Journal of Nucleic Acids 7 DNA (genotype ) mRNA Protein (phenotype) Substrate Product PURE system Volume marker Reaction product P0: Population of unilamellar liposomes 105 C104 S F f tyo103 i s n ten102 I 2D dot plot of scattering light intensity P0 101 101 102 103 104 105 Intensity of SSC r rek 102 a m um101 e l o v Io f 100 F 10−1 101 2D dot plot of ﬂuorescence intensity P1 102 103 104 105 FI of reaction product One of the data points corresponds to a liposome P1: Population of unilamellar liposomes exhibiting catalytic activity Figure 5: Characterization of in-liposome protein synthesis using an FACS. DNA, PURE system, ﬂuorogenic substrate, and ﬂuorescent volume marker are encapsulated in a giant unilamellar liposome (left). Subpopulation of unilamellar liposomes is represented by P0 in 2D dot plot of scattering light intensity (middle). Catalytic activity of enzymes expressed in liposomes is conﬁrmed in P1 region of 2D dot plot of ﬂuorescence intensity obtained by FACS measurement (right). underlying this technique is that a mutant gene library is compartmentalized as a single molecule into cell-sized liposomes (giant liposomes) and a protein variant is syn- thesized from the encapsulated mutant gene through the PURE system in each liposome. Liposome-based IVC has two primary advantages over emulsion-based IVC. Unlike W/O emulsion droplets, liposomes are directly loaded onto an FACS apparatus when they are analyzed for gene screening (no reemulsiﬁcation process is required for FACS analysis). The catalytic activity of an enzyme expressed in a liposome is quantitatively evaluated using an FACS when a giant unilamellar liposome is utilized for enzyme synthesis and catalytic activity expression. An FACS measurement collects signals from two or more diﬀerent ﬂuorescence colors (a ﬂuorescent volume marker and ﬂuorogenic substrate) from individual liposomes simultaneously and quantitates the liposome size and reaction product concentration, both of which are necessary for quantitative evaluation of catalytic activity. In addition, membrane proteins can be inserted into the phospholipid bilayer membrane of a liposome when giant unilamellar liposome is utilized for membrane protein synthesis. Membrane protein incorporated into lipid bilayer membrane is a prerequisite for quantitative evaluation of membrane protein function and subsequent genetic screen- ing. We ﬁrst performed a pilot experiment for liposome- based IVC and demonstrated that the technique is promising for genetic screens [24]. Two GFP variants, GFPuv2 and GFPuv5, were used in the pilot experiment, and they were encoded in the pETG2tag and pETG5tag vectors, respectively. GFPuv5 emits a ﬂuorescent signal eight times higher than GFPuv2 when excited at 488 nm. A mixture of the pETG2tag and pETG5tag DNA at molar ratio of 0.85 : 0.15 was compartmentalized into giant liposomes with the PURE system and a ﬂuorescent volume marker. Giant liposomes were prepared by FDEL method (Section 3). After incubation for GFP synthesis, the liposomes were measured for ﬂuorescent signals from the translated GFP as well as volume marker and sorted using the higher ﬂuorescent intensity of GFP and a certain liposome size. The pETG5tag was enriched over 10-fold from the initial genetic mixture when the liposomes were collected from two liposome subpopulations; one subpopulation ranged from 1.4fL to 6.7fL and the other ranged from 6.7fL to 13fL. Therefore, the genotype- (GFP gene-) phenotype (GFP) link was securely constructed in individual liposomes, which encapsulated a single copy of DNA. Thus, the pilot experiment successfully showed that GFP genes encapsulated in a liposome can be screened for the ﬂuorescence intensity from GFP emission. However, we anticipated the following technical issue, which can be caused by multiple compartments and lamella in giant liposomes prepared by the FDEL method [34]. The issue is underestimation of catalytic activity where a gene is expressed only in a subset of the multiple compartments in a giant liposome. This yields an inaccurate evaluation of catalytic activity in individual liposomes and lower enrichment in the gene of interest. In addition, translocation of a membrane protein into the membrane of a multilamellar liposome is a technical hurdle for detection of a functional membrane protein. To solve these problems, we used a giant unilamellar liposome for liposome-based IVC. A giant unilamellar liposome was prepared using the inverted- emulsion method (Section 3.2). We constructed a genetic screening system composed of in vitro protein synthesis encapsulated within a giant unilamellar liposome and an FACS (Figure 6). A mock genetic library for β-glucuronidase