A series of conditional shuttle vectors for targeted genomic integration in budding yeast

Abstract

The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications.

Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration

“…After transformation into yeast, we identified a strain bearing a single integrant of the YIp construct and confirmed that strains bearing a YCp produced relatively similar fluorescence levels (data not shown). As previously observed (Chou et al, ; Gnügge et al, ; Jensen et al, ), strains bearing YIp or YCp constructs produce dissimilar histograms of GFP expression when assayed by flow cytometry (Figure a). Even in plasmid‐selecting medium, a proportion of cells transformed with a YCp (blue histogram) appear to lose the plasmid and are optically dark (peak to the far left).…”

“…Those plasmids, in turn, have been transformed into thousands of yeast strains, including strains bearing empty pRS40x vectors for use as controls. Potential users of the ICE plasmids series should be aware, though, that a tremendous amount of effort has been put into updating and redesigning yeast shuttle vectors (Chee & Haase, ; Chou et al, ; Fang et al, ; Frazer & O'Keefe, ; Gnügge et al, ; Jensen et al, ; Lian et al, ). These include both updates to the pRS40x series, such as the removal of restriction sites from yeast selectable markers to increase the number of unique cutters in the multiple cloning site (MCS; Chee & Haase, ), as well as completely redesigned vector backbones (Gnügge et al, ).…”

“…Although YCps are acceptable for many applications and can often facilitate screening schemes, in some instances, they pose significant drawbacks. CEN / ARS elements confer incomplete stability upon a plasmid, leading to heterogeneity in plasmid copy number, even within clonal populations (Chou, Patel, & Gartenberg, ; Gnügge, Liphardt, & Rudolf, ; Hieter, Mann, Snyder, & Davis, ; and below). Conversely, constructs integrated into the genome using yeast integrating plasmids (YIps) are thought to be more stable, ensuring less varied levels of gene expression between clones.…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…[44] Furthermore, yeast homologous recombination has recently been used to assemble conditional shuttle vectors for yeast chromosomal integration. [50] The autonomous replication sequence and centromere of the constructed shuttle vectors are flanked by loxP sites targeted by Cre site-specific recombinase. In the extrachromosomal form, shuttle vectors can be used as backbones for yeast homologous recombination-mediated DNA assembly.…”

“…Expression of Cre recombinase initiates excision of the autonomous replication sequence and centromere-harboring cassette which leads to integration of the vector into the chromosome at a targeted locus. [50] In vivo DNA assembly in B. subtilis…”