e49 Nucleic Acids Research, 2005, Vol. 33, No. 5 efﬁcient tool for screening large DNA libraries and reporter cell populations. In order to use cell-sorting for library screening, ﬂuorescent marker reporting strategies need to be further developed. Traditional sequencing vectors based on lacZ activity are incompatible with cell-sorters. Several groups have previously created cloning vectors containing ﬂuorescent reporters. Inouye et al. (9) developed a plasmid vector that directs the production of green ﬂuorescent protein (GFP) under a con- stitutive promoter. When an insert is present, the successful translation of GFP is prevented, and colonies lack ﬂuores- cence. Similarly, Roessner and Scott (10) developed a plasmid vector containing the uroporphyrinogen III methyltransferase (cobA) marker gene that produces a ﬂuorescent product within Escherichia coli when an insert is not present. These strategies work well for colonies grown on solid media, but negative screening (the absence of ﬂuorescence) turns out to be a poor criterion for selection using cell-sorters. Clonal E.coli popu- lations exhibit substantial stochastic variations in phenotype (11). The degree of cell-to-cell variability in protein expres- sion can make it impossible to distinguish between weak ﬂuorescent cells and non-ﬂuorescent cells. Background parti- cles inherent to culture media are also weakly ﬂuorescent and are difﬁcult to segregate from non-ﬂuorescent bacteria. For sorting to be a viable approach, desired bacterial cells must have a positive ﬂuorescent signal. In this study, we demonstrate a dual-ﬂuorescence system that controls the intrinsic variations in reporter protein expres- sion. The novel vector pGRFP was developed to report the integration of cloned inserts by shifting the ratio of green to red ﬂuorescence. In the native vector, a fusion protein composed of GFP and rapidly maturing DsRed-T3 ﬂuorescent protein (12) is expressed. Cloning of DNA within the linker region between GFP and DsRed abrogates DsRed translation and results in enhanced green to red ﬂuorescence ratio within transformed E.coli. In this paper, this approach is successfully validated in real-world applications using DNA from the sea urchin Strongylocentrotus purpuratus. The described dual- ﬂuorescence method allows for accurate and efﬁcient clone characterization using ﬂow cytometry. The adaptability of this method to a diverse range of biological assays could expand its role to an even greater number of biological investigations. MATERIALS AND METHODS Vector construction The pGRFP vector is shown schematically in Figure 1A. A 1990 bp pUC backbone containing the origin of replication, an AmpR marker and a lac promoter was PCR ampliﬁed. A consensus E.coli Shine–Dalgarno sequence (13–15) and a 7 bp spacer were introduced using a 50 tail on the reverse primer. All vector construction PCRs were performed using Pfu Turbo (Stratagene). The PCR product was cleaned, cut with AatII (NEB) and treated with calf intestinal alkaline phosphatase (CIAP; NEB). The GFPmut3.1 gene (16) was PCR ampliﬁed from pGFPmut3.1 (Clontech) excluding the stop codon. The reverse primer introduced an additional 56 bp tail containing a six amino acid linker sequence, M13 forward priming site, stop codon, BsmI site and AatII site. The 868 bp phosphorylated PCR product was cut with AatII. PAGE 2 OF 7 A B Figure 1. (A) The components of the pGRFP plasmid vector. The main components are the pUC origin of replication, ampicillin resistance marker and a fused GFP-DsRed gene separated by a linker. The linker region is shown with six amino acid linkers (SGSGSG and GSGSGS) on either side, M13 forward and reverse priming sites, and EcoRV, NotI and SalI sites. (B) Flow- cytometry configuration for dual-fluorescence quantification and sorting. A 488 nm laser excites the fluorescent proteins in individual E.coli suspended in the flow stream. The flow cytometer is configured to trigger either on forward scatter or GFP fluorescence. The fluorescence is split using a 550 nm dichroic long pass beam splitter. The green fluorescence is filtered through a 560 nm short pass filter before detection. The red fluorescence passes through a 590 nm long pass filter before detection. Both DNA fragments were gel puriﬁed and ligated together with T4 DNA Ligase (Invitrogen) to generate a precursor vector. The ligation products were electrotransformed into Electromax DH10B cells (Invitrogen) and plated onto Luria– Bertani (LB)–ampicillin. Colonies expressing GFP were picked, miniprepped (Qiagen) and partially sequenced. A clone with expected sequence was digested with BsmI (NEB) to destroy the GFP stop codon, treated with Mung Bean nucle- ase (NEB) and 50 dephosphorylated. To construct the DsRed portion, the rapidly maturing DsRed-T3 mutant (12,17) gene was PCR ampliﬁed from vec- tor obtained from Bevis and Glick (12). The upstream primer introduced NotI, EcoRV and SalI sites as well as an M13 reverse priming site and an additional six amino acid linker. The downstream primer contained a StuI site after the DsRed stop codon. The PCR product was cleaned and treated with DpnI (NEB). The product was further gel puriﬁed and blunt ligated to the precursor vector. Transformed colonies express- ing both GFP and DsRed were selected by ﬂuorescence microscopy after 24 h growth. The pGRFP vector was further