Gene syntaxes modulate gene expression and circuit behavior on plasmids.

Achieving consistent and predictable gene expression from plasmids remains challenging. While much attention has focused on intra-genetic elements like promoters and ribosomal binding sites, the spatial arrangement of genes within plasmids-referred to as gene syntax-also plays a crucial role in shaping gene expression dynamics. This study addresses the largely overlooked impact of gene syntaxes on gene expression variability and accuracy. Utilizing a dual-fluorescent protein system, we systematically investigated how different gene orientations and orders affect expression profiles including mean levels, relative expression ratios, and cell-to-cell variations. We found that arbitrary gene placement on a plasmid can cause significantly different expression means and ratios. Genes aligned in the same direction as a plasmid's origin of replication (Ori) typically exhibit higher expression levels; adjacent genes in the divergent orientation tend to suppress each other's expression; altering gene order without changing orientation can yield varied expression. Despite unchanged total cell-to-cell variation across different syntaxes, gene syntaxes can also influence intrinsic and extrinsic noise. Interestingly, cell-to-cell variation appears to depend on the reporter proteins, with RFP consistently showing higher variation than GFP. Moreover, the effects of gene syntax can propagate to downstream circuits, strongly affecting the performance of incoherent feedforward loops and contributing to unpredictable outcomes in genetic networks. Our findings reveal that gene syntaxes on plasmids modulate gene expression and circuit behavior, providing valuable insights for the rational design of plasmids and genetic circuits.