Global Analysis of Promoter Architecture in E. coli

Countless industries adapt E. coli for the production of heterologous resources, such as biofuels, insulin, and other therapeutics. Successfully engineering E. coli for these purposes requires the construction of well designed and strictly regulated gene expression networks. In prokaryotes, promoters are the key determinants of gene expression and allow for its flexible regulation. This regulation is mediated by transcription factors that bind specific motifs within the promoter sequence. Variability in the positioning, sequence, and combination of these motifs is extensive and allows for the fine-tuning of downstream gene expression. While this variability allows for precise modulation of gene expression, the resulting complexity makes it difficult to accurately predict the relationship between a promoter’s sequence and its function. Here, we demonstrate a high throughput reporter assay that enables rapid exploration of promoter sequence space. In this assay, synthesized promoters are engineered to express uniquely barcoded transcripts, integrated into a quiescent locus within the E. coli genome, and their expression is measured simultaneously using RNA-Seq. Using this approach, we characterize the complexity of endogenous E. coli promoters by measuring the expression of over 17,000 loci reported to initiate transcription. We find that a majority of reported transcription start sites (TSSs) are transcriptionally inactive in multiple growth conditions and identify sequence elements responsible for transcriptional activity and differential expression in E. coli. Using this approach, we can rapidly test large libraries of designed promoters, thereby improving our understanding of promoter sequence-function relationships and facilitating the development of sequence based models for predicting promoter activity.