1.3 Protocol for Adding Cell Extract into Vesicles
- Glass vial 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
- 10ml of dodecane
- 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
- 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 20mg/ml in chloroform, ≥99.0%
Preparing the lipid-oil suspension for the inner leaflet
- Place 25 µl of the 20 mg/ml DOPC or POPC solution in a glass vial. (Equipment should generally be made of glass and not plastic so as to prevent adsorption of lipid molecules to plastic surface)
- 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 debris into the lipid film)
- Put the vial in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
- Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml.
- Place the vial containing the suspension in the ice bath.
- Sonicate suspension for 30 min (Pulse 1, ~10 Amp). (This is to disperse the phospholipids)
- Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in dodecane.
- Small tubes
- Aluminium foil
- 20µl pipette + tips
- 200µl pipette + tips
- 1000µl pipette + tips
- Tabletop centrifuge
- Vortex machine
- Syringe + stainless steel needle
- Glass slide + cover slip
- Optical microscope with phase contrast
- Fluorescent microscope
- Commercial S30 E.coli extract
- E.coli complete amino acid mixture
- S30 premix without amino acid
- S30 extract circular
- Nuclease-free water
- Plasmid DNA wtih pTet-GFP construct
Preparation of Reaction
- Add 5µl of E.coli complete amino acid mixture into a tube.
- Then add 20µl of S30 premix without amino acid.
- Next add 15µl of S30 extract circular.
- Add an appropriate volume of plasmid DNA depending on DNA concentration. (How many DNA molecules are needed in this step?)
- Finally add nuclease-free water to bring final volume to 100µl.
Formation of mono-layer vesicles
- Add 1µl of reaction into another tube containing 200μl of lipid-oil suspension.
- Vortex gently for a few seconds. (This is to break up the small aqueous droplet to form an extract-oil emulsion)
- Leave to stand for a few minutes. (Microdroplets will be stabilized by a monolayer of phospholipids at the oil–extract interface)
Formation of bi-layer vesicles
- Place 50μl of the extract-oil emulsion on top of 25μl S30 premix with amino acids.
- Leave to stand for a few minutes. (A monolayer of phospholipids will form at the interface of the biphasic solution)
- Centrifuge at 120 x g for 10 min.
(Note: Alternatively, centrifuge at 30 x g for 20 min)
Collecting the vesicles:
- Using a syringe with a stainless steel needle, collect some of the S30 premix.
- Expel some of the premix to remove all air from the syringe and needle. (Expelling most of the S30 premix would ensure a less diluted solution of vesicles)
- With the tip of the needle in the aqueous phase, gently expel the premix contained in the syringe. (This prevents the extraction of the lipid-oil suspension when the needle is plunged into the tube)
- Gently recirculate the solution several times.
- Aspirate most of the solution into the syringe, and remove the needle from the solution. (Be careful not to aspirate the lipid-oil suspension)
- Wipe the tip of the needle clean.
- Unload the vesicle suspension into a tube and store in the dark.
- Use optical microscopy to check that the vesicles obtained were not deformed or aggregated.
Ideally, the protocol should yield a few hundreds of vesicles and aggregates of 1 to a few tens of micrometers diameter. (Caution: Over-exposure of light under the microscope would bleach the GFP!!)
- Time required for Day 1: ~ 2h; for Day 2: ~2h.
- The protocol is based on A vesicle bioreactor as a step toward an artificial cell assembly by Vincent Noireaux and Albert Libchaber.
- Modifications to protocol:
- The original protocol uses mineral oil instead of dodecane
- The original protocol uses egg lecithin instead of DOPC or POPC
- The original protocol does not use Span 80