User:Cheuk Ka Tong/Sandbox5/Protocols
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
- 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.
- Add 1mM IPTG to cell culture to express T7 RNA polymerase.
- Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
- Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
- 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
- Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
- Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.
Retaining the cell extract
- Centrifuge the crude lysate at 30,000RCF for 30min at 4°C.
- Carefully remove the top layer of the supernatant (lipid layer) and the pellet and centrifuge again.
- Shake the final supernatant at 100rpm.
- Gradually add 3ml of the pre-incubation solution to 10ml of the supernatant.
- Pre-incubate the supernatant with gentle shaking at 37°C for 80min. This degrades endogenous genetic content (DNA and mRNA).
- 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.
- Centrifuge the retained extract at 4000RCF for 10min at 4°C to obtain the supernatant.
- 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
- Total time required: ~ 3 days.
- The protocol is based on Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al.
- Modifications to protocol:
- The original protocol uses creatine phosphate and creatine kinase instead of phosphoenolpyruvate and pyruvate kinase.
- The original protocol does not use protease inhibitors in the pre-incubation solution.
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
- 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.
- Add 1mM IPTG to cell culture to express T7 RNA polymerase.
- Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
- Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
- 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
- Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
- Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.
Retaining the cell extract
- Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
- Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
- Briefly pre-incubate the recovered supernatant at 37°C for 30min.
- Divide resulting S12 extract into small aliquots and store at -80°C.
Notes
- Total time required: ~ 3 days.
- The protocol is based on Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system by Kim DM et al.
- We did not prepare any E. coli S12 extract because of the lack of reagents and limited budget.
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
- Place 125µl of the 20mg/ml DOPC solution in a 100ml glass bottle.
- With the plastic tubing and 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film.
- Put the bottle in a desiccator connected to a vacuum for 1h.
- Add 50ml of mineral oil to reach a final lipid concentration of 0.05mg/ml.
- Set the sonicator probe to pulse 1, timer at 30mins.
- Put the bottle containing the suspension in the ice bath.
- Secure the sonicator probe inside the bottle, and set the amplitude to a reading of 10 when it is sonicating.
- Sonicate the suspension for 30mins.
- 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)
- Separate about 5ml of the lipid-oil suspension into a glass container. This is for the interface preparation.
- Prepare a 10ml solution A with 100mM NaCl and 5 mM Tris buffer at pH 7.4.
- Prepare solution B by adding a suitable quantity of reporter to 1ml of solution A.
- 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.
Preparing the interface (to be done while the emulsion is mixing)
- Place 2ml of lipid-oil suspension over 3ml of solution A in a 1-inch-diameter centrifuge tube.
- Leave for 2–3h for lipids to achieve the coverage of the interface surface.
Forming the vesicles
- Pour 100µl of the inverted emulsion over the interface.
- Centrifuge at 120g for 10min.
Collecting the vesicles
- Using a 5ml 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.
(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.