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

Results Summary

22nd August '07

• Having followed our normal and modified protocol strictly, we finally saw results of GFP enclosed around a circular barrier. We are not entirely convinced that these are vesicles as not many of these "vesicles" have been found, although we could attribute it to perhaps fundamental flaws in our protocol. In addition, we got word from Oscar, our resident vesicle specialist at Imperial College, that vesicles and miniature air bubbles are very hard to discern even with years of expertise, and the only true way of determining vesicle formation is to use fluorescent-tagged phospholipids.

• Another thing that was noted was also the indiscriminate clumping of GFP that was observed. Previously we have used 10X GFP standard stock solution as we could not visualize it under the fluorescence microscope. However, GFP, in excess concentration compared to phospholipid amounts, are thought to aggregate together instead of forming vesicular monolayers in our emulsion (and disrupt vesicle formation). This is strongly suggested to be the primary reason for the massive amounts of fluorescence observed under the microscope that contribute to fluorescent noise. Alternatively it has been suggested that it would be better if we reduce this concentration, focussing instead on observing lower amounts of fluorescence, but making sure that whatever is observed is clearly indicative of GFP fluorescing within vesicles, which is the function of what a marker is supposed to achieve.

• POPC, dodecane and Span 80 has arrived from sigma, which we will start preparation tomorrow.

20th August '07

• 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.