Team:Potsdam/Results
Our results - dCas9
For the dCas9-project we designed a high copy and a low copy plasmid, which should be transformed together in E.coli to do metabolic channelling. We finished our high copy plasmid, so the plasmid with the target sequences for our signal guide RNAs. The plasmid is shown in the figure below:
Figure1: Finished high-copy plasmid
Our low copy plasmid includes the fusion of the LacI-repressor with dCas9, our enzymes IAAM and IAAH fused with the RNA-binding proteins MS2 and PP7, respectively and the sgRNAs. We finished the pre-plasmids, LacI-dCas9 in pExA2 shown in Figure 2 and IAAM-MS2-IAAH-PP7 (IAA-fusion) in pSB4A5 shown in Figure 3. We had very big problems with the assembly of the IAA-fusions which cost us six and a half weeks. The next steps to first clone the LacI-dCas9 also in the pSB4A5 with the IAA-fusions and second clone the sgRNA cassettes as last step in this vector, too, couldn’t be finished.
Figure1: Finished high-copy plasmid
Our low copy plasmid includes the fusion of the LacI-repressor with dCas9, our enzymes IAAM and IAAH fused with the RNA-binding proteins MS2 and PP7, respectively and the sgRNAs. We finished the pre-plasmids, LacI-dCas9 in pExA2 shown in Figure 2 and IAAM-MS2-IAAH-PP7 (IAA-fusion) in pSB4A5 shown in Figure 3. We had very big problems with the assembly of the IAA-fusions which cost us six and a half weeks. The next steps to first clone the LacI-dCas9 also in the pSB4A5 with the IAA-fusions and second clone the sgRNA cassettes as last step in this vector, too, couldn’t be finished.
Figure 2: Part for the low copy plasmid: LacI-dCas9 in pExA2
Part 3: Part of the low copy plasmid:IAAM-MS2-IAAH-PP7 in pSB4A5
Nevertheless we started GC-MS measurements to check the auxin production of the control plasmids. Unfortunately, these measurements were started after the wiki freeze and will be only presented during our presentation and later be added here.
Our results - LLPS
The Metabolic Channeling experiments with Liquid-Liquid Phase Separation (LLPS) were conducted in yeast cells (S. cerevisiae). Yeast was chosen because of its average cell diameter of about 5-10 µ1, which is significantly larger than the diameter of E. coli cells (1-3 µm2). Yeast was hoped to be big enough to enable the formation of droplets. Such experiments have already been successfully conducted in HeLA cells[4], which have an average diameter of more than 20 µm3.
Transformation-associated recombination (TAR) was used to achieve a homologous recombination between the vector and inserts in the yeast cells. The vector pYES2-CT, which works in both yeast and E. coli, was used for that. It is depicted in the following figure 1.
Transformation-associated recombination (TAR) was used to achieve a homologous recombination between the vector and inserts in the yeast cells. The vector pYES2-CT, which works in both yeast and E. coli, was used for that. It is depicted in the following figure 1.
Figure 1: Vector pYES2-CT, which works in both yeast and E. coli.
The experiment was then conducted with the following steps: First, Ddx4-YFP was synthesized by IDT with overhangs to the vector. In a next step Ddx4-YFP was cloned into the vector using TAR. This lead to the following final construct in the yeast cells, as depicted in figure 2.
Figure 2: Final construct of Ddx4-YFP in pYES2-CT.
It was then tested, if droplet formation due to the Ddx4-YFP was possible in the yeast cells. For that, fluorescence intensity images and bright field images were taken from yeast cells expressing the Ddx4-YFP and wild type yeast cells (YPH500 strain). The results are shown in figure 3. In the fluorescence intensity image of the yeast cells expressing Ddx4-YFP (induction of protein expression with galactose) bright dots can be seen, indicating the formation of fluorescing droplets. A comparison to the corresponding bright field image shows, that those droplets formed in the cytoplasm of the yeast cells. The wild type yeast cells show a small amount of background fluorescence, but no significant droplet like structures could be seen. It was therefore shown, that droplets do form in the yeast cells in which the Ddx4-YFP gene was inserted via TAR.
Figure 3: Fluorescence intensity images and bright field images of yeast cells expressing Ddx4-YFP (induction of protein expression with galactose) and wild type yeast cells. Significant droplet formation can be seen in the cytoplasm of yeast cells expressing Ddx4-YFP.
After confirming that droplet formation in yeast cells is possible with Ddx4, the genes responsible for auxin synthesis were cloned into the vector. Ddx4-IaaM and Ddx4-IaaH were synthesized by IDT and cloned into the vector via a Gibson Assembly and then transformed into yeast. The resulting vector is shown in figure 4.
Figure 4: pYES2 vector with Ddx4-IaaH and Ddx4-IaaM inserted via Gibson Assembly.
GC-MS measurements to analyze the auxin production were then conducted on yeast cells expressing the Ddx4-IaaM and Ddx4-IaaH. These measurements are currently being evaluated and results will not be ready before the wiki freeze. Those results will however be discussed in the upcoming talk at the Giant Jamboree in Boston.
As control, experiments with just IaaM and IaaH without Ddx4 were done. For this, the coding DNA sequences of IaaM and IaaH were first amplified out of the synthesized Ddx4-IaaM and Ddx4-IaaH constructs used before. After that, IaaM and IaaH were separately cloned into the vector using Gibson Assembly. In a next step, the full IaaH cassette was amplified and inserted into the vector with IaaM via Gibson Assembly. The resulting vector is depicted in figure 5.
As control, experiments with just IaaM and IaaH without Ddx4 were done. For this, the coding DNA sequences of IaaM and IaaH were first amplified out of the synthesized Ddx4-IaaM and Ddx4-IaaH constructs used before. After that, IaaM and IaaH were separately cloned into the vector using Gibson Assembly. In a next step, the full IaaH cassette was amplified and inserted into the vector with IaaM via Gibson Assembly. The resulting vector is depicted in figure 5.
Figure 5: pYES2 vector with both IAA- cassettes inserted via Gibson Assembly.
On these yeast cells, GC-MS measurements are being done to determine the amount of auxin being produced by the cells. Again, results will not be ready before the wiki freeze, but are to be discussed in the upcoming talk.
[1] Naito, Y., Toh-e, A. and Hamaguchi, H.-o. (2005), In vivo time-resolved Raman imaging of a spontaneous death process of a single budding yeast cell. J. Raman Spectrosc., 36: 837–839.
[2] Reshes, Galina et al. “Cell Shape Dynamics in Escherichia Coli.” Biophysical Journal 94.1 (2008): 251–264.
3 Park, Sunyoung et al. “Morphological Differences between Circulating Tumor Cells from Prostate Cancer Patients and Cultured Prostate Cancer Cells.” Ed. David T. Eddington. PLoS ONE 9.1 (2014).
4 Timothy J. Nott et al., “Phase Transition of a Disordered Nuage Protein Generates Environmentally Responsive Membraneless Organelles”, Molecular Cell 57, March 5, 2015, 936–947.
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