Rose and Limonene Fragrance

Rose Fragrance

The plasmids from Guo et al. pET28a-KDC-YjgB-ARO8 and pET28a-ATF1, used in this study, were sucessfully confirmed by sequencing the plasmids at GATC (Figure 9 and 10). The rose genes KDC-YjgB-ARO8 and ATF1 were sucessfully amplified by PCR, using specific overlap primer for psB1K3 backbone. The agarosegel analysis (Fig.11) showed expected ties in the range between 4000-5000 kDa for KDC-YjgB-ARO8 and another tie at 1600 kDa for ATF1. The arabiniose-inducable promotor pBAD (BBa_K206000 from the iGEM kit plate) could also be amplified by PCR. The purified PCR product showed a discrete tie at 130 kDa. Additionally another PCR was perfomed, to couple the pBAD promotor to the gene complex with KDC-YjgB-ARO8. The Verification by agarosegel electrophoresis showed that the coupling wasn't sucessfull.

The next step was to fuse the fragments pBAD, KDC-YjgB-ARO8 and ATF1 by Gibson Assembly and insert them into the psB1K3 backbone.

The assembled rose plasmids were sucessfully transformed into DH5alpha E.coli cells. Unfortunately, despite sucessfull transformation, the rose plasmid genes couldn't be confirmed by colony PCR (Figure 13). Subsequent studied also showed no success. To confirm the rose plasmid restriction digests were performed. Therefor the assembled rose plasmids were cut with EcoRI and SpeI (Figure 14). The restriction digest failed both times. Sequencing analysis of the assembled rose plasmid by GATC showed that only a part of ATF1 and kanamycin resistence was inserted in E.coli. Despite further investigations and efforts it wasn't possible to assemble all four rose genes with a pBAD promotor and transform them into E.coli, like described by Guo et al.