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.
Fluorometer + PC
Reagents and Materials
1 x 96 Fluorometer Plate (See through bottom)
Sticky plate lid
1x10ml of Autoclaved LB media in a 200ml flask Containing suitable antibiotics
1mg/ml of GFPmut3b
Collect 10ml of LB media (containing suitable antibiotics) into a 100ml flask. Inoculate the media with a single colony from a B.subtilis plate (transformed with correct construct) and grow overnight at 37oC.
Collect the overnight culture from the incubator, remove 1ml and measure the O.D.600. Calculate the amount of the overnight culture required to return the cells to an O.D.600 of 0.5 in 10ml of LB media, use the following calculation:
Volume of Overnight Culture (X) = (0.5/O.D.600)*10ml
Volume of fresh Culture (Y) = 10-X (Overnight Culture)
Return the cultures to the incubator and grow until an O.D.600 of 1 is reached.
(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.)
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.
Incubate at 37oC for 15 minutes with occasional mixing - Lysis should be indicated by a change in the viscosity of the culture.
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
Repeat this dilution three times to enable us to average out any errors.
Once the lysis is complete we are ready to carry out the dilutions of GFPmut3b in lysed B.subtilis,
Then follow the plate loading schematic below being careful to avoid bubbles and mix solutions thoroughly before adding to the plate!!!!!
Place in the plate reader and load up the protocol,