmut3b in lysed cell culture.

8. The fluorescence of the GFP dilutions was measured in the Wallac Victor3 multi-well fluorimeter.

Data processing

9. the data plotted against the mass of GFP in each dilution.

10. A straight line (R2 = 0.99) was fit to the data.