Deng Yijie, Maurais Hannah E, Etheridge Kai, Sarpeshkar Rahul
Thayer School of Engineering, Dartmouth College, Hanover, NH, 03755, USA.
Departments of Engineering, Microbiology & Immunology, Physics, and Molecular and Systems Biology, Dartmouth College, Hanover, NH, 03755, USA.
J Biol Eng. 2025 Mar 27;19(1):25. doi: 10.1186/s13036-025-00493-0.
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.
从质粒中实现一致且可预测的基因表达仍然具有挑战性。虽然很多注意力都集中在基因内元件如启动子和核糖体结合位点上，但质粒内基因的空间排列（即基因句法）在塑造基因表达动态方面也起着关键作用。本研究探讨了基因句法对基因表达变异性和准确性这一在很大程度上被忽视的影响。利用双荧光蛋白系统，我们系统地研究了不同的基因方向和顺序如何影响表达谱，包括平均水平、相对表达比率以及细胞间差异。我们发现，质粒上任意的基因位置都可能导致显著不同的表达均值和比率。与质粒复制起点（Ori）方向相同排列的基因通常表现出更高的表达水平；处于相反方向的相邻基因往往会相互抑制对方的表达；在不改变方向的情况下改变基因顺序会产生不同的表达。尽管不同句法下细胞间的总体差异不变，但基因句法也会影响内在和外在噪声。有趣的是，细胞间差异似乎取决于报告蛋白，红色荧光蛋白（RFP）始终比绿色荧光蛋白（GFP）表现出更高的差异。此外，基因句法的影响可以传播到下游回路，强烈影响非相干前馈回路的性能，并导致遗传网络中出现不可预测的结果。我们的研究结果表明，质粒上的基因句法会调节基因表达和回路行为，为质粒和遗传回路的合理设计提供了有价值的见解。
