Transformation

After confirmed assembly, the amilCP expression cassettes were then transformed into Aspergillus oryzae RIB40. They were plated onto transformation media using no selectable conditions, as none appropriate were available. Nonetheless, some strains, mainly those transformed using linearized BBa_K2799011, exhibited a change in spore coloration. Possible transformants exhibited green and white coloured spores, which Aspergillus oryzae plated on PDA does not, instead exhibiting yellow hued spores, see fig. 4:

We, however, suspected that this difference in coloration might be an artifact of different media. Therefore, we plated the WT strain onto transformation plates as well, see fig. 5. Here it showed light-green spore formation, but no white or dark-green spores. This was confirmed under a microscope (data not shown).

Some variations in the color of transformants were additionally observed with some being a dark green and some showing a more yellowish hue, although all being green, see fig. 6:

Based on this we decided to confirm integration into the genome of transformants by PCR. Genomic DNA was purified from possible transformants exhibiting both the dark green and white spores. PCR was performed on the gDNA using primers (SeqP_AmilCP_FW and SeqP_CaMVT_BW) targeting a 227 bp region from the end of amilCP to the middle of the terminator, see fig. 7. Two samples was taken from each of the plates in fig. 6. One from a green area and one from a white are. Clear bands was produced from samples taken from green plates (1.1-2.2), while yellowish plates did not show any bands (3.1-4.2), thus indicating the darker green color as a possible byproduct of amilCP expression. No difference was seen between green and white spores from plate 1 and 2.

Next, we repeated the PCR on sample 1.1-2.2, this time including two gDNA samples prepared from scrapings of WT plates, see fig. 8. Again clear bands were seen from samples 1.1-2.2, however, the WT isolates did not produce any bands demonstrating that the band is produced due to integration and not just because of suitable binding regions for the primers present in the genome of A. oryzae.

To confirm integration in the positively identified transformants (1.1-2.2) samples have been submitted for Sanger sequencing using primer pointing into the constructs. Additionally, samples have also been submitted with primers pointing out of the construct in order to identify the site of integration in the genome. The primers used are: SeqP_AmilCP_FW, SeqP_CaMVT_FW and SeqP_CaMVT_BW. We are currently awaiting the sequencing results.