The recircularized plasmid is then rescued from yeast transformants that show robust protein expression in the yeast cultures on the same day.
The use of rice chi11, encoding a chitinase, as a co-transgene along with tlp produced a tlp chi11 co-transformant that showed enhanced resistance against R. solani than the ones that express tlp or chi11 transgene alone.
The only transformant that showed a significant growth rate (with an approximate doubling time of 3 h) was the one encoding the K617A mutant (Fig. 4).
In the present study, different IBE-producing transformants of C. acetobutylicum that showed high isopropanol excretion capacities have been constructed.
However, the relatively low number of bands in each restriction digest of genomic DNA from the two transformants that are shown in Figure 7D and from four other transformants that were analyzed, suggests that there are only ~1 3 integrations of the co-transformed plasmid, so a high gene dosage through a large number of integrations can be excluded.
As shown in Table 1, the estimated number of copies of each transgene was identical to that shown previously by our group (when the transformants were generated).
Transformants were obtained that showed significantly higher secreted protein yield, with a resulting heterologous protein activity that ranged between 10% and 373% higher compared to the parental strains when grown in complex media.
Out of 192 transformants that were screened, four showed a single 2490 bp amplicon, which confirmed that the deletion construct had integrated into the KU80 locus (Fig. S3B).
In addition to the pPmr3-transformants, the aphVIII gene was detected on Southern blots with genomic DNA from transformants that were produced with plasmid paphG (data not shown).
Transformants that were generated through co-transformation with plasmid pHsp70A-GLuc were also analyzed qualitatively for luciferase activity, and several of these transformants showed chemiluminescence (data not shown).
