Fig. 1

Impact of gene syntaxes on the population means and ratios of gene expression. The arrangement and orientation of GFP and RFP genes on a ColE1 plasmid backbone were systematically varied. Both fluorescent protein genes were constructed with identical cassettes, comprising constitutive promoters, RBS sequences, and upstream DNA regions. The mean expression levels of the reporter proteins and the GFP/RFP expression ratios were analyzed across different gene arrangements. GFP and RFP fluorescence were measured in separate channels using flow cytometry; thus, the GFP/RFP ratios represent the relative expression levels of GFP and RFP in the cells rather than their absolute expression levels. To minimize variations, GFP/RFP ratios were calculated at the single-cell level before averaging the population means of GFP/RFP. Data are presented as mean ± SD, based on eight replicates. Significant differences (P < 0.05) were analyzed by ANOVA followed by Tukey’s HSD post-hoc tests, and are indicated by different letter(s) within each column. Values that do not share a common letter are significantly different from each other. For example, values with different letters (e.g., “a” vs. “bc”) are significantly different while those sharing the same letter are not significantly different (e.g., “a” vs. “ab”)