IGEM:IMPERIAL/2007/Projects/In-Veso/Implementation/Protocol1.3: Difference between revisions

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===Day 1===
===Day 1===
====Equipment====
====Equipment====
* 100ml glass beaker x1
* Glass vial x1
* Gilsson pipette (200µl) + pipette tips
* Gilsson pipette (200µl) + pipette tips
* Nitrogen tap
* Nitrogen tap
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* 25ml Glass pipette x1
* 25ml Glass pipette x1
* Sonicator with medium-sized probe
* Sonicator with medium-sized probe
* 25°C incubator  
* 25°C incubator
 
====Reagents====
====Reagents====
* 10ml of dodecane
and
* 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
* 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
* Mineral oil
or
''(Note: Alternatively, use POPC and dodecane)''
* 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 20mg/ml in chloroform, ≥99.0%
 
 


'''Preparing the lipid-oil suspension for the inner leaflet'''
'''Preparing the lipid-oil suspension for the inner leaflet'''
# 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)'''
# 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 debis into the lipid film)'''
# 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 beaker in a desiccator connected to a vacuum for 1hr. '''(This is to remove the chloroform)'''
# Put the vial in a desiccator connected to a vacuum for 1hr. '''(This is to remove the chloroform)'''
# Add 50 ml of mineral oil to reach a final lipid concentration of 0.05 mg/ml
# Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml.
# Place the beaker containing the suspension in the ice bath
# 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)'''
# 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 oil
# Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in dodecane.
 


===Day 2===
===Day 2===
====Equipment====
====Equipment====
* Magnetic stirrer
* Small tubes
* Aluminium foil
* Aluminium foil
* 20µl pipette + tips
* 200µl pipette + tips
* 200µl pipette + tips
* 1000µl pipette + tips
* 1000µl pipette + tips
* 50ml 1-inch diameter glass centrifuge tube
* Tabletop centrifuge
* 1-inch diameter tabletop centrifuge
* Vortex machine
* 5ml syringe + long 16-gauge stainless steel needle x1
* Syringe + stainless steel needle
* Test tube x1
* Glass slide + cover slip
* Optical microscope with phase contrast
* Fluorescent microscope


====Reagents====
====Reagents====
* Solution A
* Commercial S30 E.coli extract
** Tris buffer
** E.coli complete amino acid mixture
** NaCl
** S30 premix without amino acid
** ddH<sub>2</sub>0
** S30 extract circular
* GFP standard Solution
** Nuclease-free water
* Plasmid DNA wtih pTet-GFP construct


'''Preparation of Solution A'''
# Prepare a 10ml solution A with 100 mM NaCl and 5 mM Tris buffer at pH 7.4


'''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.


'''Emulsifying the Aqueous Solution'''
# Separate about 5 ml of the lipid-oil suspension into a glass container. '''(For the interface preparation)'''
# Prepare 10X GFP standard solution with Solution A to form 1ml aqeous solution.
# Add 250 µl of the aqeous solution to the 45ml lipid-oil suspension in mineral oil.
# Wrap the beaker in aluminium foil. '''(This is to prevent bleaching of GFP)'''
# Gently stir the mixture with a magnetic stir bar for 3 hours.


 
'''Formation of mono-layer vesicles'''
'''Preparing the interface''' (While emulsion is mixed)
# Add 1µl of reaction into another tube containing 200μl of lipid-oil suspension.
# Place 2 ml of lipid-oil suspension over 3 ml of solution A in a 1-inch-diameter centrifuge tube.  
# Vortex gently for a few seconds. '''(This is to break up the small aqueous droplet to form an extract-oil emulsion)'''
# Leave for 2–3 h for lipids to achieve the coverage of the interface surface '''(>3h and the lipid may start to clump together)'''
# Leave to stand for a few minutes. '''(Microdroplets will be stabilized by a monolayer of phospholipids at the oil–extract interface)'''
''(Note: This step can been modified to use 2ml of emulsified solution instead of the lipid-oil suspension)''




'''Formation of bi-layer vesicles'''
'''Formation of bi-layer vesicles'''
# 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)''
# Place 50μl of the extract-oil emulsion on top of 25μl S30 premix with amino acids.
# Centrifuge at 120 x g for 10 min
# 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)''
''(Note: Alternatively, centrifuge at 30 x g for 20 min)''




'''Collecting the vesicles:'''
'''Collecting the vesicles:'''
# Using a 5-ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
# Using a syringe with a stainless steel needle, collect some of the S30 premix.
# 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)'''
# 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 solution contained in the syringe. '''(This prevents the extraction of the lipid-oil suspension when the needle is plunged into the tube)'''
# 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 buffer several times.
# 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)'''
# 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.
# Wipe the tip of the needle clean.
# Unload the vesicle suspension into a test tube and store in the dark.
# 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 ~10<sup>9</sup> vesicles of 1µm diameter. '''(Caution: Over-exposure of light under the microscope would bleach the GFP!!)'''
# 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!!)'''


===Notes===
===Notes===
* Time required for Day 1: ~ 2h; for Day 2: ~4h.
* Time required for Day 1: ~ 2h; for Day 2: ~2h.
* The protocol is based on [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12963816 Engineering Asymmetric Vesicles] by Sophie Pautot, Barbara J. Frisken, and D. A. Weitz.
* The protocol is based on [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=15591347 A vesicle bioreactor as a step toward an artificial cell assembly] by Vincent Noireaux and Albert Libchaber.
*Modifications to protocol:
*Modifications to protocol:
** The original protocol uses anhydrous 99:1 dodecane:silicone oil solution instead of mineral oil
** The original protocol uses mineral oil instead of dodecane
** The original protocol uses POPC instead of DOPC phospholipids
** The original protocol uses egg lecithin instead of DOPC or POPC
** The original protocol sonicates the suspension in a cleaning sonic bath for 30 min
** The original protocol does not use Span 80
* Use of GFP as a visual signal may require osmolarity considerations

Latest revision as of 04:59, 3 September 2007

1.3 Protocol for Adding Cell Extract into Vesicles

Day 1

Equipment

  • 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

Reagents

  • 10ml of dodecane

and

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

or

  • 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 20mg/ml in chloroform, ≥99.0%


Preparing the lipid-oil suspension for the inner leaflet

  1. 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)
  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 debris into the lipid film)
  3. Put the vial in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
  4. Add 10 ml of dodecane to reach a final lipid concentration of 0.05 mg/ml.
  5. Place the vial 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 dodecane.

Day 2

Equipment

  • 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

Reagents

  • 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

  1. Add 5µl of E.coli complete amino acid mixture into a tube.
  2. Then add 20µl of S30 premix without amino acid.
  3. Next add 15µl of S30 extract circular.
  4. Add an appropriate volume of plasmid DNA depending on DNA concentration. (How many DNA molecules are needed in this step?)
  5. Finally add nuclease-free water to bring final volume to 100µl.


Formation of mono-layer vesicles

  1. Add 1µl of reaction into another tube containing 200μl of lipid-oil suspension.
  2. Vortex gently for a few seconds. (This is to break up the small aqueous droplet to form an extract-oil emulsion)
  3. 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

  1. Place 50μl of the extract-oil emulsion on top of 25μl S30 premix with amino acids.
  2. Leave to stand for a few minutes. (A monolayer of phospholipids will form at the interface of the biphasic solution)
  3. Centrifuge at 120 x g for 10 min.

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


Collecting the vesicles:

  1. Using a syringe with a stainless steel needle, collect some of the S30 premix.
  2. 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)
  3. 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)
  4. Gently recirculate the solution 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 tube and store in the dark.
  8. 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!!)

Notes

  • 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
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