title: Construction of a gene screening system using giant unilamellar liposomes and a fluorescence-activated cell sorter.
abstract: We have constructed a gene screening system composed of an in vitro transcription-translation system encapsulated within giant unilamellar liposomes and a fluorescence-activated cell sorter (FACS), which allows high-throughput screening of genes encoding proteins of interest. A mock gene library of β-glucuronidase (GUS) was compartmentalized into liposomes at the single-molecule level, and liposomes exhibiting green fluorescence derived from hydrolysis of the fluorogenic substrate by the synthesized enzyme were sorted using FACS. More than 10-fold enrichment of GUS gene with higher catalytic activity was obtained 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 experimentally obtained and theoretical values were in agreement. Using this method, 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.
authors.affiliation: Yomo Dynamical Micro-scale Reaction Environment Project, ERATO, Japan Science and Technology, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Nishikawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sunami
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Matsuura
authors.forename: Norikazu
authors.initials: N
authors.lastname: Ichihashi
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yomo
mesh_terms: D002474:Cell-Free System / Q000378:metabolism; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics*; D006868:Hydrolysis; D008856:Microscopy, Fluorescence; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D053835:Unilamellar Liposomes; D005966:Glucuronidase