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Figure 1 | Microbial Cell Factories

Figure 1

From: Small, synthetic, GC-rich mRNA stem-loop modules 5′ proximal to the AUG start-codon predictably tune gene expression in yeast

Figure 1

The Sfi I restriction enzyme cloning site severely inhibits gene expression in yeast. A. A schematic diagram of the cloning strategy used to over-express heterologous ORFs in S. cerevisiae. PDR5 (orange) was cloned as Sfi I/Not I or Pac I/Not I fragments into pABC1 (the pBluescriptIISK(+) vector backbone is light green and the multiple cloning site of the transformation cassette light blue). pABC1-PDR5 was digested with Fse I and Asc I to release the 7.4 kb transformation cassette [PDR5 promoter (green)-ORF (orange)-PGK1 terminator (blue)-URA3 marker (purple)-PDR5 downstream region (green)]. The transformation cassettes were gel purified and used to transform S. cerevisiae AD to Ura+. B. Effect of 5′ UTR on Pdr5p activity. Strains expressing Pdr5p were created either using pABC1 (SfiI-PDR5, SfiI-PacI-PDR5) or pABC3 (PacI-PDR5), as previously described [14], and ∆PDR5 (AD) and wt-PDR5 (AD124567u-) were used as negative (0% Pdr5p expression) and positive (100% Pdr5p expression) controls, respectively. The 32 nucleotides upstream of the ATG start-codon for each construct are shown. Sfi I and Pac I restriction sites are underlined and the nucleotides that differ from the wild-type PDR5 5′ UTR are shown in bold type. The right hand column lists the MICFLC values for the strains. The MICFLC values for three independent transformants were measured and did not vary by more than one dilution. C. SDS-PAGE of plasma membrane proteins (30 μg) isolated from the strains listed in B including AD/PDR5 (this strain is AD with its wild-type PDR5 locus restored). The black arrowhead indicates Pdr5p and the white arrowhead indicates the prominent plasma membrane proton pump protein Pma1p. SP = AD/SP-PDR5-URA3; S = AD/S-PDR5-URA3; P = AD/P-PDR5-URA3.

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