Rose and Limonene 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 KDC gene which encoded 2-keto acid decarboxylase has a gene size of 1908 bp. YjgB has been identified as aldehyde reductase in E.coli and has a size of 1020 bp. The gene ARO8 which encode aminotransferase has a gene size of 1503 bp. The alcohol acetyltransferase ATF1 from S.cerevisae has a size of 1578 bp. 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. Our rose fragrance construct is shown in Figure 12. The assembled rose plasmids were sucessfully transformed into DH5alpha E.coli cells (figure). Unfortunately, despite sucessfull transformation, the rose plasmid genes couldn't be confirmed by colony PCR (Figure 16).
Subsequent studies 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). For SpeI we would expect a band at 3076 bp and 5267 bp. The gel in Figure 14 shows two bands, but not in the expected range. After digest with EcoRI we expected three bands at 3349 bp, 1942 bp and 3052 bp. That could not be confirmed either (Figure 15). For the uncut roseplasmid we would expect a band in a molecular range of 8343 bp, the agarosegel-analysis showed bands in the range of 4000-5000 bp, which was clearly to low. In the end a sequencing analysis of the assembled rose plasmids 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.