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
• 15ml tubes
• 96 well plates

Reagents

• M9 Minimal Media supplemented with 0.2% Casamino acids and 0.5% glucose
• 1M IPTG solution

Protocol

Day 1 PM:

Inoculation of cells

• Inoculate single colonies of E. coli cells into 15ml falcon tubes containing 5 ml of the pre-warmed (37°C) supplemented M9 medium with ampicilin (20 ug/ml). Grow the cultures for O/N with spinning at 70 rpm.

Day 2 AM:

Things needed

• Multiwell Plate reader
• 96 well plate
• Lysozyme solution

Monitering of OD and fluorescence

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

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

3)After the OD reaches 0.7 (should be immediately), add to a final concentration of 1mM of IPTG and mix well. Include negative and positive controls.

4)Take out 200ul of cell solution and add to a 96 well plate. Measure the fluorescence and absorbance using the test protocols pHTOP10 and IPTG_RFP_AB.

5) Add 4μl of the lysis solution drop by drop, mixing gently in between drops.

6) Keep inside the multi-plate reader for 30 min. Lysis should be indicated by a change in the viscosity of the culture. Then, measure the fluorescence and absorbance again.

7)Repeat the two sets of sampling of absorbance and fluorescence measurement every hour during mid-exponential growth for 6 hours.

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

8) 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.