Cited by 235Microbial Metabolic Engineering and BioproductionGene Regulatory Network AnalysisBacterial Genetics and Biotechnology

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

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Farasat, Iman, Kushwaha, Manish, Collens, Jason, Easterbrook, Michael, Guido, Matthew, Salis, Howard M·2014·Molecular Systems Biology

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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.

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  • Large libraries of promoters, terminators, and RBS calculators have been developed and characterized for model organisms like E. coli, dramatically increasing the control of metabolic pathways and allowing efficient pathway refactoring and design of genetic circuits.

    “Large libraries of promoters (Temme et al., 2012a), terminators (Chen et al., 2013b), and RBS calculators (Farasat et al., 2014) have been developed and characterized for model organisms like E. coli, dramatically increasing the control of metabolic pathways and allow efficient pathway refactoring and design of genetic circuits (Temme et al., 2012a,b).”

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Research works printed but not linked: 1

  1. Espah Borujeni A Channarasappa AS Salis HM Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding, and sliding at upstream standby sites Nucleic Acids Res 2014 42 2646 2659 24234441 PMC3936740

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Authors
Farasat, I., Kushwaha, M., Collens, J., Easterbrook, M., Guido, M., & Salis, H. M.
Affiliations
Pennsylvania State University (US)
Corresponding author
Salis, Howard M
Venue
Molecular Systems Biology
Published
2014-06-01
Volume
10
Issue
6
Pages
731-731
ISSN
1744-4292 (ISSN-L)
OpenAlex
W2164028644
Cited by
235
References
89
Citations by year
2026: 16 · 2025: 13 · 2024: 10 · 2023: 14 · 2022: 11 · 2021: 16 · 2020: 19 · 2019: 29 · 2018: 19 · 2017: 25 · 2016: 29 · 2015: 29 · 2014: 4
FWCI
11.90
Type
journal article
Language
en
Open access
Locations
  • Molecular Systems Biology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Warwick Research Archive Portal (University of Warwick) (acceptedVersion)
  • HAL (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Office of Naval Research (N00014‐13‐1‐0074)
  • NSF Career Award (CBET‐1253641)
  • DARPA Young Faculty
  • Penn State Institute
  • NSF Research
  • Amazon AWS Research Grant
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
synthetic biology, kinetic modeling, metabolic pathway optimization, plasmid expression, bacterial hosts, biosynthetic pathway, combinatorial designs
MeSH terms
Algorithms, Bacteria, Bacterial Proteins, Biophysics, Models, Theoretical, Plasmids, Genome, Bacterial, Computational Biology

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