IGEM:IMPERIAL/2009/M3/Assays/GFPciplambda

GFP measurement

Aims

• We want to show that on coupling cI to the lambda promoter, cI represses the promoter activity.
  
• By linking F2620 to cI (TetR-LuxR-pLux) and the lambda promoter to the the E0240 testing construct (RBS-GFP-TT), we can vary the HSL input. This varies the expression level of cI, and consequently the transcriptional activity of lambda promoter can be determined.
• PoPS output indirectly through a quantitative measurement of flourescence

Assay

To characterise the cI-lambda promoter interaction, the expression level of cI will be varied by varying HSL input concentration.

The consequent activity of the lambda promoter will be measured by the fluorescence over time using a fluorimeter at multiple time points.

After subtracting the background fluorescence, the activity of the lambda promoter can be reported in Relative Fluorescence Units(RFU).

Equipment

• Spectrophotometer
• Fluorimeter
• 96 well plates

Reagents

• M9 Medium

Protocol

1. Single colonies of E. coli cells containing the test construct on the vector backbone were inoculated into three 17 mm test tubes containing 5 ml of pre-warmed (28°C) supplemented M9 medium with kanamycin (20 ug/ml).

2. Cultures were grown in 17 mm test tubes for approximately 20 hrs at 37°C with spinning at 70 rpm.

3. We then diluted the cultures 1:100 into 5 ml of pre-warmed fresh media and the cultures were grown for approximately 4 hours under the previous conditions (17 mm tubes, 28°C, spinning at 70 rpm).

4. After 4 hours, we measured the OD600 of a 500 ul aliquot from each culture on a WPA Biowave Spectrophotometer. This will tell you how many cells you have.

5. There are 6 setups made, one with no HSL added, and the other 4 ranging from 1uM to 1mM, as well as a positive control.

6. Based on the OD measurement from step 4, the cultures were diluted to the same OD (0.07) in 6 setups of 5 ml of pre-warmed fresh media and grown for one hour at 28°C.

7. We then transferred three 200 ul aliquots from each culture into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner).

8. We incubated the plate in a multi-well fluorimeter (Perkin Elmer) at 28°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (485 nm excitation filter, 525 nm emission filter, 0.1 seconds, CW lamp energy 12901 units), and shaking (3 mm, linear, normal speed, 15 seconds). (Just measures GFP – fluorescence over time).

9. Background absorbance was determined by measuring wells containing only media. This should be subtracted from subsequent absorbance readings.(Blank).

10. Background fluorescence was determined at different ODs from the fluorescence of control cells without a GFP expressing vector. (Control)

11. Measurements were taken from an approximately 30 min period in mid-exponential growth.