Preparation of cell-sized water-in-oil droplets
This is a protocol for the encapsulation of the TX-TL cell-free protein expression system in the liposomes developed by Ishiwata et al., Kate Adamala, and Vincent Noireaux. The protocol was used by Malin Jonsson, Miroslav Gasparek (summer 2017 interns), and Zoila Jurado (PhD student at Caltech) to express the GFP in the liposomes in the summer of 2017 in the Murray Lab at Caltech. Although author of this page (Miroslav Gasparek) used this protocol only two times, under the supervision of Dr Shaobin Guo, the protocol delivered the results shown in the picture. I assume that the protocol might have been improved since the summer of 2017, I thought that it still would be worth of sharing.
PREPARATION OF LIPID DRY FILM
1. Dissolve POPC in chlorofom to a final concentration of 65mM (use total 100mg of lipids) in a glass vial.
2. Label membrane with 0.01% Rhodamine (Lissamine rhodamine B) by adding 40μL of 1% Rhodamine B into the 4mL of chloroform and POPC in the glass vial. Mix.
3. Dispense the lipid solution into glass test tubes, 30uL per tube.
4. Place the glass test tubes under a fume hood, preferably in the dark.
5. Evacuate overnight at room temperature in the dark to completely remove chloroform. Use the lipid dry films within a month.
PREPARATION OF LIPID-OIL MIXTURE
6. Mix mineral oil by gentle inversion just before use.
7. Collect a lipid dry film sample.
8. Place 982μL of mineral oil into the glass test tube to the lipid film.
9. Dissolve the lipid dry film by vortexing (for about 10sec).
10. Heat at 50°C for 10min using a dry bath incubator.
11. If the lipid film remains at the bottom of the tube; repeat steps 8 and 9.
12. Incubate overnight with shaking at 37°C. This can be stored longer (few days) with shaking. The concentration of lipids in oil is 1mM.
ENCAPSULATION OF TX/TL INTO WATER IN OIL DROPLETS
13. Add 30μL of the TX/TL reaction (complete with plasmid etc. to a total volume of 30μL) to the lipid-oil mixture.
14. Vortex the lipid-oil + TX/TL mixture for 15sec, then cool down to 4°C for 10min with gentle shaking (cold room shaker) to create an emulsion. The droplet size can be controlled by optimizing the power and duration of vortexing.
15. Load the mixture on top of 250uL of centrifuge buffer (PBS with 150 mM sucrose) in 1.5mL Eppendorf tubes. Wait for the interface to stabilize and flatten.
16. Centrifuge 20,000g in 20min at 4°C.
17. Remove carefully the mineral oil and pipet liposomes from the bottom of the centrifuge buffer layer where the vesicles have been formed.
18. Wash liposomes three times with buffer (whatever your final reaction will be in, for instance 50mM HEPES pH 8 + 300mM sucrose). Wash step centrifugation: 15,000g for 5 min at 4°C.
19. Incubate at 29°C for between 6-12h for protein expression.
PREPARATION FOR MICROSCOPY
1. Place two pieces of double-sided adhesive tape (thickness: ~100 μm) onto a silicone-coated coverslip with a ~8 mm gap between them.
2. Place a normal coverslip on top of the gap, to produce a chamber of 18 mm × 8 mm × 100 μm.
3. Perfuse the water-in-oil emulsion into the created chamber.
4. Seal the chamber with Valap (a mixture of Vaseline, lanolin, and paraffin at equal weight ratios) to prevent flow and evaporation of water from the droplets.
5. Observe the water-in-oil droplets using an 60x optical microscope with oil.
PREPARATION FOR LUMINESCENCE MEASUREMENTS
1. Add 3uL of Quench Mix to the 30 uL of vesicle+TXTL sample. (100uL of Quench Mix is prepared by mixing 3uL of 100xTriton, 16.5uL of DNase, 16.5 uL of RNase, 50uL of DNase Buffer, 14uL of water.)
2. Incubate the samples with the Quench Mix in a dry bath at 37°C for 15 min.
3. Add 30uL of Steady-Glo Luciferase Assay (mixed buffer and powder according to manufacturer’s instructions) to 30uL of sample onto the Biotek plate.
4. Measure the luminescence using the Biotek (plate reader).
Protocol obtained from Kate Adamala
JMP3 protocol obtained from the MurrayWiki and written by Vincent Noireaux
Miyazaki, M, Chiba, M, Ishiwata, S. Preparation of cell-sized water-in-oil droplets for in vitro reconstitution of biological processes in cellular compartments. (2015) Nature. DOI: 10.1038/protex.2015.029