Fig. 2

Gene orientation relative to the plasmid origin of replication affects gene expression profiles. (A) The designs of two plasmids with opposite gene orientations of GFP on a high-copy-number plasmid with ColE1 as Ori. Codirectional orientation means the transcriptional direction of a gene (e.g., GFP) is the same as the Ori of the plasmids while the head-on gene orientation indicates the opposite transcriptional direction to the plasmid Ori. The orientation of a gene is indicated on a plasmid map as either clockwise or counterclockwise. (B) The mean GFP expression between codirectional and head-on gene orientation. (C) Cell-to-cell variation of GFP expression among the two gene orientations. (D) Contribution of the upstream region to the transcription of GFP gene. Distance to promoter is the number of base pairs away from the transcription start site (TSS), which is indicated by the 0 on the axis; the negative sign denotes upstream of the promoter. The high peaks indicate potential promoters. (E) The plasmid design of gene orientations relative to pSC101 Ori on a low-copy-number plasmid backbone. Codirectional-T means that a terminator was inserted upstream of the reporter gene (AraC-RFP fusion protein) to control for the upstream transcriptional activity. (F) The mean AraC-RFP expression among three constructs. (G) Cell-to-cell variation of gene expression among the three constructs. (H) Contribution of upstream DNA sequences to the transcription of AraC-RFP. Note that transcription rates in (D, H) were evaluated using the Promoter Calculator, a reliable computational tool for identifying promoters and predicting their activity. Values are presented as mean ± SD (n = 8). ****p < 0.0001; *** p < 0.0001; ** p < 0.01; *p < 0.05; ns, not significant