title: Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae.
abstract: The ability to rapidly customize an expression vector of choice is a valuable tool for any researcher involved in high-throughput molecular cloning for protein overexpression. Unfortunately, it is common practice to amend or neglect protein targets if the gene that encodes the protein of interest is incompatible with the multiple-cloning region of a preferred expression vector. To address this issue, a method was developed to quickly exchange the multiple-cloning region of the popular expression plasmid pET-28 with a ligation-independent cloning cassette, generating pGAY-28. This cassette contains dual inverted restriction sites that reduce false positive clones by generating a linearized plasmid incapable of self-annealing after a single restriction-enzyme digest. We also establish that progressively cooling the vector and insert leads to a significant increase in ligation-independent transformation efficiency, demonstrated by the incorporation of a 10.3 kb insert into the vector. The method reported to accomplish plasmid reconstruction is uniquely versatile yet simple, relying on the strategic placement of primers combined with homologous recombination of PCR products in yeast.
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, United States.
authors.forename: Glen
authors.initials: G
authors.lastname: Gay
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, United States.
authors.forename: Drew T
authors.initials: DT
authors.lastname: Wagner
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, United States.
authors.forename: Adrian T
authors.initials: AT
authors.lastname: Keatinge-Clay
authors.affiliation: Department of Molecular Biosciences, University of Texas at Austin, Austin, TX 78712, United States. Electronic address: darren.gay@utexas.edu.
authors.forename: Darren C
authors.initials: DC
authors.lastname: Gay
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D005822:Genetic Vectors / Q000235:genetics*; D059765:Homologous Recombination; D016133:Polymerase Chain Reaction / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics*
chemical_list: D017931:DNA Primers
keywords: Homologous recombination; Ligation-independent cloning; Plasmid assembly; Plasmid reconstruction