Synthesis of L0 and L1 gene constructs

Text for the two column thingy here

Expression of YFP-containing constructs

The yellow fluorescent protein YFP was used as a fluorescent tag in some of our constructs. The goal of using YFP as a tag was to be able to measure enzyme expression and secretion and also being able to screen for successful mutants using YFP as a marker. Additionally we wanted to use a YFP expressing C. reinhardtii to analyse locus effects on expression. We were able to successfully transform a YFP expressing C. reinhardtii with a construct of our own design. But measuring YFP in C. reinhardtii turned out to be a great challenge, because of the strong interaction of the algae with light (photo systems, pigments, chlorophyll and light antennae). More information on our process measuring YFP can be found in our measurements page.

The successful transformation of other clones containing YFP turned out to be problematic, due to problems unrelated to the YFP. For the secretion results click here (link). And for the PETase results click here (link).

We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP expressing C. reinhardtii clones in comparison to wild type. The results showed that our clone exhibited a higher fluorescence intensity at 528 nm than the WT (YFP emission peak) and the fluorescence spectrum of YFP confirmed the presence of the yellow fluorescent protein. This YFP expressing clone also allowed us to characterize the light induction of the PsaD promoter by doing a time resolved measurement of the fluorescence intensity. During this measurement we exposed C. reinhardtii cultures which contained our YFP construct to different growth conditions. One Dark, and another synchronized growth (10 hours dark, 14 hours illuminated). Then, we started a time resolved fluorescence intensity measurement in the dark, with a WT control. After approximately four hours, we activated a light source and exposed the cultures to light, thus activating the light inducible PsaD promoter. Our results showed that for the dark and synchronized cultures containing the YFP construct a peak in fluorescence intensity could clearly be seen after the light induction. This proved the light induction of the PsaD promoter. If you are interested in this measurement, please visit the page of our YFP mVenus construct in the iGEM registry here.

Fig. - Fluorescence intensity of C. reinhardtii WT and a YFP carrying clone, in decreasing optical density of the cell culture. Excitation at 490 nm and emission measurement at 528 nm. The results clearly show that the fluorescence of the YFP expressing clone is higher than the autofluorescence of the WT algae. Fig. - YFP emission spectrum of a C. reinhardtii clone with YFP with an emission maximum at approximately 530 nm. Difference spectrum of WT and YFP spectra Fig. - Time resolved measurement of the YFP fluorescence intensity. Light induction of the PsaD promoter (white area). It can clearly be seen that at the time of illumination the fluorescence intensity of YFP increases, indicating a light induced expression of the protein.