Gene Syntax Found to Influence Expression and Stability in Synthetic Plasmids.

In a breakthrough study, researchers at Dartmouth College have discovered that the arrangement of genes within synthetic plasmids—termed gene syntax—plays a critical role in determining how genes are expressed and interact within cells. Much like how word order affects meaning in language, the sequence and orientation of genes can influence expression strength, consistency, and crosstalk with neighboring genes, even when the underlying DNA sequences remain unchanged.

Plasmids are small, circular DNA molecules that function independently of a cell’s primary chromosomes. Commonly found in bacteria, plasmids are widely used in biotechnology to introduce new genes into cells or to build synthetic biological systems. However, one of the long-standing challenges in this field has been the unpredictability of gene behavior, despite using well-characterized genetic components such as promoters and ribosomal binding sites.

The Dartmouth team sought to understand this variability by focusing solely on gene syntax while keeping all other variables constant. In the study, published in the Journal of Biological Engineering, scientists engineered a series of plasmids using E. coli NEB 10-beta cells, systematically altering the order and orientation of fluorescent reporter genes. All regulatory sequences, including promoters and ribosomal binding sites, were kept identical across constructs.

Seven different plasmid configurations were created to isolate the impact of gene syntax. Using flow cytometry, researchers measured gene expression across more than 90,000 bacterial cells per sample, allowing for a robust analysis of expression variability.

The results revealed significant differences. Expression levels of GFP (green fluorescent protein) varied by more than 1.8 times depending on its placement within the plasmid, while RFP (red fluorescent protein) expression fluctuated by up to 1.56 times. Additionally, the ratio between the two reporter genes shifted by as much as 1.4-fold.

Orientation also proved critical: genes aligned with the plasmid’s origin of replication showed consistently stronger expression than those oriented against it. Tandem gene arrangements led to higher expression levels, whereas convergent and divergent orientations—where genes face each other or away from a central point—resulted in reduced activity. Surprisingly, even when potential sources of transcriptional interference were eliminated, divergent arrangements still suppressed expression in both directions.

This study highlights gene syntax as a key factor in synthetic biology, offering a new lens for understanding and optimizing genetic circuit performance.

Source:https://phys.org/news/2025-04-gene-syntax-shown-variability-synthetic.html

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