Efficient search, mapping, and optimization of multi‐protein genetic systems in diverse bacteria

Abstract

Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs.

Automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth

“…In all, 59 base pairs corresponding to a putative constitutive promoter within the cpcG2 promoter region52 were deleted ( P cpcG2Δ59 ), and the sfgfp reporter was exchanged for a gfpmut3 reporter with a synthetic RBS designed using the RBS Library Calculator53.…”

Synthetic biology to access and expand nature's chemical diversity

“…The scope of this review is to cover recent advances in synthetic biology in the light of how they will impact the field of NPs, including the following technologies:

Abstraction of genetic functions into “parts.” There has been an emphasis on creating genetic parts, such as promoters, that generate precise levels of gene expression 29–32 . There has been focus on generating large libraries of well-characterized parts and the development of biophysical/bioinformatics models to predict part behavior 33–36 . Part libraries for different organisms will aid the transfer of BGCs between hosts 37–41 .

Large-scale construction technologies .

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“…There has been focus on generating large libraries of well-characterized parts and the development of biophysical/bioinformatics models to predict part behavior 33–36 . Part libraries for different organisms will aid the transfer of BGCs between hosts 37–41 .…”

High-throughput evaluation of synthetic metabolic pathways

“…Farasat et al . developed a sequence-expression-activity mapping method to find optimal expression conditions with desired activity for a carotenoid biosynthetic pathway 31 . In a first step, an RBS calculator is used to make a library that spans a large range of protein expression space.…”

“…1b). Alternative ways to explore sequence-flux space include computational predictions from small training sets 29–31 (Fig. 1c) or high-throughput screening or selection techniques 32–36 (Fig.…”