Assemble Base Cell

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.

Protocol¶

Prepare Stock Buffers and Lipids¶

• 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.

• Prepare sugar stock stock solutions

  • 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 ddH2O 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 for 15 s 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 0.8 mL 3M glucose stock solution to a 15 mL tube.

    • Add ddH2O water to a final volume of 3 mL.

    • Vortex vigorously to mix.