Team:Stuttgart/Rose and Limonene Fragrance
Esterases and Lipases
Enzyme activity assay: Esterases
In the following figures enzyme activities of the supernatant of the esterases EstCS2 and LIpB are shown. All results were obtained from biological triplicates. The detailed method for the activity determination is shown here: LINK At the N-terminal end of the esterase EstCS2 a signal peptide (PelB) is added. Therefore the gene induction leads to enzyme expression and the expression of the signalpeptide and the secretion of the enzyme is enabled.
Results
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). The rose genes KDC-YjgB-ARO8 and ATF1 were sucessfully amplified by PCR. The agarosegel analysis (Fig.10) 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.
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