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Flow cytometric detection of bacteria expressing gfp and dsred genes
Mikael Maksimow*; K Hakkila; J Kurittu; M Karp; M Virta
Department of Biotechnology, University of Turku, Tykistökatu 6, 6th Floor, Turku Fin-20520, Finland *(email@example.com)
Mutant forms of Aequorea victoria green fluorescent protein (GFP) are commonly used reporter proteins in both eukaryotic and bacterial cells. GFP mutant (EGFP) having excitation and emission maxima at 488 nm and 509 nm, respectively, is suitable for flow cytometric applications. Recently cloned red fluorescent protein from Discosoma striata (DsRed) has excitation and emission peaks of 558 nm and 583 nm. Despite of different excitation maxima, both proteins can be efficiently excited by 488 nm argon laser. In this study we used pEGFP and pDsRed plasmids (Clontech) containing egfp and dsred genes under the control of lac promoter. For negative control we used pUC19 plasmid. Host strain used, Escherichia coli MC1061, expresses fluorescent protein genes constitutively, because it lacks lacI repressor gene. Bacteria were cultured at 37°C to stationary phase in M9-medium supplemented with hydrolyzed casein, and analyzed by Coulter Epics XL flow cytometer using argon laser for excitation and 525 nm and 620 nm filters for detection. Our data shows that bacteria expressing egfp or/and dsred genes can be detected by flow cytometry, which should enable the sorting of bacteria according to expression of those genes.
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