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

Results Summary

3rd September '07

Vesicles found in Sample 2. Left: Several vesicles under white light. Right: Another pair of vesicles. In both cases, the fluorescence was present and visible, but VERY faint. The camera was unable to register the fluorescence - even with 4 seconds of exposure. Gamma and saturation enhancements did not reveal any colour.

• The possibility of preparing a 10ml lipid-oil suspension without overnight incubation was explored. Two suspensions were prepared: DOPC/dodecane, and POPC/dodecane. These were then used to prepare emulsions with 100x diluted GFP. However, due to a labelling mistake, it was impossible to distinguish between the two in all subsequent work. Both samples produced vesicles, with similar results.

Problems encountered

• A labelling mistake made it impossible to compare results between POPC and DOPC suspensions.

Conclusions

• The success obtained without the overnight incubation step during the suspension preparation indicates that the overnight incubation is not necessary. All preparations can be carried out in the same day.
• Reducing the volume of suspension being produced from 50ml to 10ml still produces vesicles. However, no comparison of yield was made between the two volumes.

24th August '07

Vesicles found in Sample 2, formed from 1ml POPC-dodecane-GFP emulsion. Left: A group of vesicles under white light. Right: The same vesicles, with fluorescence. The image was enhanced with gamma correction and full saturation.
Vesicles found in using Span-80 and mineral oil. Left: A group of vesicles in the Span80-Mineral Oil-GFP emulsion (not in aqueous solution). Right: A group of vesicles in Sample 3, formed from the Span80-Mineral Oil-GFP emulsion on the left.

The use POPC/dodecane and Span-80/Mineral Oil suspensions was investigated in their ability to emulsify a 100x diluted GFP solution.

• POPC/dodecane produced vesicles encapsulating GFP, provided that the interface had been formed before the emulsion was added. Neglecting to form the interface yielded a low number of vesicles, without any fluorescence. Strangely, samples taken from the emulsion showed very few vesicles. This may have arised from the method and time of collection - the vesicles may have sedimented by the time the sample was collected, and collection was made from an area far from the region of sedimentation.
• Span-80/Mineral Oil produced what looked like vesicles, but they were very mobile and did not contain GFP. However, the emulsion did show numerous fluorescent vesicles, suggesting that the problem occurred somewhere between the interface and sampling steps.
• On one occasion, a large Span-80 vesicle was seen budding into two smaller ones. This possibly indicates that Span-80 has such a strong surface tension reduction effect that the lowest energy state is in fact one with a larger surface area. It is possible, then, that the interface formation step is not a stable condition for Span-80, as it requires the formation of a flat surface. If this is true, then Span-80 would induce the budding off, or swelling, of vesicles at the interface - possibly towards the aqueous phase - in order to obtain a lower energy state. This would explain why the vesicles seen were not fluorescent - they were formed enclosing the interface solution, not the emulsified one.

Problems encountered

• GFP still aggregates outside vesicles, even with 100x dilution of the stock solution. It is not clear if these aggregates enter the solution without ever being encapsulated in vesicles, or if they enter in vesicles which then break and release the aggregates.

Solutions proposed

• Performing a control experiment, where the GFP solution (instead of emulsion) is added directly over the interface, may help determine how these aggregates enter the sampled solution. If they enter without ever being encapsulated, then changing the protocol to improve encapsulation efficiency would solve the problem. If they are released by bursting vesicles, then changing the protocol to improve vesicle stability would be the solution.

Conclusions

• The POPC/dodecane combination is suitable for forming vesicles encapsulating GFP.
• Span-80 has a high adsorption rate, and is a very good surfactant for stabilising emulsions. It might be possible to combine it with phospholipids when there are difficulties stabilising emulsions. This possibility needs to be investigated further by carrying out experiments comparing yield and quality of different surfactant mixtures.

22nd August '07

Microscope pictures from sample 4 (2ml emulsion, centrifuge for 20min at 30x g). Left: A fluorescent object. Right: The same object under white light.
Strange structures. Left: Aggregates in the oil-lipid emulsion. Right: Air-water interface and an unexpected 'bubbly' structure in the aqueous region.

• 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.