Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks

• Main Authors: Yeung, Enoch (Author), Martin, Kyle B. (Author), Ng, Andrew H. (Author), Beck, James L. (Author), Murray, Richard M. (Author), Dy, Aaron James (Contributor), Del Vecchio, Domitilla (Contributor), Collins, James J. (Contributor)
• Other Authors: Institute for Medical Engineering and Science (Contributor), Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Mechanical Engineering (Contributor), Massachusetts Institute of Technology. Plasma Science and Fusion Center (Contributor)
• Format: Article
• Language: English
• Published: Elsevier BV, 2018-12-17T15:09:39Z.
• Subjects: Article
• Online Access: Get fulltext

 Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability.