But measurements of YFP in C. reinhardtii turned out to be a great challenge, because of the strong interaction of the algae with light (by their photosystems, pigments, chlorophyll and light antennae). More information on our process of measuring YFP can be found on our Measurement page.

We were able to successfully measure YFP fluorescence intensity and fluorescence spectra of YFP-expressing C. reinhardtii clones in comparison to the wild type (WT). 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 was exposed only to the dark, the other to synchronized growth lighting conditions (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.

1. L1c-Psad-ARS-YFP-RbcS2
2. L1c-Psad-ARS-YFP-SP₂₀-RbcS2
3. L1c-AR-ARS-YFP-RbcS2

The first and third construct were designed for measuring the influence of arylsulfatase secretion signal (ARS) on mVenus secretion, expressed by two different promoters. The first and second constructs could be compared to see how the SP₂₀ glycomodule increased the secretion driven by the ARS peptide. The level 1 constructs were transformed into C. reinhardtii via electroporation. After two weeks of growth on TAP-agar plates containing paromomycin, the present clones were picked for colony PCR or sequencing.

To see if and how our different constructs worked, we conducted supernatant measurements. For each positive clone 50 ml flasks were prepared 4 days in advance. Of each flask 150 µl were transferred to a black 96-well plate and sequentially diluted up to 1:128. These flasks were then centrifuged for 10 min with a speed of 2000 rpm at 4°C. The supernatant was separated from the pellet in a fresh falcon tube for measurement. Before starting another centrifugation process, 12 x 150 µl of each supernatant were transferred to a black 96-well plate. This step was repeated after the second centrifugation, creating a third well plate. All three well plates were then analyzed under the plate reader to measure for fluorescence emission.

Unfortunately, we were unable to measure fluorescence in any of the supernatants. For every well in each plate, absorbance was measured at a wavelength of 680 nm to check the supernatants for possible cell debris or leftover Chlamys and absorbance scans were performed to scan for the absorption by YFP. Additionally, we measured the fluorescence with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The fluorescence intensity, plotted against the absorbance of the medium, can be seen in Fig. 4. Here we see that the fluorescence intensity correlates with the optical density of the medium, which explains the fluctuations in fluorescence intensity due to the presence of residual cells or debris in the medium. It was not possible to assess with certainty if there was YFP present in the medium.

Nevertheless, we were able to prove that YFP was present in vesicles near the membrane area of one of our YFP mVenus secretion clones (BBa_K2984030). This was done by imaging C. reinhardtii cells using confocal microscopy (Fig. 4.1). Although we were not able to measure YFP in the medium surrounding the algae, which is difficult, as can be read in our measurements page, it is possible to assume that secretion did occur if we observe the confocal micrsocopy images. The presence of the vesicle strongly suggests secretion of YFP mVenus.

Additionally, we can verify that the ARS secretion signal works in principle during the screening of one of our CRISPR/Cas9 transformations, where secretion is used to color positive clone blue (Fig. 5). By knocking out the SNRK gene, which regulates the expression of the arylsulfatase, uncontrolled secretion of this enzyme takes place. By adding a special pigment to the colonies, the knockout colonies become blue, demonstrating the function of the ARS secretion signal.

Furthermore, b< transforming such an enzyme into a C. reinhardtii strain these transformants gain a selection advantage in comparison to the wild type. Trough coupling of PtxD to PETase or MHETase we could avoid the problem of C. reinhardtii cells, which lost PETase/MHETase activity by expulsion of the plasmid prevailing.

Based on these advantages we want to transform the PtxD gene into C. reinhardtii. To determine the impact of phosphate and phosphite on growth of C. reinhardtii, we tested different strains under several media conditions. As a reference we used the TAP medium containing phosphate. We further tested growth in a medium without phosphate (TA medium) and in TAP medium supplemented with phosphite (TAPi).

The first graph shows the growth curve of the wildtype strain SAG11-32b. The cells growing on TAP media show growth up to OD 2.3 [R.U.]. Cultures growing on phosphorus-deficient media (TA) grow similarly. This is probably due to the phosphate storage of C. reinhardtii in the cytosol (Vered Irihimovitch et al., 2008).

In comparison, the cultures growing on phosphite (TAPi) media show strongly reduced growth. This is explained by the fact that phosphonate acts as a potent enzyme inhibitor which competes with its structural analogues for binding the enzyme's active sites (White & Metcalf, 2007). In conclusion, the metabolism of phosphate is inhibited by phosphite in C. reinhardtii wild type cells.

To show how the C. reinhardtii mutant strain UVM4 (mutant without cell wall) can handle phosphate starvation, cultures were grown in TA medium for 9 days and were then used to inoculate the cultures on TAPi medium. The graph shows three replicates of UVM4 cultures that were cultivated in phosphate-depleted medium (blue, red, green). These cells were not able to grow at all but if these were rescued in TAP medium they were able to grow again. Cells that grew on TAP medium as a control displayed normal growth. This experiment shows that C. reinhardtii cannot grow under phosphate starvation if the storages of phosphate were previously depleted.

To see if our Cas9 assays work in general, we first used the easy screenable SNF-related serine/threonine-protein kinase (SNRK2.2) locus. Inactivation of the SNRK2.2 gene leads to the constitutive expression of arylsulfatase, which gets secreted into the medium. The presence of arylsulfatase can easily monitored with a color reaction. By cleaving the sulfate group from the colourless substrate 5-bromo-4-chloro-3-indolyl-sulfate (X-SO₄) the dye indole blue is formed (Fig. 8). This blue-green screen poses as an efficient method of finding CRISPR/Cas9-directed insertion mutants.

The first construct we inserted into the SNRK2.2 locus was the level 1 construct L1c-Psad-YFP-Rbcs2. This experiment delivered us our first positive YFP-expressing clone exhibiting a robust expression of mVenus. Then, we performed a colony PCR with SNRK2.2 locus-specific primers to amplify the insert. The SNRK2.2 specific amplification without an insert would lead to bands with a length of approximately 200 bp. If the insert was successfully inserted into the SNRK2.2 gene, we expected a band of approximately 2000 bp. The PCR results showed that the insertion of the YFP construct into the SNRK2.2 locus was only partly successful (Fig. 9). We can see a bigger band at around 5000 bp. The YFP-expressing clone is labeled on Fig. 9 as A4. We sent the bands of a length of 5000 bp to sequencing but obtained only overlayed signals from a mix of PCR products. So the SNRK2.2 target site was cut by CRISPR/Cas9 but we have no certainty if the YFP construct is inserted along with other sequences, inserted twice or not inserted at all.

After confirming that we can insert our YFP construct at targeted regions into the genome we now thought of loci that would boost the expression of our transgene. We chose two of the most highly expressed genes in C. reinhardtii, PSAD and RBCS2 and designed guide RNAs for CRISPR/Cas9 to insert our YFP-constructs into the 3'UTR region of these genes. By inserting our construct into these loci we hoped for an increased expression of our YFP mVenus.

After the transformation of our algae with CRISPR/Cas9 ribonucleoproteins we performed colony PCRs as a screening method to examine if the insertion into the loci was successful. For this, we designed primers that bind to the PSAD and RBCS2 genes to investigate if the insertion worked as planned.

The PCR result, seen on Fig. 10, shows the analysis of 96 clones that were transformed to target a YFP construct into PSAD and RBCS2. Only for the wild type (WT) probe of RbcS2 a clear PCR band can be seen at the expected length. No clear bands can be observed in the other samples. This indicates that there was a problem with the PCR parameters and fragments were not amplified correctly. Due to the fact that we did this experiment at the end of our project we did not have enough time to do further screening or transformations to identify high expression loci in C. reinhardtii.

We cultivated a C. reinhardtii clone which was successfully transformed with our YFP mVenus construct and showed abundant expression of the protein. We wanted to investigate in which locus our transgene was inserted, so we planned a RESDA PCR.

The RESDA PCR method is a PCR method designed to identify the insertion locus of transgenes. The method was developed by González-Ballester and his colleagues and is based on the amplification of a transgene from the genome using primers that bind on recurrent restriction sites in the genome (González-Ballester et al. 2005). To amplify the transgene, one primer that binds to the insert is used and another primer that binds to one of the recurrent enzymes in the genome, in hope that the insert is close enough to a restriction site to be amplified. The amplification is done through two different rounds where the second round uses nested primers to narrow down the specificity of the amplification. After the amplification the fragment can be sequenced to determine its locus.

Our goal was to determine the locus of insertion of our YFP mVenus construct, to see if we could use this locus for future directed insertion of transgenes with CRISPR/Cas9. Additionally, we also performed the RESDA-PCR with a luciferase-expressing clone kindly provided by the research group of Prof. Dr. Hegemann of the Humboldt University of Berlin.

The PCR was made with primers for four different restriction sites. These are AluI, PstI, SacII and TaqI. We performed the PCR with a temperature gradient and with different primer concentrations to check for optimal conditions of amplification. Unfortunately, we were not able to observe any bands on the agarose gel after our RESDA-PCR (Fig. 1). We repeated the PCR twice, varying the conditions each time to optimize the formation of bands. Still, we were not able to reproduce the results described by González-Ballester et al. (2005). This means that we were unfortunately unsuccessful to determine the locus of insertion of our YFP expressing clone.