Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons.

mRNA degradation is a central process that affects all gene expression levels, and yet, the determinants that control mRNA decay rates remain poorly characterized. Here, we applied a synthetic biology, learn-by-design approach to elucidate the sequence and structural determinants that control mRNA stability in bacterial operons. We designed, constructed, and characterized 82 operons in Escherichia coli, systematically varying RNase binding site characteristics, translation initiation rates, and transcriptional terminator efficiencies in the 5' untranslated region (UTR), intergenic, and 3' UTR regions, followed by measuring their mRNA levels using reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays during exponential growth. We show that introducing long single-stranded RNA into 5' UTRs reduced mRNA levels by up to 9.4-fold and that lowering translation rates reduced mRNA levels by up to 11.8-fold. We also found that RNase binding sites in intergenic regions had much lower effects on mRNA levels. Surprisingly, changing the transcriptional termination efficiency or introducing long single-stranded RNA into 3' UTRs had no effect on upstream mRNA levels. From these measurements, we developed and validated biophysical models of ribosome protection and RNase activity with excellent quantitative agreement. We also formulated design rules to rationally control a mRNA's stability, facilitating the automated design of engineered genetic systems with desired functionalities.

Automated design of thousands of nonrepetitive parts for engineering stable genetic systems.

Engineered genetic systems are prone to failure when their genetic parts contain repetitive sequences. Designing many nonrepetitive genetic parts with desired functionalities remains a difficult challenge with high computational complexity. To overcome this challenge, we developed the Nonrepetitive Parts Calculator to rapidly generate thousands of highly nonrepetitive genetic parts from specified design constraints, including promoters, ribosome-binding sites and terminators. As a demonstration, we designed and experimentally characterized 4,350 nonrepetitive bacterial promoters with transcription rates that varied across a 820,000-fold range, and 1,722 highly nonrepetitive yeast promoters with transcription rates that varied across a 25,000-fold range. We applied machine learning to explain how specific interactions controlled the promoters' transcription rates. We also show that using nonrepetitive genetic parts substantially reduces homologous recombination, resulting in greater genetic stability.

Simultaneous repression of multiple bacterial genes using nonrepetitive extra-long sgRNA arrays.

Engineering cellular phenotypes often requires the regulation of many genes. When using CRISPR interference, coexpressing many single-guide RNAs (sgRNAs) triggers genetic instability and phenotype loss, due to the presence of repetitive DNA sequences. We stably coexpressed 22 sgRNAs within nonrepetitive extra-long sgRNA arrays (ELSAs) to simultaneously repress up to 13 genes by up to 3,500-fold. We applied biophysical modeling, biochemical characterization and machine learning to develop toolboxes of nonrepetitive genetic parts, including 28 sgRNA handles that bind Cas9. We designed ELSAs by combining nonrepetitive genetic parts according to algorithmic rules quantifying DNA synthesis complexity, sgRNA expression, sgRNA targeting and genetic stability. Using ELSAs, we created three highly selective phenotypes in Escherichia coli, including redirecting metabolism to increase succinic acid production by 150-fold, knocking down amino acid biosynthesis to create a multi-auxotrophic strain and repressing stress responses to reduce persister cell formation by 21-fold. ELSAs enable simultaneous and stable regulation of many genes for metabolic engineering and synthetic biology applications.

An Automated Pipeline for Engineering Many-Enzyme Pathways: Computational Sequence Design, Pathway Expression-Flux Mapping, and Scalable Pathway Optimization.

Engineering many-enzyme metabolic pathways suffers from the design curse of dimensionality. There are an astronomical number of synonymous DNA sequence choices, though relatively few will express an evolutionary robust, maximally productive pathway without metabolic bottlenecks. To solve this challenge, we have developed an integrated, automated computational-experimental pipeline that identifies a pathway's optimal DNA sequence without high-throughput screening or many cycles of design-build-test. The first step applies our Operon Calculator algorithm to design a host-specific evolutionary robust bacterial operon sequence with maximally tunable enzyme expression levels. The second step applies our RBS Library Calculator algorithm to systematically vary enzyme expression levels with the smallest-sized library. After characterizing a small number of constructed pathway variants, measurements are supplied to our Pathway Map Calculator algorithm, which then parameterizes a kinetic metabolic model that ultimately predicts the pathway's optimal enzyme expression levels and DNA sequences. Altogether, our algorithms provide the ability to efficiently map the pathway's sequence-expression-activity space and predict DNA sequences with desired metabolic fluxes. Here, we provide a step-by-step guide to applying the Pathway Optimization Pipeline on a desired multi-enzyme pathway in a bacterial host.