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

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(New page: ===Preparation of Vesicles Containing Cell Extract === The protocol is based on [http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=12963816 Engineering Asymmetric Ve...)
 
 
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===Preparation of Vesicles Containing Cell Extract ===
==1.3 Protocol for Adding Cell Extract into Vesicles==
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.


====Materials====
===Day 1===
=====Equipment=====
====Equipment====
* Sonicator with medium-sized probe
* Glass vial x1
* Gilsson pipette (200µl) + pipette tips
* Nitrogen tap
* Nitrogen tap
* 1000µl pipette tip
* Desiccator connected to a vacuum
* Desiccator connected to a vacuum
* 25ml Glass pipette x1
* Sonicator with medium-sized probe
* 25°C incubator
* 25°C incubator
* Magnetic stirrer
* 120 x g centrifuge (1-inch tubes)
* 200µl pipette
* 1000µl pipette
* 50ml glass tube
* 100ml glass bottle


=====Chemicals and reagents=====
====Reagents====
* 50ml Mineral oil
* 10ml of dodecane
* 125µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
and
* 10ml ddH2O
* 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
* Tris buffer
or
* NaCl
* 1-Palmitoyl,2-oleoyl-sn-Glycero 3-phosphocholine (POPC) 20mg/ml in chloroform, ≥99.0%
* REPORTER - will probably be GFP (Determine quantity)


=====Supplies=====
* 1-inch diameter glass centrifuge tube
* 5-ml syringe
* long 16-gauge stainless steel needle
* Ice bath
* 1ml pipette tip
* Plastic tubing


====Procedure====


'''Preparing the lipid-oil suspension for the inner leaflet:'''
'''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.


# Place 125 µl of the 20 mg/ml DOPC solution in a 100-ml glass bottle
===Day 2===
# With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
====Equipment====
# Put the bottle in a desiccator connected to a vacuum for 1hr
* Small tubes
# Add 50 ml of mineral oil to reach a final lipid concentration of 0.05 mg/ml
* Aluminium foil
# Set the sonicator probe to pulse 1, timer at 30mins
* 20µl pipette + tips
# Put the bottle containing the suspension in the ice bath
* 200µl pipette + tips
# Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating
* 1000µl pipette + tips
# Sonicate the suspension for 30mins
* Tabletop centrifuge
# Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil
* Vortex machine
* Syringe + stainless steel needle
* Glass slide + cover slip
* Optical microscope with phase contrast
* Fluorescent microscope


'''Emulsifying the acqueous solution:''' (while the interface settles)
====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


# Separate about 5 ml of the lipid-oil suspension into a glass container (for the interface preparation)
# Prepare a 10ml solution A with 100 mM NaCl and 5 mM Tris buffer at pH 7.4
# Prepare solution B by ADDING REPORTER (Determine quantity) to 1ml of solution A.
# Add 250 µl of solution B to the 45ml lipid-oil suspension in mineral oil
# Gently stir the mixture with a magnetic stir bar for 3 hours


'''Preparing the interface:''' (to be done while the emulsion is mixed)
'''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.


# Place 2 ml of lipid-oil suspension over 3 ml of solution A in a 1-inch-diameter centrifuge tube.
# Leave for 2–3 h for lipids to achieve the coverage of the interface surface


'''Forming the vesicles:'''
'''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)'''


# Pour 100 µl of the inverted emulsion over the interface
# Centrifuge at 120 x g for 10 min


'''Collecting the vesicles:'''
'''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)''


# Using a 5-ml syringe with a long 16-gauge stainless steel needle, collect some of solution A
# Expel some of the solution to remove all air from the syringe and needle
# With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe
# Gently recirculate the buffer several times
# Aspirate most of the solution into the syringe, and remove the needle from the solution
# Wipe the tip of the needle clean
# Unload the vesicle suspension into its final container


Use optical microscopy to check that the vesicles obtained were not deformed or aggregated.
'''Collecting the vesicles:'''
 
# Using a syringe with a stainless steel needle, collect some of the S30 premix.
=====Time Required=====
# 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)'''
* The lipid-oil suspension preparation takes about 2 hours, before being left overnight (with a 1hr waiting period 15min into the procedure).
# 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)'''
* The remainder of the procedure takes another 4 hours, with one 2hr waiting period after an initial 1hr preparation.
# Gently recirculate the solution several times.
* Total working time in the lab is around 3 hours.
# 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!!)'''


====Notes====
===Notes===
* The original protocol uses anhydrous 99:1 dodecane:silicone oil solution instead of mineral oil
* Time required for Day 1: ~ 2h; for Day 2: ~2h.
* The original protocol uses POPC instead of DOPC phospholipids
* 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.
* The original protocol sonicates the suspension in a cleaning sonic bath for 30 min
*Modifications to protocol:
* Do not use rubber tubing in the nitrogen evaporation - these emit debris into the lipids
** The original protocol uses mineral oil instead of dodecane
* This procedure should form around 10^9 vesicles with 1µm diameter
** The original protocol uses egg lecithin instead of DOPC or POPC
* Use of salt in the solution A preparation may require osmolarity considerations
** The original protocol does not use Span 80
* The reporter in solution B is optional - the vesiles may be visible without it
* Use of GFP as a visual signal may require osmolarity considerations
* The interface should settle for more than 2 hours, but less than 3 - more than three and the lipids begin to clump.

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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