IGEM:IMPERIAL/2007/CFS/Design/Protocols

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Cell-Free Systems




Protocol for Preparation of E. coli S30 Extract

Day 1

Equipment

  • 37°C shaking incubator
  • 1L conical flasks x 3
  • Pipette fillers + pipettes (5ml, 10ml and 25ml)
  • Spectrometer + cuvettes
  • Weighing scale
  • Centrifuge + 150ml centrifuge tubes
  • Pipettes + pipette tips (20µl, 200µl and 1000µl)

Reagent

  • 2xYT medium
  • IPTG
  • Buffer A
    • 10mM Tris-acetate (pH 8.2)
    • 14mM Mg-acetate
    • 60mM K-lutamate
    • 1mM dithiothreitol (DTT)
    • 0.05% (v/v) 2-mercaptoethanol (2-ME)

Procedure

Growing the cells

  1. Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
  2. Add 1mM IPTG to cell culture to express T7 RNA polymerase.
  3. Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
  4. Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
  5. Centrifuge and weigh the wet cell pellets before storing them at -80°C.

(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)

Day 2

Equipment

  • Pipette filler + pipettes (5ml, 10ml and 25ml)
  • Weighing scale
  • French press + French press cell
  • Centrifuge + 50ml centrifuge tubes
  • Pipette + pipette tips (20µl, 200µl, 1000µl)
  • 37°C shaking incubator
  • Dialysis membrane with molecular weight cut-off of 10,000
  • Magnetic stirrer
  • 4°C cold room

Reagent

  • Buffer B
    • 10mM Tris-acetate (pH 8.2)
    • 14mM Mg-acetate
    • 60mM K-glutamate
    • 1mM DTT
  • Pre-incubation solution
    • 293.3mM Tris-acetate (pH 8.2)
    • 2mM Mg-acetate
    • 10.4mM ATP
    • 4.4mM DTT
    • 0.04mM amino acids
    • 16.9mM phosphoenolpyruvate
    • 0.77U/ml pyruvate kinase

(Note: For the ATP regenerating system in the pre-incubation solution, phosphoenolpyruvate and pyruvate kinase are used instead of creatine phosphate and creatine kinase. This is due to cost considerations.)

Procedure

Lysing the cells

  1. Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
  2. Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.

Retaining the cell extract

  1. Centrifuge the crude lysate at 30,000RCF for 30min at 4°C.
  2. Carefully remove the top layer of the supernatant (lipid layer) and the pellet and centrifuge again.
  3. Shake the final supernatant at 100rpm.
  4. Gradually add 3ml of the pre-incubation solution to 10ml of the supernatant.
  5. Pre-incubate the supernatant with gentle shaking at 37°C for 80min. This degrades endogenous genetic content (DNA and mRNA).
  6. Dialyze the pre-incubated sample for 45min each at 4°C against 50 volumes of buffer B using a membrane with molecular weight cut-off of 10,000. Repeat the dialysis step three times.
  7. Centrifuge the retained extract at 4000RCF for 10min at 4°C to obtain the supernatant.
  8. Divide resulting S30 extract into small aliquots and store at -80°C.

(Note: Protease inhibitors are added to pre-incubation solution to prevent degradation of proteins required for gene expression.)

Notes


Protocol for Preparation of E. coli S12 Extract

Day 1

Equipment

  • 37°C shaking incubator
  • 1L conical flasks x 3
  • Pipette fillers + pipettes (5ml, 10ml and 25ml)
  • Spectrometer + cuvettes
  • Weighing scale
  • Centrifuge + 150ml centrifuge tubes
  • Pipettes + pipette tips (20µl, 200µl and 1000µl)

Reagent

  • 2xYT medium
  • IPTG
  • Buffer A
    • 10mM Tris-acetate (pH 8.2)
    • 14mM Mg-acetate
    • 60mM K-lutamate
    • 1mM dithiothreitol (DTT)
    • 0.05% (v/v) 2-mercaptoethanol (2-ME)

Procedure

Growing the cells

  1. Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
  2. Add 1mM IPTG to cell culture to express T7 RNA polymerase.
  3. Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
  4. Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
  5. Centrifuge and weigh the wet cell pellets before storing them at -80°C.

(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)

Day 2

Equipment

  • Pipette filler + pipettes (5ml, 10ml and 25ml)
  • Weighing scale
  • French press + French press cell
  • Centrifuge + 50ml centrifuge tubes
  • Pipette + pipette tips (20µl, 200µl, 1000µl)
  • 37°C shaking incubator
  • Dialysis membrane with molecular weight cut-off of 10,000
  • Magnetic stirrer
  • 4°C cold room

Reagent

  • Buffer B
    • 10mM Tris-acetate (pH 8.2)
    • 14mM Mg-acetate
    • 60mM K-glutamate
    • 1mM DTT

Procedure

Lysing the cells

  1. Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
  2. Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.

Retaining the cell extract

  1. Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
  2. Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
  3. Briefly pre-incubate the recovered supernatant at 37°C for 30min.
  4. Divide resulting S12 extract into small aliquots and store at -80°C.

Notes


Protocol for Vesicle Formation

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 adding a suitable quantity of reporter to 1ml of solution A.
  4. Add 250µl of solution B to the 45ml lipid-oil suspension in mineral oil.
  5. 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.

(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 reporter in solution B is optional. The vesicles may be visible without it.
  • The interface should settle for more than 2h, but less than 3h. More than 3h causes the lipids to clump.
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