Development of a Rapid, Single-Cell Method of Recombinant Clone Screening Using Flow Cytometry

Fluorescence activated cell sorting methods have been adapted to accelerate the isolation of recombinant bacterial clones in the past decade. Previous studies have established methods for sorting bacterial cells transformed with plasmids encoding for one fluorescent reporter protein, however, to our knowledge, no system has been reported using more than one fluorescent marker. In this proof-of-concept study, we construct pGSBB, a dual-reporter plasmid encoding both enhanced green fluorescent protein (egfp) and mCherry that would enable high-throughput quantification and cell-sorting of bacterial transformants using flow
cytometry and fluorescence activated cell sorting. We showed that E. coli transformed with pGSBB variants can be visualized as discrete populations of cells expressing either GFP, mCherry, both reporters, or neither using flow cytometry. Future researchers may leverage this construct to expedite conventional cloning protocols that use phenotypic-based screens requiring overnight colony growth.