A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting

Abstract

Molecular biology critically depends upon the isolation 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 heterogeneity 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 variability. 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 containing 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 successfully 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.

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.

Figure 2

Fluorescence microscopy image of E.coli containing pGRFP with cloned BAC library. The green cells are GFP+/DsRed− and contain plasmids with successfully inserted fragments from the library. The orange colored cells are GFP+/DsRed+ and contain native pGRFP vector without inserts.

Figure 3

(A) Flow-cytometry bivariate dot plot of E.coli expressing GFPmut3.1 from a constitutive promoter. Cells were picked from a single colony and cultured overnight. Because of the large cell-to-cell variation of fluorescence from a single fluorophore, it is difficult to use a single fluorophore as an indication of the presence or absence of a cloned insert. (B) Dot plot shows a population of E.coli containing native pGRFP. All cells are observed expressing both GFP and DsRed. (C) Dot plot showing a S.purpuratus BAC library cloned into the pGRFP vector. A second population of cells that are GFP+/DsRed− appears owing to loss of function of the DsRed half of the fusion protein. This population contains cells with successfully incorporated inserts. This reporter system clearly distinguishes between insert containing and non-insert containing E.coli.

Figure 4

Gel image showing PCR amplifications of inserts from 46 cultures grown from single sorted GFP+/DsRed− cells containing members of a S.purpuratus shotgun library. All 46 cultures yielded a PCR product consistent with a cloned insert. The majority of these inserts were in the 1.5–3 kb size range selected during initial library construction.