Assemble Base Cell

Overview¶

Base Cells are liposome-based synthetic cells that can perform transcription and translation.

In this protocol, you will make the necessary precursors for liposome encapsulation, and then assemble and encapsulate Cytosol that expresses Green Fluorescent Protein (GFP). The Base Cell is deployed in a gluocose outer solution.

Successfully built Base Cells will start dark and then increase in green fluorescence over time as GFP is produced.

Protocol¶

Prepare Stock Solutions¶

Prepare lipids in mineral oil

• Add 1 mL of mineral oil to the 1.8 mL glass vial using a 1 mL pipette.

• Add the lipids in the above Table to the glass vial on top of the mineral oil using the appropriate glass syringe.

• Briefly vortex the lipid-oil mixture for 5 seconds 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 hours.

• In a glass bottle, add 4 mL of chloroform using a glass 10 mL serological pipette.

• Clean syringes by rinsing with chloroform 5 times into an empty glass bottle. Store the syringes with the plunger removed inside the fume hood for 3–4 hours to allow remaining chloroform to evaporate.

• After 4 hours of incubation at 55°C in a dry bath in a fume hood, allow the lipid-oil mixture to cool to room temperature for 10-15 minutes.

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

• Dispose of chloroform waste following applicable chemical safety guidelines.

Assemble Outer Solutions¶

• Prepare 1.5 mL microcentrifuge tubes labelled with the appropriate reaction.

• Mix glucose stock solution and water according to the following table:

Assemble Cytosol Reactions¶

• Remove all components listed in the Composition Table above from appropriate cold storage.

• Thaw reagents on ice.

• Prepare 1.5 mL microcentrifuge tubes, on ice, to assemble reactions into.

• For a given reaction, assemble by adding the volume of reagents from the Composition Table in the order listed. Pay special attention to the handling of the Cytosol components:

  • Vortex SMix: Ensure thorough mixing; 10s vortex / 10s rest on ice; should be transparent with no visible precipitate; and add to the reaction tubes.

  • Vortex or pipette mix tRNA, and add to the reaction tubes.

  • Vortex or pipette mix PMix, and add to the reaction tubes.

  • Do NOT vortex ribosomes: gently pipette mix or flick the tube, and add to the reaction tubes.

  • Add remaining reactions in the order they appear

• Mix the master mix thoroughly by pipetting up and down 10–15 times until it appears homogeneous and clear.

• Close lids on the microcentrifuge tubes and briefly spin down to eliminate bubbles.

• Pipette out 10 µL of the reaction for osmolarity check using a Vapor Pressure Osmometer before starting encapsulation.

• Hold assembled reactions on ice until ready for encapsulation.

Encapsulate Cytosols into Liposomes¶

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

  • T—transfer

  • L—liposomes

• Add 300 uL of the appropriate glucose outer solution to each of the tubes labelled T.

• Add 150 uL of the lipid-oil mixture (at room temperature) on top of each assembled Cytosol reaction.

• Emulsify the lipid-oil and Cytosol reaction by running the tube along a row of empty slots on the 1.5 mL tube rack. Run it down 20–30 times until the solution forms a stable emulsion with an even milky color.

• Immediately layer each emulsion over the outer solution. Slowly pipette the entire emulsion 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:

  • Remove the oil layer and lipid debris from the top of each T tube by gently pipetting with a 1000 uL pipette set to 800 uL.

  • Gently pipette mix the pellet 10-15 times with the outer solution.

  • Extract liposomes by pipetting 50-100 uL 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.

• Pipette the liposomes into a well on a 384-well glass bottom plate. If the density appears too high under the microscope, dilute the liposomes with the outer solution for better data analysis.

• Begin measurement.

Return reagents to their appropriate storage locations.

• Add a black dot to the lid of each of Cytosol component. The number of dots indicates freeze–thaw cycles.

Downloads¶