MAT a KanMX4-deletion strains were transformed with one of the 11 gene insertion cassettes constructed with the GFP insertion module, and transformants were selected via the URA3 marker.
The overall FAME contents of the two transformants were similar with one another, and with WT, under normal culture conditions (Table 2).
In these cases it is important to identify promising candidates for further breeding at an early stage, and hence being able to distinguish transformants with low copy numbers (one or two) from those with high copy numbers is sufficient.
One of the transformants with highest expression was grown on a 5-l bioreactor and induced with methanol for 48 h.
The crosses utilized a common GAL4 driver line in combination with one of the eight RNAi lines (transformant ID: 21806, 26416, 33207, 36641, 43632, 47145, 49795, 49796) developed by the Vienna Drosophila RNAi Center (VDRC).
A 26 bp duplication of the T-DNA border region was detected in one of the transformants, with a 12 bp filler DNA fragment of unknown origin (Fig. 5B).
When the pHFGE-1 with one or the other of these foreign genes were transformed into H. fabianii J640 u-1, the transformants (named HF-AAMY and HF-XYN, respectively) showed α-amylase and xylanase activities respectively.
An ascospore isolate with one copy of the overexpression plasmid was used for further crosses with transformants carrying the knockout construct at the homologous locus to establish a transformant carrying both the deletion background of sec22 and an ectopically integrated overexpression construct.
Strains with one additional copy of the expression cassette were chosen for further analysis and named according to the promoter region and cellulase expressed (e.g., t-cel7b for a transformant expressing cel7b under the tef1 promoter region).
Co-cultivation for 60 hours yielded more transformants than at 48 hours; however, more transformants with growth defects tended to emerge on the SM plates.
