FIG 4.

Evaluation of autonomously replicating activity and plasmid retention through repeated subculture in P. pastoris. CBS7435 harboring pUC19-Cen2-EGFP (Cen2) was grown for 24 h (starting from an initial OD₆₆₀ of 0.1) in BMGY medium supplemented with 200 μg/ml Zeocin. The cells then were subcultured (initial OD₆₆₀ of 0.1) to fresh selective medium every 24 h. GFP expression in individual cells was measured by flow cytometry (FCM) at 24-h intervals, and the histogram data from a total of 10,000 cells at each time point are shown. Three different transformants were analyzed, and one typical histogram is displayed.