Design and synthesis of synthetic UP elements for modulation of gene expression in Escherichia coli

To limit both cost and environmental impact, microorganisms are exploited by synthetic biologists for ecologically friendly ways to produce biochemicals essential to both industry and health. Synthetic biologists and metabolic engineers work to hijack the metabolism of accommodating hosts in order to produce useful chemicals. The alteration and optimization of endogenous and heterologous metabolic pathways require careful balancing and tuning of gene expression. While there are numerous ways to control gene expression in the cell, by far the most common way to regulate expression is transcriptionally through the promoter. In this work we explore a particular region of the bacterial promoter, the UP element, as an effective way modulate gene expression in Escherichia coli. We use both rational and library design of UP elements to search the upstream sequence space of the core promoter, rrnD. Using FACS and flow cytometry, we screen multiple libraries to identify a group of UP elements capable of modulating expression in E. coli. Additionally, we explore the effects of modifying sequences adjacent and upstream to the UP element site. We report that expression strength can be tuned both positively and negatively through the modifications in the UP element sequence and through modifications of sequences upstream and adjacent of the UP element. The strongest sequence identified, TP-24, amplifies our core promoter strength up to 30-fold based on GFP fluorescence. Our final selection of UP elements activate rrnD from 8 –– 26 fold, each stronger than the commonly used IPTG inducible promoter, Ptac1. This work provides a novel and minimal way to control promoter strength in E. coli expression without major alteration to the core promoter.Cellular and Molecular Biolog