Simultaneous detection of bacteria expressing GFP and DsRed genes with a flow cytometer.

Abstract

In this study, Escherichia coli cells producing red fluorescent protein of Discosoma sp. (drFP583 DsRed) were investigated with flow cytometry by using 488 nm excitation. We also studied whether green fluorescent protein (GFP) and drFP583 could be detected simultaneously from a single bacterial cell. Plasmids pDsRed and pEGFP were used for the production of drFP583 and enhanced GFP, respectively, in E. coli MC1061 cells. To produce enhanced GFP and drFP583 simultaneously, plasmids pG9R and pG19R were constructed. These encode tandem fusions of enhanced GFP and drFP583 to ensure similar production levels for both proteins. Bacteria producing enhanced GFP and drFP583 were found to be brightly green and red fluorescent, respectively. Production of enhanced GFP and drFP583 fusion proteins resulted in bacteria that emitted both green and red fluorescence, which was detected easily by a flow cytometer using single laser excitation. Previously reported tetramerization of drFP583 did not restrict its use as a reporter gene, although it maturated significantly slower than enhanced GFP. The results show that enhanced GFP and drFP583 proteins can be detected simultaneously from single bacteria with a standard flow cytometer with simple optical configuration.