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.

An improved system for performing stable integration of multi‐gene pathways in yeas...

Source: FEMS Yeast Research - October 10, 2014 Category: Research Authors: Michael S. Siddiqui, Atri Choksi, Christina D. Smolke Tags: Research Article Source Type: research