title: Directed evolution of genetic parts and circuits by compartmentalized partnered replication.
abstract: Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression.
authors.affiliation: 1] Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA. [2].
authors.forename: Jared W
authors.initials: JW
authors.lastname: Ellefson
authors.affiliation: 1] Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA. [2].
authors.forename: Adam J
authors.initials: AJ
authors.lastname: Meyer
authors.affiliation: 1] Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA. [2] Applied Research Laboratories, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Randall A
authors.initials: RA
authors.lastname: Hughes
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Joe R
authors.initials: JR
authors.lastname: Cannon
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Brodbelt
authors.affiliation: 1] Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA. [2] Applied Research Laboratories, University of Texas at Austin, Austin, Texas, USA. [3] Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas, USA. [4] Center for Systems & Synthetic Biology, University of Texas at Austin, Austin, Texas, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
mesh_terms: D017123:Bacteriophage T7 / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D019020:Directed Molecular Evolution; D004926:Escherichia coli; D011401:Promoter Regions, Genetic; D012343:RNA, Transfer / Q000235:genetics; D012441:Saccharomyces cerevisiae; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism; D014369:Tryptophan-tRNA Ligase / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; D014764:Viral Proteins; D012343:RNA, Transfer; D019914:Taq Polymerase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D014369:Tryptophan-tRNA Ligase