title: A system for multilocus chromosomal integration and transformation-free selection marker rescue.
abstract: Yeast integrating plasmids (YIPs) are a versatile tool for stable integration in Saccharomyces cerevisiae. However, current YIP systems necessitate time- and labor-intensive methods for cloning and selection marker rescue. Here, we describe the design, construction, and validation of a new YIP system capable of accelerating the stable integration of multiple expression constructs into different loci in the yeast S. cerevisiae. These 'directed pop-out' plasmids enable a simple, two-step integration protocol that results in a scarless integration alongside a complete rescue of the selection marker. These plasmids combine three key features: a dedicated 'YIPout' fragment directs a recombination event that rescues the selection marker while avoiding undesired excision of the target DNA sequence, a multifragment modular DNA assembly system simplifies cloning, and a new set of counterselectable markers enables serial integration followed by a transformation-free marker rescue event. We constructed and tested directed pop-out YIPs for integration of fluorescent reporter genes into four yeast loci. We validated our new YIP design by integrating three reporter genes into three different loci with transformation-free rescue of selection markers. These new YIP designs will facilitate the construction of yeast strains that express complex heterologous metabolic pathways.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Siddiqui
authors.forename: Atri
authors.initials: A
authors.lastname: Choksi
authors.forename: Christina D
authors.initials: CD
authors.lastname: Smolke
mesh_terms: D015870:Gene Expression; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012641:Selection, Genetic
keywords: counterselectable markers; genome editing; in vitro DNA assembly