Construction of a gene screening system using giant unilamellar liposomes and a fluorescence-activated cell sorter

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.