IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.1

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Contents

1.1 Protocol for Empty Vesicle Formation

Day 1

Equipment

  • 100ml glass beaker x1
  • Gilsson pipette (200µl) + pipette tips
  • Nitrogen tap
  • 1000µl pipette tip
  • Desiccator connected to a vacuum
  • 25ml Glass pipette x1
  • Sonicator with medium-sized probe
  • 25°C incubator

Reagents

  • 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
  • Mineral oil

(Note: Alternatively, use POPC and dodecane)

Preparing the lipid-oil suspension for the inner leaflet

  1. Place 125 µl of the 20 mg/ml DOPC solution in a 100-ml glass beaker. (Equipment should generally be made of glass and not plastic so as to prevent adhesion of lipid molecules to plastic surface)
  2. Using plastic tubing and a 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film. (Rubber tubes are not recommended as they are more likely to emit debis into the lipid film)
  3. Put the beaker in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
  4. Add 50 ml of mineral oil to reach a final lipid concentration of 0.05 mg/ml
  5. Place the beaker containing the suspension in the ice bath
  6. Sonicate suspension for 30 min (Pulse 1, ~10 Amp). (This is to disperse the phospholipids)
  7. Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil


Day 2

Equipment

  • Magnetic stirrer
  • 200µl pipette + tips
  • 1000µl pipette + tips
  • 50ml 1-inch diameter glass centrifuge tube
  • 1-inch diameter tabletop centrifuge
  • 5ml syringe + long 16-gauge stainless steel needle x1
  • Test tube x1

Reagents

  • Solution A
    • Tris buffer
    • NaCl
    • ddH20


Preparation of Solution A

  1. Prepare a 10ml solution A with 100 mM NaCl and 5 mM Tris buffer at pH 7.4


Emulsifying the Aqueous Solution

  1. Separate about 5 ml of the lipid-oil suspension into a glass container. (For the interface preparation)
  2. Add 250 µl of solution A to the 45ml lipid-oil suspension in mineral oil. (This is the aqeous solution that would be encapsulated in the vesicles)
  3. Gently stir the mixture with a magnetic stir bar for 3 hours.


Preparing the interface (While emulsion is mixed)

  1. Place 2 ml of lipid-oil suspension over 3 ml of solution A in a 1-inch-diameter centrifuge tube.
  2. Leave for 2–3 h for lipids to achieve the coverage of the interface surface (>3h and the lipid may start to clump together)

(Note: This step can been modified to use 2ml of emulsified solution instead of the lipid-oil suspension)


Formation of bi-layer vesicles

  1. Pour 100 µl of the inverted emulsion over the interface. (Note: This step is omitted if 2ml of emulsified solution is used instead of the lipid-oil suspension)
  2. Centrifuge at 120 x g for 10 min

(Note: Alternatively, centrifuge at 30 x g for 20 min)


Collecting the vesicles:

  1. Using a 5-ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
  2. Expel some of the solution to remove all air from the syringe and needle. (Expelling most of the Solution A would ensure a less diluted solution of vesicles)
  3. With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe. (This prevents the extraction of the lipid-oil suspension when the needle is plunged into the tube)
  4. Gently recirculate the buffer several times.
  5. Aspirate most of the solution into the syringe, and remove the needle from the solution. (Be careful not to aspirate the lipid-oil suspension)
  6. Wipe the tip of the needle clean.
  7. Unload the vesicle suspension into a test tube.
  8. Use optical microscopy to check that the vesicles obtained were not deformed or aggregated. Ideally, the protocol should yield ~109 vesicles of 1µm diameter.


Notes

  • Time required for Day 1: ~ 2h; for Day 2: ~4h.
  • The protocol is based on Engineering Asymmetric Vesicles by Sophie Pautot, Barbara J. Frisken, and D. A. Weitz.
  • Modifications to protocol:
    • The original protocol uses anhydrous 99:1 dodecane:silicone oil solution instead of mineral oil
    • The original protocol uses POPC instead of DOPC phospholipids
    • The original protocol sonicates the suspension in a cleaning sonic bath for 30 min
  • Use of salt in the solution A preparation may require osmolarity considerations
  • Use of GFP as a visual signal may require osmolarity considerations
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