IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Results1.2

Results Summary

• So far, there has been no conclusive evidence of GFP solution that is encapsulated within vesicles. To date, our best result is given as green fluorescence around the border of a ~10 μm vesicle (we don't know why), and strong fluorescence that is observed in miniature circles of < 1μm diameter, but no conclusive argument that those are vesicles.

• We are admist modifiying the protocol, especially at the magnetic stirring, interface formation, and centrifugation stages. This is because we found conclusive results of monolayer vesicle formation containing GFP diluted solution at the emulsion phase, but no vesicles that contain GFP at the latter phase.

• Problems encountered:
  • Dessicator not working properly. We discovered it only a couple of days into this phase of the experiment (and rectified the problem), but as it affected the preliminary preparation of our vesicles, we did not yield good results with those samples.
  • We diluted GFP to 200X, then 100X, before realizing that we could not visualize both dilutions of GFP (faint fluorescence for 100X) on the fluorescence microscope. Since then, we dilute GFP standard solution to 10X, which gives us a clear signal.
  • We also did not know the light microscope will bleach GFP. Slides last at most 10 minutes under light microscopy.

• Solutions proposed:
  • Use of POPC and dodecane instead that is common across literature.
  • Use of aluminium foil to avoid GFP bleaching.
  • Experimentation on the protocol itself to suit our reagents and specifications.