IGEM:IMPERIAL/2007/Notebook/2007-9-4

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7

Construction of pT7-GFP

 * 1) Picked colonies to grow in 5ml LB + Kan
 * 2) Cultures left to grow at 37&deg;C overnight

GFP Calibration Curve
More concentrations were carried out for the calibration curve, these give a range of concentrations tested: The calibration curve can be found Here
 * 3.7uM, 1.8uM, 1.uM, 0.5uM, 0.1uM, 0.05uM, 0.025uM, 0.0125uM

Vesicles
Formation of Vesicles

2160&mu;l of Solution A-CE was prepared with the following components:
 * 583&mu;l of S30 cell extract (home made)
 * 1080&mu;l of cell extract buffer
 * 36&mu;l of rNTP solution
 * 112&mu;l of Pyruvate Kinase solution
 * 349&mu;l of ddH2O

360&mu;l of Solution B-CE was prepared with the following components:
 * 351&mu;l of Solution A-CE
 * 4&mu;l of GFP solution
 * 5&mu;l of DNA solution (Ptet with GFP)

The remaining 1.8ml of Solution A-CE was then diluted 10x to form 18ml of Solution A-CEd

The following 3 emulsions were prepared:
 * 50&mu;l of Solution B-CE into 8ml of POPC/dodecane suspension
 * 50&mu;l of Solution B-CE into 8ml of DOPC/dodecane suspension
 * 250&mu;l of Solution B-CE into 47,8ml of POPC/dodecane suspension

11 samples were created in total (note there is no Sample 9):


 * Sample Noireaux: Produced from the Noireaux protocol. Solution B-CE was added to 200&mu;l taken from the 50ml POPC/dodecane suspension prepared  the day before.
 * Sample 1: 2ml from the 10ml POPC/dodecane suspension was used to prepare the interface over 3ml of Solution A-CEd. 1ml of emulsion was added 2 hours later, centrifuged at 120x g, and collected.
 * Sample 2: 2ml from the 10ml DOPC/dodecane suspension was used to prepare the interface over 3ml of Solution A-CEd. 1ml of emulsion was added 2 hours later, centrifuged at 120x g, and collected.
 * Sample 3: Prepared the day before and left overnight before being collected. Sample not centrifuged.
 * Sample 4: Prepared the day before and left overnight before being collected. Sample not centrifuged.
 * Sample 5: 2ml from the 50ml POPC/dodecane suspension was used to prepare the interface over 3ml of Solution A-CEd. 1ml of emulsion was added 2 hours later, centrifuged at 120x g, and collected.
 * Sample 6: 2ml from the 10ml POPC/dodecane emulsion was poured over 3ml of Solution A-CEd (with no prepared interface), and left to rest for 2 hours, before being centrifuged at 120x g for 10 minutes and collected.
 * Sample 7: 2ml from the 10ml DOPC/dodecane emulsion was poured over 3ml of Solution A-CEd (with no prepared interface), and left to rest for 2 hours, before being centrifuged at 120x g for 10 minutes and collected.
 * Sample 8: 2ml from the 50ml POPC/dodecane emulsion was poured over 3ml of Solution A-CEd (with no prepared interface), and left to rest for 2 hours, before being centrifuged at 120x g for 10 minutes and collected.
 * Sample 10: 2ml from the 10ml POPC/dodecane emulsion recycled from the day before was left stirring overnight, and then was poured over 3ml of Solution A (with no prepared interface). It was left to rest for 2 hours before being centrifuged at 120x g for 10 minutes and collected.
 * Sample 11: 2ml from the 10ml DOPC/dodecane emulsion recycled from the day before was left stirring overnight, and then was poured over 3ml of Solution A (with no prepared interface). It was left to rest for 2 hours before being centrifuged at 120x g for 10 minutes and collected.

Samples 1-11 were produced using protocol 1.2.

Samples 1, 2, 5, 6, 7, 8 were prepared from an interface formed on top of 3ml of Solution A-CEd (see above).

Results

All 11 samples prepared today were scrutinised under the microscope. Here are the results (scroll down to the bottom for images.):


 * Sample Noireaux: No vesicles were found. There were lots of GFP aggregates.
 * Sample 1: Plenty of vesicles were found, but with very weak fluorescence inside. There were lots of GFP aggregates.
 * Sample 2: No vesicles were found, but one large fluorescent blob was present. There were lots of GFP aggregates. A strange, unidentified texture of objects was found.
 * Sample 3: One possible vesicle or blob found, with no fluorescence inside. E.coli contamination. Strange vesicle coalescence around a large GFP aggregate (vesicles faintly fluorescent).
 * Sample 4: Lots of coalescence, with faint fluorescence inside.
 * Sample 5: Flocculated vesicles, with very faint fluorescence inside.
 * Sample 6: Well formed vesicles, and some coalescence, with faint and not-so-faint fluorescence inside.
 * Sample 7: Rare but well formed vesicles, with faint fluorescence inside.
 * Sample 8: Best results. High count of well formed vesicles, with not-so-faint fluorescence inside.
 * Sample 10: Single large coalesced blob, with faint fluorescence, in spite of dense aggregate. No external aggregates.
 * Sample 11: Many 2&mu;m fluorescent vesicles, with very good enclosure - very few external aggregates. Large group of coalescence blobs.

Preparations No preparations were made today.