|An example of flocculated vesicles, found in Sample 5. The vesicles were faintly fluorescent.|
|A large GFP aggregate surrounded by coalescing faintly fluorescent vesicles, found in Sample 3.|
- An attempt at enclosing cell extract inside vesicles was made today. Two different suspension mixtures were used: DOPC/dodecane, and POPC dodecane. The latter was prepared with both 50ml and 10ml volumes. All three suspensions were used to produce two samples each: one with a prepared interface, and one without - for a total of six samples.
- The best results were obtained from the 50ml POPC/dodecane suspension (Samples 5 and 8). Sample 5 was produced with previously prepared interface, while Sample 8 was produced with no prepared interface. Both samples had good results, but Sample 8 was better. Sample 5 had flocculated fluorescent vesicles, while those of Sample 8 were well dispersed and more numerous.
- The 10ml POPC/dodecane suspension gave mixed results. Fluorescent vesicles were present and numerous in both samples, but the sample produced without a prepared interface showed much more coalescence.
- The 10ml DOPC/dodecane suspension gave poor results. In the sample produced with a prepared interface, no vesicles at all were found. The other sample had very few vesicles, but those were well formed and faintly fluorescent.
- In addition to using the older Procol 1.2, we made a first attempt with the [[IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.3 | Noireaux protocol. The sample produced had many GFP aggregates, but no vesicles at all.
- We also recycled some of the work done the the day before. The 10ml DOPC/dodecane and POPC/dodecane emulsions (with GFP only) were left stirring overnight, and used to produce samples without a prepared interface.
- Sample 10, made with POPC/dodecane, produced no vesicles, but a strange large coalescence blob.
- Sample 11, made with DOPC/dodecane, produced many small fluorescent vesicles, and showed excellent enclosure - there were very few GFP aggregates outside thev esicles. It also contained a large group of coalescence blobs.
- Samples 3 and 4, prepared the day before, had not been completed. They were left to sediment overnight, and collected in the morning without centrifugation.
- Sample 3, made with POPC/dodecane, was contaminated with E.coli, and showed a strange coalescence structure.
- Sample 4, made with DOPC/dodecane, had many fluorescent coalescence blobs.
- The number of vesicles produced was smaller than that produced when only GFP is being encapsulated, and there was much more coalescence.
- There was too much fluorescence outside vesicles when using the cell extract. It was not clear whether this fluorescence came from the added GFP, if it was inherent in the cell extract, or if it resulted from expression of GFP by the cell extract.
- Contamination was present in one of the samples, and it was not clear where the contamination came from.
- The compensation for osmotic pressure, achieved through diluting the cell extract 10x (Solution A-CEd), may not have been enough. Moreover, it also contained cell extract (but without DNA).
- Different combinations of surfactants will betried to improve yield and reduce coalescence. Changes to the solutions used may also be considered.
- The cell extract will be examined under the fluorescence microscope, both with and without the DNA used in the experiments above. This should indicate whether the excessive fluorescence observed was due to the cell extract or the added GFP.
- Better contamination precautions will be taken, and the cleaning process will be reviewed in order to identify possible contamination routes.
- An inert compensation for osmotic pressure must be found. In order to achieve this, however, the osmotic pressure due to the enclosed solution must be found. Experiments elucidating the effects of osmotic pressure must be carried out.
- The results obtained from stirring emulsions overnight (Samples 10 and 11) suggest that the interface adsorption rate of both DOPC and POPC is not very high. It is possible that with a longer and gentler stirring period, better encapsulation and form can be obtained. This probably has two effects - it mixes the phospholipid into the suspension, and allows more time for phospholipds to adsorb at the interface between the aqueous and oil phases.
- POPC/dodecane works better than DOPC/dodecane. This is evident from comparing the results from Samples 1 and 6 to Samples 2 and 7.
- It is possible to reduce the volume of suspension being produced from 50ml to 10ml. However, comparing Samples 1 and 6 to Samples 5 and 8 reveals that the stirring of the 10ml emulsion needs to be adjusted in order to obtain the same yield.
- The increase in coalescence found when enclosing cell extract (as compared to GFP) indicates that there is a greater surface tension in the vesicles formed. Adjusting the solutions, or tweaking the surfactants used may solve this problem and improve yield and stability.