Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks

Area of Science:

• Synthetic biology
• Molecular biology
• Genetics

Background:

• Synthetic gene expression is sensitive to intragenic context.
• Intergenic compositional context effects on gene expression are less understood.
• Gene arrangement (orientation) is a key intergenic factor.

Purpose of the Study:

• To investigate the impact of intergenic gene orientation on synthetic gene expression levels.
• To compare expression of genes in convergent, divergent, and tandem orientations.
• To elucidate the role of supercoiling and transcriptional interference in mediating these effects.

Main Methods:

• Comparing gene expression levels for convergent, divergent, and tandem gene arrangements.
• Inducing single or dual gene expression.
• Modeling transcriptional interference and supercoiling effects.
• In vitro supercoiling relaxation experiments using gyrase.

Main Results:

• Convergent gene arrangements resulted in up to 400% higher expression, increased ultrasensitivity, and broader dynamic range compared to divergent or tandem arrangements.
• Gene orientation effects persisted regardless of whether one or both genes were induced.
• Transcriptional interference, mediated by supercoiling, was identified as the likely cause.
• Gyrase treatment eliminated orientation-dependent expression differences.

Conclusions:

• Intergenic gene orientation is a critical determinant of synthetic gene expression levels.
• Convergent arrangements enhance expression by mitigating transcriptional interference.
• Supercoiling plays a key role in mediating these orientation effects.
• Exploiting supercoiling via gene arrangement can improve synthetic gene network performance, as demonstrated in a rebuilt toggle switch.