Team:DTU-Denmark/Results-amilCP
amilCP Expression
Assembly
AmilCP was acquired from the distribution kit and assembled into pSB1C3 with different fungal/eukaryotic promoters – the two Aspergillus nidulans derived promoters pTrpC and pGpdA as well as the cauliflower mosaic virus 35S promoter - using 3A assembly. Afterwards the terminator from the cauliflower virus was also added via 3A assembly. Correct transformants were identified by colony PCR and Sanger sequencing using the verification primers, VF2 and VR (See figure 1).
Figure 1: Lav et billede af den relevante gel og inkluder et udklip af sekvensen.
Transformation
After confirmed assembly the amilCP expression cassettes were then transformed into Aspergillus oryzae RIB40. Due to the lack of appropriate selectable conditions, none were used. Nonetheless some strains exhibited a darkened spore coloration indicating a possible transformation, see figure 2.
Figure 2: Tag et billed til sammenligning fra de lineariserede pCaMV
Genomic DNA was purified from the strains identified as possible transformants. Integration of the amilCP sequence was then confirmed by PCR using VF2 and VR, as well as sequence specific primers, see figure 3.
Figure 3: Tag gel-billeder med
Brick construction
To test whether the integration, and possible expression of amilCP, would lead to a visual change in materials produced, a small brick was inoculated using spores from the identified transformants. However, this did not produce any visual obvious change in brick color, see figure 4:
Figure 4: Tag et billede af murstenen af svamp