4 Journal of Nucleic Acids W/O emulsion droplets (gene expression) Double emulsion droplets (screening) Oil Water “Membrane proteins cannot be reconstituted into surfactant layer of w/o emulsion” “Genotype- phenotype linkage is broken in a double emulsion droplet for FACS sorting” Water Surfactant layer Phospholipids DNA IVTT . . . Reaction mixture for protein synthesis Oil Surfactant layer Inner aqueous phase (reactor for protein synthesis) Lipid bilayer membrane (matrix for membrane protein) Figure 3: The technical limitations of emulsion-based IVC due to the “W/O emulsion” and “double emulsion” structures. Liposomes DNA Figure 4: Liposomes as a platform for in vitro protein synthesis. Reaction mixture for protein synthesis constitutes an inner aqueous phase of liposome, in which protein is synthesized from a DNA. Membrane protein synthesized inside liposome can be embedded into a lipid bilayer membrane. 1964 [26]. They observed dried samples from an aqueous dispersion of lecithin by electron microscopy and discovered a spherical structure with a 4.4 nm thick lipid layer of lamellae. “Liposome” is a term for a phospholipid vesicle and was proposed by Sessa and Weissmann in 1968 [27]. This term is generally accepted. Since the ﬁrst report by Bangham, liposomes have been utilized in various biophysical and biochemical studies, including model membranes, microre- actors, supramolecular assemblies for biomimetic systems, and drug carriers for drug delivery systems [28]. Currently, liposome-related studies are motivated by a growing interest in “synthetic cells” [29] and the “origin of life” [29], both of which are intended to address how living things might emerge from nonliving matter [30]. The recent trend in liposome-related studies regards liposomes as a protocell model in which biochemical reactions inside a living cell are executed by ﬁlling liposomes with the required components. In vitro protein synthesis in liposomes and its application to genetic screening (liposome-based IVC) are examples of bioengineering as well as liposome-related studies. 3.2. Preparation Methods for Cell-Sized Liposomes. Lipo- somes are diverse in size (from several tens of nm to hundreds of μm in diameter), lamellarity (singly lamellar or multilamellar), and internal structure (single compartment or multiple compartments). This diverse structure depends