“Hello, world” PURE Liposomes

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Nucleus Hello World Liposomes Protocol v0.1.2.pdf

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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	Prepare stock buffers and lipids	1 h	4 h	Buffers and lipids may be prepared in advance and used for experiments on subsequent days.
2	Assemble PURE reactions	1 h	1 h
3	Encapsulate liposomes	2 h	2 h
4	Measure and image	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.

<aside> <img src="/icons/wrench_gray.svg" alt="/icons/wrench_gray.svg" width="40px" /> Materials and Equipment

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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]

<aside> <img src="/icons/iterate_gray.svg" alt="/icons/iterate_gray.svg" width="40px" /> Step 1: Prepare Stock Buffers and Lipids

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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 480 uL 25 mg/mL POPC.
- [ ] Rinse the syringe with ethanol.
- [ ] Add 15 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 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.