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Overview
PURE liposomes are the basis of a synthetic cell that can perform the fundamental operations of biology: transcription, and translation.
In this protocol, you will make the necessary precursors to creating liposomes, assemble a PURE cell-free reaction that will express Green Fluorescent Protein (GFP), and encapsulate it within a liposome. For the outer solution, you will use the same simple sugar solution that was used in 
Buffer-in-Buffer Liposomes.
Successfully built PURE liposomes will start dark and then increase in green fluorescence over time as GFP is produced.
There are four key stages to making PURE liposomes:
Step | Process | Hands-on Time | Total Time | Notes |
1 | 1 h | 4 h | Buffers and lipids may be prepared in advance and used for experiments on subsequent days. | |
2 | 1 h | 1 h | ||
3 | 2 h | 2 h | ||
4 | 1 h | 6–12 h | Total time depends on the exact experiment and incubation conditions. GFP expression should be seen over the first 6 hours at 37C.
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Materials and Equipment
Name | Product | Manufacturer | Part # | Price | Link |
Buffers | |||||
Glucose | D-(+)-Glucose, 99% | Thermo Scientific | A16828-36 | $41.65 | [link] |
Sucrose | Sucrose, 99% | Thermo Scientific | A15583-36 | $41.65 | [link] |
Lipids | |||||
POPC | 16:0-18:1 PC 25 mg/mL | Avanti Lipids | 850457C-500mg | $435.00 | [link] |
Liss-Rhod-PE | 18:0 Liss Rhod PE 1 mg/mL | Avanti Lipids | 810179P-1mg | $273.47 | [link] |
Mineral Oil | Mineral oil, mixed weight | Thermo Scientific | AC415080010 | $53.40 | [link] |
Glass Syringe 250 uL | Hamilton | 14-815-238 | $150.15 | [link] | |
PURE | |||||
PURE | PURExpress | NEB | E6800S | $295.00 | [link] |
RNase Inhibitor | RNase Inhibitor, Murine | NEB | M0314S | $81.00 | [link] |
DNA | pT7-plamGFP PURE | Nucleus v0.1.0-001 DNA | Well E3 | [link] |
Step 1: Prepare Stock Buffers and Lipids
Stock buffers may be prepared ahead of time, and stored for months. Lipids may also be prepared in advance and stored at 4C for at least 1 month. If you are making buffers and lipids at the same time, begin with the lipids so that they can evaporate while the buffers are being prepared.
Prepare lipids in mineral oil
Clean glass syringes.
Pour a small amount of 95% ethanol into a glass container (e.g. a 10 mL beaker).
Assemble the glass syringe and prime it by drawing ethanol into the glass syringe, then empty into a waste bottle.
Weight 2.67 g (or pipette 3.25mL) mineral oil into a glass test tube or beaker
Add the lipids to the mineral oil using the appropriate glass syringe:
Add 250 uL 25 mg/mL POPC.
Rinse the syringe with ethanol.
Add 10 uL 1 mg/mL Liss Rhod PE.
Rinse the syringe with ethanol.
Evaporate the chloroform from the lipid–oil mixture:
Place lipid beaker in a 55°C dry bath in a fume hood.
(optional) Shield with aluminum foil to protect from light.
Evaporate uncovered for 3 h.
Clean syringes and store with plunger removed
Let the sample cool to room temperature in the fume hood.
If lipids settle towards the bottom, stir them using a 10 uL pipette tip.
Aliquot the lipid–oil mixture into 1.5 mL aliquots, in glass jars.
The lipid–oil mixture can be stored for a week or more at room temperature, or up to one month at 4C.
Invert gently 3 times before use. Make sure the solution is not cloudy.
Prepare sugar stock stock solutions
Buffer | Target Concentration (M) | MW (kDa) | Weight (g) | Final Volume (mL) |
3M Glucose Stock | 3.0 | 180.16 | 5.40 | 10.0 |
2M Sucrose Stock | 2.0 | 342.3 | 10.27 | 15.0 |
Make 3M glucose stock solution:
Combine 5.40 g glucose and 5.0 mL ddH2O in a 50 mL tube.
Heat for 15 s in a microwave and mix vigorously to dissolve material completely.
Add additional MilliQ water to achieve a final volume of 10 mL.
Filter sterilize while warm using a 0.22 um filter and store at -20 C.
Make 2M sucrose stock solution:
Combine 10.27 g sucrose and 5.0 mL ddH2O in a 50 mL tube.
Heat and mix vigorously to dissolve material completely.
Add additional ddH2O to achieve a final volume of 15 mL.
Filter sterilize using a 0.22 um filter and store at -20 C.
Prepare outer buffer
Make a 800 mM glucose outer solution:
Add 1.6 mL 3M glucose stock solution to a 15 mL tube.
Add MilliQ water to a final volume of 6 mL.
Vortex vigorously to mix.
Step 2: Assemble PURE Reactions
PURE reaction setup
Component | Volume (per reaction) (uL) | Notes |
PURE Solution A | 16 | PURE energy solution: small molecules |
PURE Solution B | 12 | PURE proteins and ribosomes |
RNAse Inhibitor | 2 | Prevents RNAse activity |
pT7-deGFP (50 fM, ~100 ng/uL) | 4 | DNA encoding green fluorescent protein |
2M sucrose | 6 | Adds density for phase-transfer and matches osmolarity |
Total | 40 |
Thaw reagents on ice and then keep on ice.
Prepare a PCR strip in a strip holder, on ice, to assemble reactions into.
For each reaction, assemble by adding the reagents from the table in the order listed:
Vortex and spin down Solution A, pipette mix, and dispense 16 uL into the reaction tube.
Briefly spin down Solution B, gently pipette mix, and dispense 12 uL. Do not vortex.
Vortex and spin RNAse Inhibitor, pipette mix, and dispense 2 uL.
Vortex and spin
pT7-deGFP DNA, pipette mix, and dispense 4 uL.Dispense 6 uL 2M sucrose and gently pipette mix the assembled reaction.
Close lids on the PCR tubes and briefly spin down to eliminate bubbles.
(optional) Transfer 10 uL of each assembled reaction to a 384-well assay plate for plate reader measurement of the reaction.
Hold assembled reactions on ice until ready for encapsulation.
Return reagents to their appropriate storage locations.
Add a black dot to the lid of each of PURE Solution A and B. The number of dots indicates freeze–thaw cycles, PURExpress functions well up to 4 cycles.
Step 3: Encapsulate PURE into Liposomes
Set up a microfuge tube rack, with three 1.5 mL microfuge tubes per liposome encapsulation:
Number the tubes per the number of reactions assembled in Step 2.
For each reaction, label the three tubes:
E—oil emulsion
T—transfer
L—liposomes
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Add 150 uL of the lipid / oil mixture to tubes labelled E.
Add 150 uL of 800 mM glucose outer solution to each of the tubes labelled T and L.
Emulsify each reaction in its appropriate oil tube:
Add 30 uL PURE reaction its matching E tube.
Mix the lipid–oil and PURE reaction by running the tube along a row of empty slots on the microfuge tube holder.
Run along the row 5–10 times, until the solution forms a stable emulsion and is an even milky color.
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Immediately layer each emulsion over the transfer solution.
Pipette 150 uL from tube E slowly down the side of the matching tube T.
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Centrifuge T tubes at 9000 g for 10 min at room temperature to pellet the liposomes:
Align the hinges of each microfuge tube towards the outside of the centrifuge rotor, so that the final pellet location will be indicated by the tube hinge.
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Extract the liposomes from each T tube:
Remove the oil layer from the top of each T tube by gently pipetting with a 1000 uL pipette set to 150 uL.
Identify location of the liposome pellet at the corner of the tube where the side wall angles toward the bottom, directly below the hinge.
Gently extract liposomes by pipetting 50 uL of pellet and outer solution from beside the pellet location.
Add the 50 uL liposome sample to the matching liposome tube, L.
Hold liposomes on ice until you are prepared to begin measurement.
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Step 4: Measure Liposomes
Prepare a 18-well imaging slide for microscopy.
Pipette 20 uL of each liposome sample into a well on the slide.
Remaining liposomes may be stored on ice or at 4C.
Configure microscope:
Set magnification to 4x.
Set up imaging conditions for brightfield, GFP fluorescence (480 nm excitation, 520 nm emission), and Rhodamine fluorescence (540 nm excitation, 580 nm emission).
Place the slide on the microscope and prepare for imaging:
Find focus by focusing on the edge of a single well in brightfield.
Locate a well containing a sample of interest.
Move to imaging resolution:
Switch to rhodamine / red fluorescence imaging.
Find focus on liposomes just above the coverslip:
Back focus out until an entire field of liposomes appear to lose focus simultaneously (they are all sitting on the coverslip).
Slowly move back in until these liposomes are in sharp focus.
Move up to a higher magnification, and find focus again as described.
Repeat until you are at an imaging magnification of 20x or 40x.
Capture images of liposomes.
Background Protocols
Prepare lipids for use in encapsulation: 
Lipid Preparation
Lipid Preparation Prepare inner and outer buffers: 
PURE inner and outer solution
PURE inner and outer solution Prepare 
Make Energy Mix
Make Energy Mix 
Resources and References
- Other Protocols
- The Nucleus protocol simplifies this previous work:The Build a Cell Phase-Transfer Liposome Protocol.
- Modified protocol from Miyazaki et al on OpenWetWare.
- Miyazaki protocol on Protocol Exchange
- Papers
- Fujii, S., Matsuura, T., Sunami, T. et al. Liposome display for in vitro selection and evolution of membrane proteins. Nat Protoc 9, 1578–1591 (2014) [link]
- Resources
Credits
- Developers
- Testers
