Building Enhancers from the Ground Up: A Synthetic Biology Approach

SummaryA challenge of the synthetic biology approach is to use our understanding of a system to recreate a biological function with specific properties. We have applied this framework to bacterial enhancers, combining a driver, transcription factor binding sites, and a poised polymerase to create synthetic modular enhancers. Our findings suggest that enhancer-based transcriptional control depends critically and quantitatively on DNA looping, leading to complex regulatory effects when the enhancer cassettes contain additional transcription factor binding sites for TetR, a bacterial transcription factor. We show through a systematic interplay of experiment and thermodynamic modeling that the level of gene expression can be modulated to convert a variable inducer concentration input into discrete or step-like output expression levels. Finally, using a different DNA-binding protein (TraR), we show that the regulatory output is not a particular feature of the specific DNA-binding protein used for the enhancer but a general property of synthetic bacterial enhancers.

Graphical AbstractFigure optionsDownload high-quality image (267 K)Download as PowerPoint slideHighlights
► Basic bacterial enhancers contain a driver, a poised promoter, and protein-binding sites
► The enhancers regulate expression by modifying the DNA's looping capability
► The enhancers translate a variable signal into discrete (step-like) expression levels
► The presence and number of protein-binding sites within the loop determine the output