To obtain α-type derivative cells from hybrid strain K76 according to the scheme illustrated in Fig. 2c, plasmid pAUR112 was introduced into strain K7A after removing plasmid pLhyS-2 K-Pa1, yielding K7A/pAUR112 transformants, which were selected on YPD + AUR plates.
To minimize positional effect of reporter gene expression, at least 1000 transformants on the membrane were collected to create a mixed population of transformants, which were cultured and assayed for the strength of GFP florescence.
This PCR used primers erm-Fwd-EcoRI and erm-Rev-HindIII and the following PCR conditions: 1 cycle of 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 45 s, and 68°C for 1 min; and a single extension of 68°C for 10 min. After electroporation of the transposon, C. perfringens strain 13 Erm-resistant transformants, which were also erm-positive by PCR, were randomly chosen for sequencing.
Both amplicons developed the expected digestion profile, demonstrating the authenticity of the two transformants, which were then selected to be further characterized in later studies.
There is considerable variation in the expression of transgenes in individual transformants, which is not due to differences in copy number [ 30].
One of these transformants, which was verified by PCR with primers PgpdA-F2 and PtraR1, was used for further genetic engineering.
A maximum of up to 28.6 U mL−1 at 120 h after induction) was achieved in P. pastoris transformants which was ~17 folds higher than that of wild type G. stearothermophilus.
Paromomycin-resistant transformants, which were generated either with paphG or pPmr3, yielded a PCR fragment of the expected size, whereas the wild type strain gave no such fragment, as expected.
Briefly, cDNA for seeding the amplifications were obtained by directly transferring into the reactions 5 μl of cDNA bacterial transformants, which were grown in 1.2 ml of 2X YT and 0.005% chloramphenicol in 96-deepwell plates for 24 hours at 37°C.
Treatment of cells in liquid cultures consisted of generating freshly saturated overnight cultures of the different yeast transformants, which were then diluted in fresh, pre-warmed galactose-containing media, incubated for 4 h to induce GAL1 gene expression, and subsequently treated with the indicated concentrations of compound including cadmium, cycloheximide and rapamycin.
