IGEM:IMPERIAL/2007/Notebook/2007-9-11

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Vesicles

Formation of Vesicles

Three commercial S30 cell extract mixes were prepared, with the ratio of 1:4:3 AA Mixture : Premix : S30 Extract. These were then used to form three reactions:

• 50μl Negative Control: 33,3μl of S30 mix, 16,7μl nuclease free water.
• 50μl Experiment: 33,3μl of S30 mix, 3,8μl P_lux GFP DNA, 12,9μl nuclease free water.
• 20μl Positive Control: 13,4μl of S30 mix, 1,5μl P_lux GFP DNA, 5,1μl nuclease free water.

Two of the three suspensions prepared the day before were used to prepare emulsions containing commercial S30 cell extract mixes:

• Experiment: 10ml emulsion with 50μl of S30 cell extract with P_lux GFP DNA
• Negative Control: 10ml emulsion with 50μl of S30 cell extract without DNA.

The third suspension was used to prepare four interfaces (2ml suspension over 3ml Solution A), to become four samples:

• Sample 1: Experiment, with circulation by syringe
• Sample 2: Experiment, without circulation by syringe
• Sample 3: Negative Control, with circulation by syringe
• Sample 4: Negative Control, without circulation by syringe

The remaining 20μl of S30/DNA reaction was used to form postitive controls:

• Concentrated Positive Control: 15μl of S30/DNA reaction
• Diluted Positive Control: 5μl of S30/DNA reaction diluted into 295μl of Solution A

AHL was then added to each of the four samples and the two positive controls above, to reach a concentration of 1μM/dm³.

Results

Samples 1-4 were looked at, as well as both emulsions before and after centrifugation, and the two positive controls.

• Emulsion - Experiment (No pictures taken)
  • Before CF: Vesicles of around 2μm diameter found, plus many artifacts.
  • After CF: Very few vesicles found, plus many artifacts.

• Emulsion - Positive Control (No pictures taken)
  • Before CF: Vesicles of around 2μm diameter found, plus many artifacts.
  • After CF: Very few vesicles found, plus many artifacts.

• Positive Controls
  • Concentrated: Faint fluorescence throughout, and some aggregates.
  • Diluted: Tiny bright specs of green fluorescence, plus some large aggregates. (See pictures below.)

• Sample 1 (Experiment): Very few vesicles found, with faint fluorescence inside. Many fluorescent aggregates.
• Sample 2 (Experiment): Vesicles found, and one with very strong green fluorescence. All other vesicles with faint green fluorescence.
• Sample 3 (Negative Control): Few vesicles found, but with no fluorescence inside. Large fluorescent object found, and possibly contaminated.
• Sample 4 (Negative Control): Many vesicles found, but with no fluorescence inside. Bacterial contamination present. Strange textures found that may be oil from the collection process. (No images.)

Diluted Positive Control: A large, brightly fluorescent object found, shown in white light, and red, green, and blue fluorescence from left to right respectively. Such objects were typical of this control sample.

Sample 1: An example of a 2μm vesicle found in Sample 1. The vesicle showed some fluorescence, but too faint to be captured by the camera. It is likely that the fluorescence observed was simply background fluorescence, and not GFP.

Sample 2: A very strongly fluorescent vesicle was found, pictured above. Such a strong fluorescence is most likely originating from GFP. The object showed blue fluorescence as well, but not red. Left: The vesicle seen in white light. Center: The vesicle seen with a green fluorescence filter. Right: The image in the center, but with full saturation and gamma correction (1.20). A video was taken as the microscope was focused above and below the vesicle: Focusing Video The video shows how vesicles look at different levels of focus, and also shows the object to the right of the vesicle as being an air bubble on a different plane.

Sample 3: A fluorescent object found in the negative control. It is not a vesicle. The images show it under white light and its fluorescence in red, green, and blue (left to right, respectively).

Sample 3: A strange object was found in the negative control. It seems to be an aggregate, flanked by what may be bacterial contamination. The darker, elongated objects on the top left showed blue fluorescence, but not green or red.

Cloning of Biobricks

1. Placed 5 ml of LB in a 15 ml tube
2. Added appropriate antibiotics into the tube
3. Picked 6 colony from the fresh overnight plate
4. Innoculated the colony in the LB media
5. Incubated overnight at 37 °C
   • 6x1 Biobricks cloned

Maxiprep of Biobricks (preparation)

1. Cells were spun down and pelleted
2. Cells stored at -20 °C for Maxiprep tomorrow