The Operon Calculator: Automated Design of Synthetic Operon Sequences Using 15 Models and Design Rules

Single and multi-cistronic operons are the central architectural unit of all natural and engineered genetic systems in bacteria, including multi-regulator genetic circuits and multi-enzyme pathways. An operon’s sequence ultimately determines the expression levels of its RNAs and proteins, though the sequence-to-function rules that dictate optimal operon design remain to be elucidated or fully tested. Currently, operons are designed by appending several pre-existing promoters, ribosome binding sites, coding sequences, and terminators. As a consequence, many engineered operons are full of overlapping, context-dependent, and undesired genetic elements that confound rational design and will inevitably break the operon’s function, such as repetitive DNA sequences, internal promoters and terminators, and highly translated internal start codons.

Here, we present a radically new algorithm, the Operon Calculator, that automatically designs whole operon sequences to yield maximum tunable control over its encoded RNA and protein expression levels, while eliminating the presence of all cryptic, undesired genetic elements that might break the operon’s function. We combine 15 biophysical models and design rules using multi-objective genetic algorithm optimization to: (i) control transcription rates using models of transcriptional regulation; (ii) quantify transcriptional elongation efficiencies; (iii) eliminate RNAse binding sites; (iv) remove internal promoters and (v) transcriptional terminators, both intrinsic and rho-dependent; (vi) control translation and (vii) translational coupling rates using models of translation; (viii) harmonize synonymous codon usage to prevent translation elongation pile-ups; (ix) remove ribosomal pause sites, unless desired; (x) remove highly translated internal start codons to prevent expression of truncated proteins; remove genetic instability elements, including (xi) repetitive sequences, (xii) recombinase sites, and (xiii) transposon insertion sites; (xiv) remove undesired restriction sites; and (xv) calculate system-level RNAP and ribosomal loads. Using systematic experimentation, we present quantitative sequence-function relationships controlling mRNA stability and the expression of truncated proteins, two key factors that are often responsible for operon failure. Finally, we demonstrate an illustrative case study where we use the Operon Calculator to redesign the Central Metabolic network of the E. coli MG1655.