Fig 1.
Encapsulation of Cell-Free Expression (CFE) and formation of lipid vesicles.
(A) HeLa mammalian CFE system is a combination of HeLa lysate, Mix 1 buffer, GADD34, T7 RNA polymerase, Mix 2 accessory factors and DNA plasmid containing T7 promoter. (B) Thin double emulsions are formed in the glass capillary microfluidic device. (C) Image of thin double emulsion formation using the glass capillary microfluidic device. (D) Lipid vesicles are formed by first forming aqueous-in-oil-in-aqueous double emulsions where phospholipids are dissolved in volatile middle phase organic solvents. CFE system was encapsulated with surfactant (PVA/pluronic) and fluorinated surfactant (F6-TAC/F8-TAC) inside the lipid vesicles. Phospholipids self-assemble to form phospholipid monolayers on the aqueous-oil and oil-aqueous interfaces. Upon solvent evaporation, lipid vesicles form.
Fig 2.
Aggregate was observed when CFE is encapsulated in the vesicle.
(Top) Buffer solution (20 mM HEPES pH 7.5, 400 mM sucrose, 2% PVA, 8% PEG) was encapsulated as the inner phase using capillary droplet microfluidics with DOPC/cholesterol dissolved in chloroform/hexane as the middle phase (see materials and methods) (Bottom) HeLa CFE (18 μl HeLa lysate, 4.5 μl Mix 1 buffer, 5.4 μl GADD34, 4.5 μl T7 RNA polymerase, 3.6 μl Mix 2 accessory factors and 3 μl MscL DNA plasmid containing T7 promoter) with 2% PVA was encapsulated with DOPC/cholesterol dissolved in chloroform/hexane as the middle phase and incubated at 32 degrees in a closed environment.
Fig 3.
CFE of MscL is solubilized in fluorinated surfactants.
(A) Schematic illustration of the solubility assay for CFE of MscL. (B) Solubility of CFE of MscL after 5 hr in the presence or absence of 0.2% of Triton X-100. (C) Solubility of CFE of MscL after 5 hr at different concentrations of fluorinated surfactant F6-TAC. (D) Formation of vesicles encapsulating mammalian CFE expressing MscL in the presence of 2% PVA and 0.6 mM F8-TAC. The vesicles were formed from 36/64 chloroform/hexane as the middle phase, and imaged in brightfield (top left) and in green fluorescence (top right). 5 μM of calcium indicator Rhod-5N and 1 mM of calcium was also encapsulated inside the vesicle (bottom left). Merged image of green and red fluorescence is shown in the bottom right. (E) Formation of HeLa lysate encapsulated vesicles formed from 40/60 chloroform/hexane in the presence of 2% PVA and 2 mM F6-TAC, imaged in brightfield (left) and in fluorescence (right).
Fig 4.
PVA surfactant caused the formation of aggregate and reduced CFE activity.
(A) Brightfield images of mammalian CFE at 0, 1, or 2% of PVA surfactant. (B) eGFP expression in HeLa CFE as a function of PVA concentration measured in microwell plates (n = 3, ±S.E.), unpaired t test comparing with 0%; *: p < 0.01; **: p < 0.001. Brightfield images of microwell corresponding to the different PVA concentration are shown on top. Yellow arrowhead points to the large aggregate in the microwell.
Fig 5.
Pluronic surfactant also caused the formation of aggregate and reduced CFE activity.
(A) Brightfield (top) and fluorescence (bottom) images of double emulsion templated vesicles (without HeLa lysate) with 2% Pluronic F-68. (B) eGFP expression in HeLa CFE at different concentrations of surfactants for Pluronic F-68 (red), F-88 (green), and F-127 (blue), unpaired t test comparing with 0%; *: p < 0.01; **: p < 0.001; ***: p < 0.0001. Inset shows brightfield microwell images at 2% of surfactant concentration with yellow arrowhead pointing to the aggregates.
Fig 6.
High concentration of mammalian Hela lysate is prone to aggregation when exposed to surfactant.
(A) (Top) Representative brightfield images of different dilution of CFE reaction with 2% PVA under bulk condition. (Bottom) Representative brightfield images of single emulsions encapsulating 2% PVA at different CFE dilutions. Yellow arrowheads denote the appearance of aggregates. (B) (Left) eGFP expression in HeLa CFE as a function of CFE dilution measured in microwell plates (n = 4, ±S.E.), unpaired t test comparing with 1X; ***: p < 0.0001. (Right) eGFP expression in HeLa CFE at different volume ratios between lysate and Mix 1 measured in microwell plates (n = 4, ±S.E.), unpaired t test comparing with 9:2.25 volume ratio; ***: p < 0.0001.
Fig 7.
Actin aggregates with PVA surfactant.
(A) Coomassie blue stained gel showing proteins in the supernatant or pellet after 10 min centrifugation at 16,100 g, in the presence of PVA surfactant at different concentrations. (B) Western blot showing the actin in the supernatant or pellet fractions at different PVA concentrations. (C) Percentage of actin measured in the supernatant and pellet fractions at different PVA concentrations (n = 3, ±S.E.), unpaired t test comparing with 0% PVA; *: p < 0.01; ***: p < 0.0001.