Abstract
A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.
Highlights
A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli
The lacZYA promoter is a classic model for gene regulation in E. coli, with many studies investigating the relationship between sequence composition and induction properties
This promoter contains two LacI dimer sites positioned at the proximal +11 and distal −82 positions relative to the transcription start site (TSS)[30,31], which flank a set of σ70 −10 and −35 elements (Fig. 1a, see WT PlacZYA)
Summary
A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. Directed evolution is a promising strategy that leverages stepwise random mutagenesis and selection to identify favorable promoters, but is generally limited to optimizing within local, evolutionarily accessible sequence space[11,12] While this black box approach can produce variants with the desired phenotype, it often requires iterative rounds of library screenings[12] and does not inform our ability to logically construct promoters. This approach enables the measurement of thousands of synthetic sequences in a single, multiplexed experiment, often using transcriptional barcodes as a readout[20,21] This paradigm has been used to empirically examine both the individual and combinatorial effects of transcription factor binding sites on gene expression in eukaryotes, improving our ability to design synthetic eukaryotic promoters with programmable responses[22,23,24,25,26,27,28,29]. There have been few similar high-throughput studies in prokaryotes
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