A family of isomeric multicopy model expression plasmids of 5526 bp was created based on pIFC3.13, a particularly stable member of the aforementioned pIFC3.X series (Hohnholz et al. 2017). These plasmids carry an expression block with the yeast-adapted yEGFP3 serving as reporter protein.
Escherichia coli and yeast strains
For cloning and DNA amplification, E. coli strain DH5α (Life Technologies, Darmstadt, Germany) was used employing standard molecular biology protocols. As we did not see a difference in transformation efficiency and segregational stability between the two common laboratory S. cerevisiae strains SY922 and BY4742 (Hohnholz et al. 2017), we focused in this study on the widely employed BY4742 (MAT
α, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0; Brachmann et al. 1998; Euroscarf collection, Frankfurt, Germany). E. coli and yeast transformation and propagation were done as described previously (Hohnholz et al. 2017).
Assembly of the yEGFP3 expression block and plasmid construction
The yEGFP3 expression block (1180 bp; Fig. 1a) consists of a fragment of the strong constitutive yeast TEF1 promoter, a yeast-adapted synthetic sequence encoding a mutated version of the Aequorea victoria GFP (S65G, S72A; Cormack et al. 1997), and the Tsynth8 synthetic transcription termination sequence (Curran et al. 2015). TEF1 promoter sequences (YPR080W; 407 bp) were derived by PCR from yeast strain SY992 (Tomlin et al. 2001) with a SacI site added to the 5′ end. yEGFP3 sequences (714 bp) were amplified from pUG34 (supplied by J. H. Hegemann, Heinrich-Heine-Universität, Düsseldorf, Germany) adding a TAG stop codon to their 3′ end. The Tsynth8 synthetic terminator (49 bp), preceded by an A, with 6 bp added to its 3′ end for a SacI site, were assembled from a pair of oligonucleotides. Oligonucleotides used in the assembly are
RH009 5′-GCATAGCAATCTAATCTAAGATGTCTAAAGGTGAAGAATTATTCACTGGTG-3′, and RH010 5′-TATGAGCTCTTTGAAAGATGATACTCTTTATTCCTACATAAGTAAATGAGTTTATATATCTATTTGTACAATTCATCCATACCATGGGTAATACC-3′. The yEGFP3 expression block was inserted in pUC18 from where it was recovered as a SacI-SacI fragment.
Cloning of the expression plasmids pIFC4.131–4.136 and yeast transformation
The SacI fragment of the yEGFP3 expression block was cloned into pIFC3.13 (Hohnholz et al. 2017) partially digested with SacI. The complete series of isomeric expression plasmids was recovered with the reporter expression block inserted in all three possible positions and, in each case, in both orientations (Fig. 1c). The yEGFP3 expression block was also inserted into pIFC3.11 in the same manner, recovering pIFC4.112. For the purpose of this work, we transformed yeast strain BY4742 with each member of the series (Fig. 1c) employing a published method (Gietz and Schiestl 2007).
Plasmid loss studies
His+ transformants of each plasmid were cultivated for 16 h in selective SDsup (fully synthethic media with dextrose as carbon source and with L-leucine, L-lysine, and uracil as supplements) and for three consecutive cycles of 24 h of cultivation (= 72 h) in SD sup starting with a 1:1000 dilution or in YPDAU (YPD supplemented with adenine and uracil) starting with a 1:4000 dilution as described for previous plasmid loss studies of the pIFC3.X plasmid family unless otherwise stated (Hohnholz et al. 2017). Plasmid loss rates were calculated in the published manner (Hohnholz et al. 2017). As we expected segregational stability to drop for the expression vectors, we followed plasmid loss for a reduced time, i.e., 3 × 24 h = 72 h instead of 5 × 24 h = 120 h (in our case more than 30 generations).
Plasmid copy numbers
DNA was prepared, and PCNs were assessed by qPCR with the PerfeCTa® SYBR® Green FastMix (Quanta, Beverly, MA, USA). Detection system and analysis software were the Mastercycler® RealPlex2 and realplex software version 2.2 (Eppendorf, Hamburg, Germany). A 113 bp fragment of the HIS3 ORF (YOR202W) contained in the plasmid was amplified, likewise a 111 bp fragment of the chromosomal single copy gene ENB1 (YOL158C) serving as internal standard (Hohnholz et al. 2017).
Reporter gene expression analysis
His+ transformants (a mixture of 10 colonies randomly chosen) were recovered from a SDsup agar plate and were grown overnight in liquid SDsup at 30 °C or for 48 h in YPDAU as described for the plasmid loss studies. Cells were harvested, washed, and suspended in a 1:1 ratio of cells to potassium phosphate buffer (50 mM, pH 6.0) containing a protease inhibitor mix (Complete, Mini, EDTA-free, Roche Diagnostics, Mannheim, Germany). Glass beads (Ø 0.25–0.5 mm, Carl Roth, Karlsruhe, Germany) were added to the suspension. Cells were lysed by vortexing at Vmax in 6 cycles of 2 min with intermittent 1 min cooling on ice. Extracts were recovered and glass beads were washed twice with 50 μL of ice cold buffer. Extracts and washings were pooled and centrifuged at 13,000 x g at 4 °C for 1 min. Supernatants were withdrawn and kept at −18 °C in aliquots for further use. Protein concentrations were assessed employing the Bradford protocol with bovine serum albumin as a standard.
Native polyacrylamide gel electrophoresis (PAGE) was carried out following the protocol of Bio-Rad (Bio-Rad Laboratories GmbH, Munich, Germany). Samples with the desired amount of protein were diluted in sample buffer (Bio-Rad) and were separated on commercial 4–20% polyacrylamide gradient gels (Bio-Rad) in running buffer (Tris-glycine; following the Bio-Rad protocol). Fluorescent bands were detected illuminating the gels with the Blue/Green LED Transilluminator (Nippon Genetics, Dueren, Germany) at 480–530 nm.
Relative fluorescence was determined using the Hitachi F-2500 Fluorescence Spectrophotometer (FL Solutions program; Hitachi High-Technologies Europe GmbH, Krefeld, Germany) with an excitation of 485 nm and an emission spectrum between 500 and 560 nm. For whole cell analyses, 5 OD600 units of cells were centrifuged, washed, and resuspended in 1.5 mL water. For protein extract analyses, 80 and 40 μg of total protein were diluted in 1.5 mL of water, respectively.