Constructing Integrative Plasmids with Yeast Markers

Plasmids in this work were constructed using pRS40B as a starting point [13 (link)]. The backbone of pRS40B (Figure 1) carries sequences relevant for transformation and maintenance in E. coli (bla, ori) and other common features (LacZα, MCS, f1 ori), as well as a TEF promoter and terminator originating from Eremothecium gossypii, known to be able to drive gene expression in various yeast species [14 (link)]. Under the control of the TEF promoter, dominant antibiotic resistance markers needed for selection of yeast transformants and plasmid maintenance in yeast cells were cloned. Markers were codon-optimized to be functional across the Saccharomycotina subphylum (Section 2.3) and synthesized de novo. All steps in the construction of integrative plasmids plasmid construction are shown in detail in Figure 1. DNA manipulations and restriction cloning were performed as in Sambrook (2001). Restriction and modification enzymes were used according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Competent cells of E. coli (NEB Stable, New England Biolabs, Ipswich, MA, USA) were prepared and electroporated as in Miller and Nickoloff (1995). Primers were synthesized by Metabion (Planegg/Steinkirchen, Germany). PCR was performed with Q5 polymerase (New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions.