Nucleic Acids Research, 2005, Vol. 33, No. 5 e49 doi:10.1093/nar/gni049 A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting Juno Choe1,2, Haiwei H. Guo3 and Ger van den Engh1,* 1Institute for Systems Biology, 1441 North 34th Street, Seattle, WA 98103-8904, USA, 2Department of Genome Sciences and 3Department of Pathology, University of Washington, Seattle, WA 98195, USA Received November 3, 2004; Revised February 14, 2005; Accepted February 22, 2005 ABSTRACT Molecular biology critically depends upon the iso- lation of desired DNA sequences. Flow cytometry, with its capacity to interrogate and sort more than 50 000 cells/s, shows great potential to expedite clone characterization and isolation. Intrinsic hetero- geneity of protein expression levels in cells limits the utility of single fluorescent reporters for cell-sorting. Here, we report a novel dual-fluorescence strategy that overcomes the inherent limitations of single reporter systems by controlling for expression vari- ability. We demonstrate a dual-reporter system using the green fluorescent protein (GFP ) gene fused to the Discosoma red fluorescent protein (DsRed ) gene. The system reports the successful insertion of foreign DNA with the loss of DsRed fluorescence and the maintenance of GFP fluorescence. Single cells con- taining inserts are readily recognized by their altered ratios of green to red fluorescence and separated using a high-speed cell-sorter for further processing. This novel reporter system and vector were success- fully validated by shotgun library construction, cloned sequence isolation, PCR amplification and DNA sequencing of cloned inserts from bacteria after cell-sorting. This simple, robust system can also be adapted for diverse biosensor assays and is amenable to miniaturization. We demonstrated that dual-fluorescence reporting coupled with high-speed cell-sorting provides a more efficient alternative to traditional methods of clone isolation. INTRODUCTION Many investigations in modern molecular biology require the construction of large DNA libraries and the subsequent isolation of clones of interest. The sequencing of the human genome necessitated the evaluation of a large number (in the order of 108) of clones (1). As many other genomes are sequenced, it is expected that the demand for high-throughput cloning capabilities will increase. Additionally, many gene regulatory networks and protein–protein interactions are being elucidated, often by means requiring the handling of thousands of individual clones. Within directed molecular evolution, improved gene products for medical and industrial purposes are being generated from large diverse mutagenesis libraries (2–4). All of these processes depend critically upon the creation of random libraries as well as rapid and reliable screening methods. Library generation followed by clone selection is effective if it can be conducted inexpensively and on a large scale. In a traditional experiment, libraries of DNA fragments are cloned into bacterial expression vectors, transformed into bacteria and grown on solid media. A phenotypic marker identiﬁes colonies that are derived from bacterial cells with desirable inserts. The commonly used lacZ system uses blue or white color to distin- guish colonies of interest in the presence of X-gal (5). For high-throughput applications, clone picking robots are used to mechanically transfer individual colonies into multi-well plates. Over the past 30 years, ﬂuorescence activated cell-sorting (FACS) machines have evolved to become powerful tools for analyzing and isolating single cells at very high rates (6). If the properties of recombinant DNA can be expressed as ﬂuores- cent tags in carrier cells, cell-sorters can isolate desirable cells at very high rates. Cell-sorters are also adept at depositing small volumes of liquid precisely within individual wells for culturing or DNA ampliﬁcation (7,8). FACS of reporter cell populations also constitutes a powerful quantitative ana- lytical system. Each individual cell is, in effect, a single micro- plate well (6). Modern high-speed cell-sorters are currently capable of quantifying multicolor ﬂuorescence and making sort decisions at the rates of up to 50 000 cells/s (6). Therefore, extending the analogy, a single cell-sorter can effectively examine and select from 15 million 96-well microtiter plates per work-shift. Cell-sorting could become an extremely *To whom correspondence should be addressed. Tel: +1 206 364 3400; Fax: +1 206 364 346; Email: engh@systemsbiology.org ª The Author 2005. Published by Oxford University Press. 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