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

Preparations

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