Difference between revisions of "Chassis/Cell-Free Systems/Vesicle/Preliminary Testing"

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#Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
 
#Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
 
#Prepare solution B by making up the cell extract reaction mixture.
 
#Prepare solution B by making up the cell extract reaction mixture.
 +
#Commercial E.coli Cell Extract: Add 120ul of cell extract + 160ul of premix + 20ul of amino acid mix minus cys + 20ul amino acid mix minus leu to an eppendorf tube.
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#Add 4ug of DNA to complete solution B.
 
#Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
 
#Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
 
#Gently stir the mixture with a magnetic stir bar for 3h.
 
#Gently stir the mixture with a magnetic stir bar for 3h.
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#Wipe the tip of the needle clean.
 
#Wipe the tip of the needle clean.
 
#Unload the vesicle suspension into its final container.
 
#Unload the vesicle suspension into its final container.
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'''Induction of expression'''
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#Add AHL stock solution to vesicle suspension, making a final AHL concentration of 100nM.
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#Wait for 3h, then place 5ul sample of vesicle suspension on the microscope slide for observation.
  
 
''(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)''
 
''(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)''

Revision as of 18:06, 26 October 2007

Vesicle-Encapsulation

Cell-Free Systems Chassis description Preparation protocol Preliminary testing

Preliminary Testing

Picture of a vesicle expressing GFP. Encapsulated within the vesicle is the Commercial E. coli S30 extract together with the DNA constuct BBa_T9002. The vesicle was seen under a phase-contrast microscope with white light (left picture) and with a filter (right picture).

Experimental protocol

Day 1

Equipment

  • Nitrogen tap + plastic tubing
  • Desiccator connected to a vacuum
  • 100ml glass bottle
  • Sonicator with medium-sized probe
  • Ice bath
  • 25°C incubator
  • Pipette + pipette tips (1000µl)

Reagents

  • 10ml dodecane
  • 12.5µl 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%

Procedure

Preparing the lipid-oil suspension for the inner leaflet

  1. Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle.
  2. With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
  3. Put the bottle in a desiccator connected to a vacuum for 1h.
  4. Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml.
  5. Set the sonicator probe to pulse 1, timer at 30mins.
  6. Put the bottle containing the suspension in the ice bath.
  7. Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating.
  8. Sonicate the suspension for 30mins.
  9. Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil.

Day 2

Equipment

  • Magnetic stirrer
  • Centrifuge + 1-inch glass centrifuge tubes
  • Pipette + pipette tips (200µl, 1000µl)
  • 50ml glass tube
  • 5ml syringe
  • Long 16-gauge stainless steel needle

Reagents

  • 10ml ddH2O
  • Tris buffer
  • NaCl
  • Reporter

Procedure

Emulsifying the aqueous solution (while the interface settles)

  1. Separate about 5ml of the lipid-oil suspension into a glass container. This is for the interface preparation.
  2. Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
  3. Prepare solution B by making up the cell extract reaction mixture.
  4. Commercial E.coli Cell Extract: Add 120ul of cell extract + 160ul of premix + 20ul of amino acid mix minus cys + 20ul amino acid mix minus leu to an eppendorf tube.
  5. Add 4ug of DNA to complete solution B.
  6. Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
  7. Gently stir the mixture with a magnetic stir bar for 3h.

Preparing the interface (to be done while the emulsion is mixing)

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

Forming the vesicles

  1. Pour 100µl of the inverted emulsion over the interface.
  2. Centrifuge at 120g for 10min.

Collecting the vesicles

  1. Using a 5ml 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.
  3. With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe.
  4. Gently recirculate the buffer several times.
  5. Aspirate most of the solution into the syringe, and remove the needle from the solution.
  6. Wipe the tip of the needle clean.
  7. Unload the vesicle suspension into its final container.

Induction of expression

  1. Add AHL stock solution to vesicle suspension, making a final AHL concentration of 100nM.
  2. Wait for 3h, then place 5ul sample of vesicle suspension on the microscope slide for observation.

(Note: Use optical microscopy to check that the vesicles obtained arenot deformed or aggregated.)

Notes

  • Time Required:
    • The lipid-oil suspension preparation takes about 2h (with a 1h waiting period 15min into the procedure), before being left overnight.
  • The remainder of the procedure takes another 4h, with one 2h waiting period after an initial 1h preparation.
    • Total working time in the lab is around 3 hours.
  • 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 30min.
  • Do not use rubber tubing in the nitrogen evaporation. This emits debris into the lipids.
  • This procedure should form around 10^9 vesicles with 1µm diameter.
  • Use of salt in the solution A preparation may require osmolarity considerations.
  • Use of GFP as a visual signal may require osmolarity considerations.
  • The interface should settle for more than 2h, but less than 3h. More than 3h causes the lipids to clump.