8 Journal of Nucleic Acids Compartmentalized library Selection Ampliﬁcation Library of mutated genes Encapsulation of mutated genes into unilamellar liposomes Protein synthesis in individual microcompartments Identifying proteins with desired properties FACS selection DNA encoding a protein with a desired property Recompartmentalization Figure 6: Flow chart for protein screening using liposome-based IVC. Library of mutated genes are compartmentalized with PURE system and other reagents into liposome. Each protein variant is synthesized from a single copy of gene in each liposome. Liposomes encapsulating the gene of interest are screened using an FACS. DNA extracted from the liposomes is ampliﬁed to be transferred to the next round of gene screening. (GUS) was compartmentalized into liposomes as a single molecule. The liposomes that exhibited green ﬂuorescence from hydrolysis of the ﬂuorogenic substrate through the synthesized GUS were sorted from the subpopulation of giant unilamellar liposomes using an FACS. More than a 10-fold enrichment of the GUS gene with a higher catalytic activity was generated when a single copy of the GUS gene was encapsulated in each liposome. Quantitative analysis of the enrichment factors and their liposome size dependencies showed that the experimentally generated and theoretical values agreed. Using this method, the genes encoding active GUS were then enriched from a gene library of randomly mutated GUS genes. Only three rounds of screening were required, which was also consistent with our theoretical estimation. The consistency between the theoretical and experimental values generated using our screening system indicates that the screening system operates as expected. 6.2. Protein Evolution Directed by Compartment Size. Here, the directed evolution of protein is discussed through the eﬀect of compartment size on protein function. Nature con- tains living prokaryotes with cell sizes that range from 0.02fL to 400fL [45]. The lower limit of the cell size is determined by the catalytic eﬃciency of enzymes, protein synthesis machin- ery, and machinery to cope with sudden environmental changes [45]. Under this theory, smaller cells could be gener- ated if the enzyme catalytic eﬃciency was greater. Naturally occurring proteins have evolved in the cell through Dar- winian selection. However, directed evolution of protein has never been discussed regarding compartment size because conventional microcompartments (emulsions) have been unsuitable for this purpose. Of the gene screening techniques for directed evolution of proteins, liposome-based IVC is the most promising technique for studying how compart- ment size inﬂuences protein evolution because the internal aqueous phase volume of the liposome is accurately evalu- ated by FACS measurement. A molecular evolution system using liposome-based IVC is an experimental approach for simulating the evolutionary process of protein function in a certain cell size. We propose a molecular evolution system for evaluating the eﬀect of compartment size on protein evolution. The system comprises a giant unilamellar liposome, GUS, and an FACS. Giant unilamellar liposomes are polydisperse in size ranging from 0.5fL to 250fL or larger, which includes the cell size discussed. GUS is a tetrameric enzyme, and GUS tetramer formation is a rate limiting step in catalytic activity expression [46]. Kinetic analysis of GUS tetramer formation in emulsion droplets showed that tetramer formation is sus- ceptible to compartment size when GUS is synthesized from a single gene in a W/O emulsion droplet [47]. Monomeric GUS is prone to assemble in a smaller compartment because tetramer formation is the rate limiting step. In our molecular evolution system, a library of randomly mutated GUS genes and the PURE system are compartmentalized in giant unilamellar liposomes. GUS variants are synthesized in individual liposomes. Liposomes exhibiting GUS catalytic activity are sorted from the subpopulation deﬁned by a certain liposome size (100fL) and green ﬂuorescence intensity above threshold value. We predict that GUS variants prone to assemble in a larger compartment (100fL) will be generated after iterative rounds of genetic screening. Our genetic screening experiment is in progress and will continue until it generates a gene encoding active GUS variants, which are ﬁt to a certain liposome size. 6.3. Adaptation of Membrane Protein Function to a Liposome Environment via Directed Evolution. Membrane proteins perform a variety of functions in cells, including material transport, signal transduction, and cell-cell contact. With recent progress in minimal cell research using liposomes and an IVTT, experimental methods for including mem- brane proteins are under development. Giant unilamellar liposomes are an ideal cell-mimetic environment because