Abstract
We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli. This set of pRSII plasmids includes new shuttle vectors that can be used with histidine and adenine auxotrophic laboratory yeast strains carrying mutations in the genes HIS2 and ADE1, respectively. Our pRSII plasmids also include updated versions of commonly used pRS plasmids from which common restriction sites that occur within their yeast-selectable biosynthetic marker genes have been removed to increase the availability of unique restriction sites within their polylinker regions. Hence, our pRSII plasmids are a complete set of integrating, centromere and 2μ episomal plasmids with the biosynthetic marker genes ADE2, HIS3, TRP1, LEU2, URA3, HIS2, and ADE1 and a standardized selection of at least 16 unique restriction sites in their polylinkers. Additionally, we have expanded the range of drug selection options that can be used for PCR-mediated homologous replacement using pRS plasmid templates by replacing the G418-resistance kanMX4 cassette of pRS400 with MX4 cassettes encoding resistance to phleomycin, hygromycin B, nourseothricin, and bialaphos. Finally, in the process of generating the new plasmids, we have determined several errors in existing publicly available sequences for several commonly used yeast plasmids. Using our updated sequences, we constructed pRS plasmid backbones with a unique restriction site for inserting new markers to facilitate future expansion of the pRS series.
Highlights
We have constructed a set of 42 plasmid shuttle vectors based on the widely used pRS series for use in the budding yeast Saccharomyces cerevisiae and the bacterium Escherichia coli
The choice of restriction sites for cloning constructs into pRS plasmids is marker-dependent and may complicate in vitro cloning. This is due to the presence of several restriction sites within the S. cerevisiae HIS3, TRP1, LEU2, URA3 and other yeastselectable marker sequences of the pRS plasmids that are found in the pBluescript/pBluescript II multiple cloning site (MCS) of the pRS plasmid backbone (Brachmann et al 1998; Eriksson et al 2004; Sikorski and Hieter 1989)
Despite the availability of plasmids that can be used with histidine auxotrophic laboratory strains of budding yeast that are his3 mutants, these plasmids cannot be used with His2 strains that are his2 mutants
Summary
Plasmid construction Standard techniques were used for DNA manipulation. Restriction enzymes were purchased from New England Biolabs, except for PfoI, which was purchased from Fermentas. We used silent mutations that preserve the amino acid sequence to mutagenize restriction sites found in the open reading frame of the yeast-selectable auxotrophic marker genes ADE2, HIS3, TRP1, LEU2, URA3, ADE1, and HIS2 (Table S1). The prototrophic yeast strain S288C (MATa SUC2 gal mal mel flo flo hap ho bio bio6) (Mortimer and Johnston 1986), known as SBY1806 in our lab, was used to verify the utility of our new pRS plasmids carrying MX4 drug resistance cassettes in PCR-mediated gene replacement (File S1). To transform the wild-type strains 15Daub and W303a using pRS/ pRSII plasmids, we used either 200 ng of integrating plasmid linearized by restriction at a unique site within the yeast-selectable prototrophic marker sequence or 50 ng of CEN/2m plasmid. The transformation of yeast with PCR-amplified MX4 drug resistance cassettes is described in detail in File S1
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