title: 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.
authors: Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.
authors: Iman
authors: I
authors: Farasat
authors: Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA.
authors: Manish
authors: M
authors: Kushwaha
authors: Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA.
authors: Jason
authors: J
authors: Collens
authors: Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.
authors: Michael
authors: M
authors: Easterbrook
authors: Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA.
authors: Matthew
authors: M
authors: Guido
authors: Department of Chemical Engineering, Pennsylvania State University, University Park, PA, USA Department of Biological Engineering, Pennsylvania State University, University Park, PA, USA salis@psu.edu.
authors: Howard M
authors: HM
authors: Salis
mesh_terms: D000465:Algorithms; D001419:Bacteria / Q000145:classification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D001703:Biophysics; D019295:Computational Biology / Q000379:methods*; D016680:Genome, Bacterial; D008962:Models, Theoretical; D010957:Plasmids
chemical_list: D001426:Bacterial Proteins
keywords: SEAMAPs; biophysical models; pathway optimization; synthetic biology