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

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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


Reagents

Media

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)


Protocol

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].



Calibration Curve5.JPG


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

Calibration Curve2.JPG