Journal of Nucleic Acids on the liposome preparation methods. However, not all sizes of liposomes are applicable as microcompartments for IVC- based gene screening due to detection limits (approximately 1 μm in diameter) in FACS measurements (see Section 5 for details). Therefore, the liposome size suitable for this experiment is similar to a cell size (larger than 1 μm in diam- eter), and such a cell-sized liposome is referred to as a “giant liposome” [31]. Giant liposomes are primarily generated using the “hydration of thin ﬁlm method,” “rehydration of freeze-dried empty liposome (FDEL) method,” or “inverted- emulsion method” [32]. In liposome-based IVC, a single DNA and an IVTT are encapsulated in the same giant liposome to link genotype and phenotype (Figure 4). Feasibility of liposome prepa- ration, encapsulation of the reactants for in vitro protein synthesis, and the internal structure of the liposome are signiﬁcantly inﬂuenced by the preparation method for the giant liposome. For the hydration of thin ﬁlm or rehydration of freeze-dried empty liposome methods of giant liposome preparation, the liposomes are formed by reconstituting dried lipid ﬁlm with a reaction buﬀer for protein synthesis. The advantage of the hydration of thin ﬁlm method is that liposomes can be prepared using various phospholipids irrespective of electrical charge. However, the disadvantage of the method is that a relatively large quantity of reaction mixture is necessary for swelling the dried thin ﬁlm of the lipids during liposome preparation, and macromolecular compounds in the reaction mixture are diﬃcult to trap in the liposomes [32]. When giant liposomes are prepared by the rehydration of freeze-dried empty liposomes, the liposome structure is suﬃciently strong to withstand the osmotic pressure change in the outer solution and the sorting operation during the FACS screen [33]. However, the giant liposomes generated by this method are unsuitable for quantitative evaluation of in-liposome protein synthesis using an FACS because certain giant liposomes have multiple compartments and lamella; thus, the liposome size measured using an FACS does not represent the compartment size for protein synthesis [34]. Consequently, liposomes with a single compartment and lamella (e.g., giant unilamellar liposomes) are required for quantitative evaluation of in-liposome protein synthesis (liposome size and product quantity) by high-throughput analysis using an FACS. The inverted-emulsion method is a preparation method for giant unilamellar liposomes [35]. This method comprises the following steps. The aqueous phase is emulsiﬁed in an oil phase containing phospholipids to prepare a water-in- oil emulsion. The emulsion is layered on an outer aqueous solution and centrifuged to sediment the emulsion droplets towards the oil-water interface where a lipid monolayer forms. The emulsion droplets generate a second lipid layer upon crossing the interface and transform into unilamellar liposomes in the outer aqueous solution. When this type of giant unilamellar liposome is applied to in vitro protein synthesis, the compartmentalized reaction mixture is sep- arated from the outer aqueous phase until the liposomes are formed. Thus, researchers can control the composition of both inner and outer aqueous phases. The inverted- emulsion method is promising for construction of a suitable 5 microcompartment to quantitatively evaluate in-liposome protein synthesis and reconstitution of membrane proteins. 4. Protein Synthesis in Liposomes 4.1. The PURE System for In Vitro Protein Synthesis. The primary components for in vitro protein synthesis are DNA encoding the protein of interest, an IVTT, and a detection reagent. These components should be encapsulated ﬁrmly in a liposome when in vitro protein synthesis is performed in liposomes (Figure 4). An IVTT is a multimolecular machine that facilitates protein synthesis from DNA in a test tube. Cell extracts from E. coli, wheat germ, rabbit reticulocytes, and insect cells have been used as IVTTs for in vitro protein synthesis [36]. However, cell extracts comprise cer- tain unknown constituents. Furthermore, proteases, DNase, RNase, and intrinsic enzymes (e.g., β-galactosidase) remain in the cell extracts, and these remnants considerably decrease the production of protein and interfere with the detection of protein function. These problems are inevitable as long as cell extracts are used for in vitro protein synthesis. To overcome these problems, we have been using the IVTT developed by reassembling the individual components for protein synthe- sis, which were extracted from E. coli cells overexpressing the protein factors with a histidine tag and thoroughly puriﬁed. This new IVTT is referred to as a “protein synthesis using recombinant elements (PURE) system” [37]. In vitro protein synthesis is a coupled reaction system comprising transcription, aminoacylation of tRNA, translation, and energy source regeneration. The PURE system includes the entire reaction system and is prepared by reconstituting protein factors, ribosomes, tRNA mixture, and substrates (20 amino acids and four nucleoside triphosphates) in a buﬀer solution. The protein factors are T7 RNA polymerase, pyrophosphatase, 20 aminoacyl-tRNA synthetases, creatine kinase, myokinase, and nucleoside diphosphate kinase in addition to 10 translation factors (three initiation factors (IF), three elongation factors (EF), three release factors (RF), and a ribosome recycling factor (RRF)). 4.2. Protein Synthesis from a Single Gene in a Liposome. Using the experimental system with liposomes, DNA, and an IVTT, in vitro protein synthesis in liposomes has been studied by a number of groups [31]. The review article by Stano et al. [31] is a comprehensive survey of biomacromolecule synthesis in liposomes for the creation of semisynthetic minimal cells and provides the most recent and comprehensive list of publications on protein synthesis inside liposomes. Thus, our primary focus is on protein synthesis that begins with a single gene in a liposome, which is a crucial part of liposome- based IVC, because genotype and phenotype must be linked for the gene screening process. Our strategy, which links genotype and phenotype inside a liposome, includes DNA that encodes the protein of interest and is encapsulated at a single molecule level with the PURE system in liposomes. Liposomes in which green ﬂuorescent protein (GFP) was translated from a single gene were successfully detected, analyzed, and sorted for a ﬂuorescence signal from GFP using an FACS [24]. β-glucuronidase catalytic activity expressed