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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.
|
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] |
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
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 |
Prepare outer buffer
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 |
pT7-deGFP
DNA, pipette mix, and dispense 4 uL.- 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
- Developers
- Testers