IGEM:IMPERIAL/2009/M3/Assays/F2620/calib/fluor/expt2

=Relating intracellular fluorescence to GFP molecules=

Aims

 * By relating intracellular [GFP] rather than extracellular [GFP] to fluorescence observed, cells do not have to be lysed. Therefore, fluorescence can be measured at various time points for a particular culture of cells.

Equipment

 * Spectrophotometer
 * 600nm absorbance filter
 * 17mm tubes
 * 96 well plates

M9 Minimal Media
Disodium Phosphate (12.0g)

Potassium dihydrogen phosphate (6.0g)

Sodium Chloride (1.0g)

Ammonium Chloride (2.0g)

Magnesium Sulphate (0.75g)

Glycerol (5.0g per L)= 54.39uM 0.5%

Glucose (0.5g per L) = 2.78mM 0.05% per 100ml

Others
IPTG (2 g)

Day 1 4PM:
 Omit this day if there are already available cells in culture and do the minimal media preparation in day 2 

Things needed

 * Minimal Media constituents
 * 17mm tubes

1) M9 Minimal Media Preparation:

 * Measure out the following reagents and dissolve them in 1000ml of sterile H20:

Disodium Phosphate = 6.0g

Potassium dihydrogen phosphate = 3.0g

Sodium Chloride = 0.5g

Ammonium Chloride = 1.0g

Glycerol (5.0g per L)= 54.39uM

Glucose (0.5g per L) = 2.78mM

2) Inoculation of cells

 * Inoculate single colonies of E. coli cells into 17mm tubes containing 5 ml of the pre-warmed (37°C) normal supplemented M9 medium with kanamycin (20 ug/ml)


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

Day 2 AM:
 Start from here if there are already available cells in culture 

Things needed

 * IPTG
 * Minimal Media constituents
 * 17mm tubes

1) IPTG solution Preparation:

 * 1g of IPTG dissolved in 4ml of dH2O (filter sterilize) to get 1M IPTG

2) Growing up of cultures

 * Dilute the cultures which are at a high cell density 1:1000 into 20 ml of fresh media in a 100ml flask and grow the cultures at 28°C in 28°C incubator O/N

Things needed

 * Spectrophotometer
 * 600nm absorbance filter

Monitering of OD and fluorescence

 * Now prepare the lysozyme solution by adding 2mg of lysozymes to 1ml of ddH2O, mix thoroughly and store on ice.


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


 * After the OD reaches 0.7 (should be immediately), transfer 200 ul aliquots from the culture into the labelled flat-bottomed 96 well plate with IPTG (all except well 1)and mix well.


 * Incubate the plate in a multi-well spectrophotometer at 28°C (water bath?) and assay with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid) and shaking (3 mm, linear, normal speed, 15 seconds)


 * Repeat the absorbance and fluorescence measurement every hour during mid-exponential growth for 6 hours


 * Determine background absorbance by measuring control well. This should be subtracted from subsequent absorbance readings.


 * For each hourly sample, add 4μl of the lysis solution, to the 200ul culture drop by drop mixing gently in between drops.


 * Incubate at 28°C for 30 minutes with occasional mixing - Lysis should be indicated by a change in the viscosity of the culture.


 * In the 96 well plate, there will be the following wells:

Well 1: 0 ul of IPTG Well 2: IPTG for 1hr Well 3: IPTG for 2hr Well 4: IPTG for 3hr Well 5: IPTG for 4hr Well 6: IPTG for 5hr Well 7: IPTG for 6hr Well 8: IPTG for 7hr

Data processing

 * From the standard curve in experiment 1, we can determine [extracellular GFP] from the normalised fluorescence reading obtained.


 * After lysing the cells, we know the [intracellular GFP] for each duration of IPTG induction by relating fluorescence to [lysed extracellular GFP].



Therefore, now we can plot [intracellular GFP] against fluorescence observed.