Journal of Nucleic Acids the lipid composition can be optimized for reconstitution of membrane proteins. Although certain water soluble proteins have been synthesized using an IVTT inside liposomes (Section 4), thus far only a few membrane proteins have been synthesized inside liposomes and reconstituted into a lipid bilayer membrane. For synthesis of membrane proteins inside liposomes, a few groups have succeeded in synthesizing and characterizing α-hemolysin for membrane permeation of nutrient molecules in giant unilamellar lipo- somes [18] as well as sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT) for lipid synthesis in liposomes [48]. We believe that through liposome-based IVC development, in vitro molecular evo- lution of membrane proteins become possible. Advantages of using liposome-based IVC on the molecular evolution of membrane proteins are expected as follows. (1) Various kinds of membrane proteins can be engineered irrespective of their toxicity threatening cells’ lives, (2) functions of membrane proteins can be evaluated under various reaction conditions and also in membranes with various lipid compositions. Thus, research on membrane proteins is entering a new stage for applications aimed at complex molecular machines, such as biosensors for a monitoring device, biochips for diagnosis, and biointerfaces for computing. 7. Conclusions In this paper, we reviewed a novel in vitro genetic screen- ing system comprising liposome-based IVC and an FACS. Liposome-based IVC is a new technique developed to link genotype and phenotype. This technique utilizes a giant unil- amellar liposome and a PURE system. A library of mutant genes and a PURE system are compartmentalized into giant unilamellar liposomes for in vitro protein synthesis. A protein variant (phenotype) translated from a single DNA is colocalized with the DNA (genotype) inside a liposome. Using an FACS for high-throughput screening, liposomes encapsulating the gene of interest are sorted from a large population of liposomes using ﬂuorescent signals generated from expression of a protein function. The genes of interest are enriched through iterative rounds of genetic screening. With the gene screening system, genetic diversity at approximately 107 can be screened in a day [25]. The diversity size is suﬃciently large for directed evolution of proteins. The system can screen various proteins including enzymes and membrane proteins. A large population of liposomes of diﬀerent sizes (from 0.5fL to 250fL) facilitates the search for a protein function that has adapted to a certain compartment size. In addition, the semipermeable character of the liposomal membrane facilitates external feeding of a liposome compartment using additional solutes. If the protein function of interest is coupled to the external solute, then the protein screen is controlled by the timing of feeding and/or solute quantity. Liposome-based IVC was successfully proven eﬀective for screening a protein function under simple conditions. However, in nature, proteins must have evolved to adapt to more complex and dynamic environments where many biochemical reactions are coupled and organized to control 9 cell behavior. This suggests that a reaction system comprising many proteins and enzymes can evolve to perform more eﬃciently and more productively. Liposome-based IVC will be a useful method for simulating versatile conditions by assembling the necessary components into a liposome reactor. 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