the Promoter Calculator - a Sequence-to-Function Biophysical Model of Transcriptional Initiation for Sigma70 Promoters with Any Sequence

Engineering synthetic promoters with precision control has remained a challenge due to our inability to predict how a promoter's sequence and DNA context controls its function and mRNA output. Here, we developed an accurate sequence-to-function model of transcriptional initiation enabling the automated design of synthetic promoters and the a priori prediction of cryptic promoters - both of which are needed to advance Synthetic Biology towards genome-scale functional design. To do this, we combined oligopool synthesis, library-based cloning, and next-generation sequencing to construct and characterize 14,206 rationally designed sigma⁷⁰ promoters in vitro. Measurements include transcriptional start site frequencies and overall mRNA levels. This approach enables highly-parallel characterization of constitutive promoter activity without confounding factors such as unintentional transcriptional regulation, non-sigma⁷⁰ activity, and mRNA decay. These measurements, in combination with machine learning, were used to parameterize a thermodynamic model quantifying the interactions controlling transcription rate. For demonstration, the “Promoter Calculator” was used to accomplish three major tasks – accurate prediction of thousands of sigma⁷⁰ promoters across various conditions, the de novo design of novel sigma⁷⁰ promoters, and the identification of cryptic promoters internal to a genetic circuit. 4,350 highly non-repetitive promoters and 6,165 genome-integrated promoters characterized in vivo were accurately predicted by the model with Spearman Rank-Order Coefficients of .68 and .78, respectively. Promoters designed de novo using the Promoter Calculator exhibited a 683-fold range in expression in vivo resulting in a Pearson Correlation Coefficient of .85. The Promoter Calculator was used to analyze a genetic circuit containing 11 circuit promoters and 29 cryptic promoters. The model identified 29 sigma⁷⁰ promoters out of 40 observed promoters (72.5% accurate), including 10/11 circuit promoters and 19/29 cryptic promoters. The Promoter Calculator facilitates context-aware, rational promoter design without relying on a fixed table of pre-characterized sequences while serving as a powerful tool in promoter identification.