IGEM:IMPERIAL/2006/Results/T9002

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Contents

02/09/06

30/08/06

28/08/06

  • The results of the T9002 assay are not useable...carry on testing.

27/08/06

  • The results of the T9002 assay are not useable...carry on testing.

25/08/06

24/08/06

23/08/06

22/08/06

  • (96-well plate was left in shaker overnight)

18/08/06

Image:180806 GFPOD T9002.xls

16/8/06

15/8/06

Aims

To run through T9002 protocol and achieve transfer function plot

Results

  • Graphs
Average T9002 response to AHL in LB over time periods 3h20, 3h40, 4h (Line)
Average T9002 response to AHL in LB over time periods 3h20, 3h40, 4h (Line)
Average T9002 response to AHL in M9 over time periods 3h20, 3h40, 4h (Line)
Average T9002 response to AHL in M9 over time periods 3h20, 3h40, 4h (Line)

Comments

  • The amplitude of change between GFP expression at 0 AHL and 10uM AHL seems to a lot smaller in comparison to the experiment on 8/8/06.
  • Flourescence decreased over the time period
    • This suggests the peak of GFP expression is even earlier than 3h
    • This could either be down to
      • A very short AHL Half-Life
      • Cell death caused by poor heat conductance and lack of shaking in the water bath (used after the first fluorescence measurement, no shaker in plate reader room!)

Conclusions

  • Repeat experiment
  • Try and find a shaker
  • Experimentally assess AHL half-life

Well Layout

14/8/06

Aims

To run through T9002 protocol and achieve transfer function plot

Extra Information

  • Due to a protocol error the cultures could not be innoculated with AHL at OD600s of 0.1. Instead they were innoculated at the following ODs:
    • T9002 LB: 0.05
    • T9002 M9: 0.03

The protocol error has since been corrected.

Results

  • Graphs
Average T9002 response to AHL in LB over time periods 4h, 4h20, 4h40, 5h (Line)
Average T9002 response to AHL in LB over time periods 4h, 4h20, 4h40, 5h (Line)
Average T9002 response to AHL in M9 over time periods 4h, 4h20, 4h40, 5h (Line)
Average T9002 response to AHL in M9 over time periods 4h, 4h20, 4h40, 5h (Line)

Comment

  • The results show that the cultures in M9 had a higher amplitude of fluorescence
  • The results seem to suggest that the cultures in M9 would be responsive to greater concentrations of AHL than those tried, in contrast to the experiment on the 8th, and MIT's experiments
    • This could be due to the innoculation OD600
  • Unsure on how to remove media background fluorescence
  • Levels of GFP generally went down over the measured time period
    • This suggests GFP had already reached it's peak of expression at an earlier time
    • It also suggests AHL half-life may be shorter than the assumed 24 hours

Conclusions

  • The experiment should be repeated at OD600 of 0.1
  • A way to mathmatically remove background flourescence should be devised
  • AHL half-life should be experimentally measured

Well Layout

8/8/06

Aims

This experiment was a basic test run of the T9002 protocol. It was also designed to give insight into whether to use LB or M9 media for our characterisations and, eventually, full system.

Results

  • Graphs
T9002 response to AHL in LB (Scatter)
T9002 response to AHL in LB (Scatter)
T9002 response to AHL in M9 (Scatter)
T9002 response to AHL in M9 (Scatter)
T9002 response to AHL in LB (Line)
T9002 response to AHL in LB (Line)
T9002 response to AHL in M9 (Line)
T9002 response to AHL in M9 (Line)

Comment

  • The results gave a curve similar to that of MIT's.
  • Cells in M9 show a much sharper transistion from the ground state to the saturated state than in LB
    • Reasons for this are of yet unkown, but should be considered in the decision to choose between LB and M9.
  • There appears to be an error at 100nM AHL for both readings in LB and M9 (not shown in line graphs)
    • This is most likely due to an experimental error such as pippetting. This should be noted for the repeat.
  • The cells in LB media showed poor growth when shown against those in M9.
    • This could be due to the cultures not being shaken for the 3.5 hours, or poor heat conductance from the water bath to the 96 well plate.
    • The protocol should be modified to reflect these concerns.
  • Both LB and M9 on their own showed greater fluorescence than those wells containg supposedly GFP producing cells
    • This could imply the cells themselves quench media fluorescence
    • This should be tested by examining blank media and media containg cells that do not fluoresce

Conclusions

  • Overall, the experiment showed the general trend we expected
  • The protocol should be altered to allow better incubation of and shaking of the 96 well plate
  • A control of non-flourescing cells should be used to establish if the cells quench media flourescence

Well Layout

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