Build a Base Cell

Overview¶

This protocol takes you from starting materials to finished Base Cells in one pass. It joins three parts that are documented on separate pages: the lipid–oil mixture for the Base Membrane: POPC/Chol, the outer solution, and an inner solution made of Base Cytosol. Encapsulation: Phase Transfer then closes the membrane around the inner solution.

Plan the day around the lipids. The lipid–oil mixture takes about 4.5 h and keeps for one week at 4 °C, so make it first, or up to a week before. Assemble the inner solution last, on ice, immediately before you encapsulate, because it starts to express as soon as it is warm.

Protocol¶

1. Prepare Lipids in Mineral Oil¶

• Add 1 mL of mineral oil to a 4 mL glass vial using a 1 mL pipette.

• Add the lipids in Lipids for 1 mL of mineral oil. to the glass vial on top of the mineral oil using the appropriate glass syringe.

• Briefly vortex the lipid-oil mixture for 5 s to mix.

• Evaporate the chloroform from the lipid–oil mixture:

  • Place glass vial in a 55 °C dry bath in a fume hood.

  • Shield with aluminum foil to protect from light.

  • Evaporate uncovered for 4 h.

• Meanwhile, clean your glass syringe.

  • Add 4 mL of chloroform to a glass bottle using a glass 10 mL serological pipette.

  • Rinse your syringe with that chloroform 5 times.

  • Dry the syringes with the plunger removed inside the fume hood for 3 h to 4 h to allow remaining chloroform to evaporate.

• Cool the lipid-oil mixture to room temperature (between 10 min and 15 min). Your lipid-oil mixture is now ready for use.

• Return the plungers to the syringes and store them in their designated location.

• Dispose of chloroform waste following applicable chemical safety guidelines.

2. Assemble the Outer Solution¶

• Mix 2 M glucose and water as below. This makes 1 mL of 1140 mM glucose, which is enough for three encapsulations.

• Read the outer solution’s osmotic concentration on the osmometer, and record it.

3. Assemble the Inner Solution¶

The inner solution is Base Cytosol with Optiprep. Each reaction is 40 µL: 10 µL goes to the osmometer, and the remaining 30 µL is what one encapsulation takes.

• Thaw SMix, tRNA, PMix, ribosomes and RNase inhibitor on ice.

• Chill one 1.5 mL microcentrifuge tube per reaction on ice.

• Resuspend each component:

  • Vortex SMix aggressively until visibly clear. Alternate 10 s vortex / 10 s rest on ice to maintain cool temperature. SMix should be transparent with no visible precipitate when ready.

  • Vortex or pipette mix tRNA.

  • Pipette mix PMix.

  • Do NOT vortex ribosomes! Gently pipette mix or flick the tube.

• Add each component to its chilled tube, in the order and volume of Inner solution, one reaction..

• Mix thoroughly by pipette (6-10x) until visibly homogeneous.

• Take 10 µL of the inner solution and read its osmotic concentration on the osmometer.

• Compare it with the outer solution’s reading from part 2. The outer solution must read 100 mOsm/kg to 150 mOsm/kg lower. If it does not, adjust its glucose concentration and read it again.

• Hold the inner solution on ice until part 4.

4. Encapsulate¶

• Allow the lipid-oil mixture to come to room temperature as you perform these next steps.

• For each liposome encapsulation, set up a 1.5 mL tube rack with two 1.5 mL microcentrifuge tubes. Number the tubes according to the number of reactions assembled. Label the two tubes for each reaction:

  • T—transfer

  • L—liposomes

• Add 300 µL of the outer solution to each of the tubes labeled T. Vortex thoroughly and spin down.

• Once at room temperature, add 150 µL of the lipid-oil mixture on top of each 30 µL inner solution.

• Emulsify the lipid-oil and inner solution by running the tube along a row of empty slots on the 1.5 mL tube rack. Drag your tube across empty slots at least 50 times until you form a stable emulsion with a homogeneous milky appearance.

• Immediately layer each emulsion on top of the outer solution by slowly pipetting down the side of the corresponding T tube.

• Centrifuge T tubes at 9000 g for 10 min at room temperature to pellet the liposomes.

• Extract the liposomes from each T tube:

  • Gently remove the oil layer and lipid debris from the top of each T tube with a 1000 µL pipette.

  • Gently resuspend the pellet by mixing by pipette 10-15 times.

  • Extract liposomes by pipetting 50 µL to 100 µL of pellet and outer solution from T and transfer liposome sample to the respective liposome tube L.

• Hold liposomes on ice until you are prepared to begin measurement.

• Transfer 5 µL liposomes and 20 µL of the outer solution onto a 384-well glass bottom plate (5x dilution). If the density of liposomes appears too high under the microscope, dilute them with outer solution to facilitate data analysis.

• Observe liposomes by fluorescence microscopy. Base Cells have red fluorescent membranes. With pOpen-deGFP in the inner solution, green fluorescence rises over time; see Base Cell for the expected kinetics.

5. Return Reagents¶

• Return the Base Cytosol components to their storage locations.

• Mark the lid of each Base Cytosol component that you used. The number of dots indicates how many freeze–thaw cycles each component has gone through.

Downloads¶