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

22th August '07

Results

Using a prepared well-dessicated, well sonicated DOPC/mineral oil suspension from yesterday,

• 2ml of suspension was taken to prepare an interface according to protocol.
• 10x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.
• Special care was taken to protect the GFP solution from light at all times.

4 samples prepared:

• Sample 1: 2ml of emulsion, no centrifuge.
• Sample 2: Following the protocol, with 2ml suspension interface and 100μl of each of two emulsions added.
• Sample 3: 2ml of emulsion, centrifuge at 120x g for 10mins.
• Sample 4: 2ml of emulsion, centrifuge at 30x g for 20mins.

Results

Light Microscopy (Magnification - 100x):

• Emulsion: Vesicles seen, presumably monolayer.
• Sample 1: Water-oil interface observed. Presumably the phospholipids have clumped altogether.
• Sample 2: Vesicles seen.
• Sample 3: Vesicles seen.
• Sample 4: Vesicles seen.

Fluorescence Microscopy (Magnification - 100x):

• Emulsion: GFP vesicles seen. Lots of GFP artefacts.
• Sample 1: No fluorescent vesicles observed.
• Sample 2: No fluorescent vesicles observed.
• Sample 3: "Vesicles" of sizes 1-10 μm found encapsulating GFP within. Lots of GFP artefacts.
• Sample 4: "Vesicles" of sizes 1-10 μm found encapsulating GFP within. Lots of GFP artefacts.

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.