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<div class="h3"><strong>Expression of uTP tagged by Fluorescent proteins</strong></div>
<p>The ultimate goal with the uTP sequences we identified is to understand and confirm whether they are indeed responsible for protein import into UCYN-A. Conventional methods to check this would require a toolbox for genetic manipulation of B. bigelowii, not yet available and beyond the scope of this project. We therefore opted for using 2 model eukaryotes for further research on uTP’s behavior, namely C. reinhardtii and S. cerevisiae, and designed an experiment to confirm uTP’s function without modifying B. bigelowii.</p>
<p>We worked off of a <em>Saccharomyces</em> and a <em>Chlamydomonas</em> backbone, pUDE1311 and pOpt2-mVenusBle respectively, in order to design constructs expressing
- fluorescent proteins (FP) tagged by known transit peptides as well as uTP. Unmodified, pUDE1311 expresses ymNeongreen and pOpt2-mVenusBle expresses mVenus, a YFP analogue. We designed 2 constructs for expression in our yeast and 3 in our algae. For our yeast, one construct had uTP inserted in the C-terminus of ymNeongreen and the other had MTS1, a mitochondrial transit peptide <a href="#cite2" style="color: #185A4F;">[2]</a>, inserted in the N-terminus of the fluorescent protein.
- For our algae, one construct had uTP inserted in the C-terminus of mVenus, while the two others had a chloroplastic (cTP, <a href="#cite3" style="color: #185A4F;">[3]</a>) and a mitochondrial transit peptide (mTP, <a href="#cite11" style="color: #185A4F;">[11]</a>) respectively, both inserted in the N-terminus of mVenus.
+ fluorescent proteins (FP) tagged by known transit peptides as well as uTP. Unmodified, pUDE1311 expresses ymNeongreen and pOpt2-mVenusBle expresses mVenus, a YFP analogue; Both express AmpR for selection on <em>E.coli</em>, while . We designed 2 constructs for expression in our yeast and 3 in our algae. For our yeast, one construct had uTP inserted in the C-terminus of ymNeongreen and the other had MTS1, a mitochondrial transit peptide <a href="#cite2" style="color: #185A4F;">[2]</a>, inserted in the N-terminus of the fluorescent protein.
+ For our algae, one construct had uTP inserted in the C-terminus of mVenus, while the two others had a chloroplastic (cTP, <a href="#cite3" style="color: #185A4F;">[3]</a>) and a mitochondrial transit peptide (mTP, <a href="#cite11" style="color: #185A4F;">[11]</a>) respectively, both inserted in the N-terminus of mVenus. Plasmid maps for our constructs and vectors can be found in our Materials and Methods page.
We planned to observe the localization of uTP in the absence of UCYN-A in these species, hypothesizing based on the dry-lab analysis detailed above that we would observe uniform diffusion in the cytoplasm. Cells transformed with the regular pUDE and pOpt plasmids as well as the known transit peptides would serve as controls showing both uniform diffusion as well as localization to organelles respectively.
-
- We used Gibson assembly to construct the uTP-FP and transit-peptide-FP plasmids, transformed
+ </p>
+ <div class="img-pagestyle">
+ <img src="https://static.igem.wiki/teams/5054/experiment-abstract.jpeg.png" alt="Fig 1: Graphical overview of the experiment plan.">
+ <figcaption>Figure 8: Overview of experiment
+ </figcaption>
+ </div>
+
+ <p>
+ We used Gibson assembly to construct the uTP-FP and transit-peptide-FP plasmids,
</p>
+ <div class="img-pagestyle">
+ <img src="https://static.igem.wiki/teams/5054/experiment-abstract.jpeg.png" alt="Fig 1: Graphical overview of the experiment plan.">
+ <figcaption>Figure 9: Example of a diagnostic colony PCR gel: Here,
+ </figcaption>
+ </div>
+
+
<div class="img-pagestyle">
<img src="https://static.igem.wiki/teams/5054/mts1.png" alt="Fig 1: Graphical overview of the experiment plan.">
- <figcaption>Figure 9: MTS1-mNeongreen yeast transformants under fluorescent confocal microscope at 1000x magnification. The clustering of fluorescence is visible and visually very similar to mitochondrial localization reported by <a href="#cite6" style="color: #185A4F;">[6]</a>.
+ <figcaption>Figure 10: MTS1-mNeongreen yeast transformants under fluorescent confocal microscope at 1000x magnification. The clustering of fluorescence is visible and visually very similar to mitochondrial localization reported by <a href="#cite6" style="color: #185A4F;">[6]</a>.
</figcaption>
</div>
<p>Due to time constraints coupled with a long culture time for selection after transformation, we were unable to image our <em>C. reinhardtii</em> transformants</p>
@@ -128,7 +142,7 @@
<p>Thanks to the generous help of Dr. Kyoko Hagino, a pioneer in research into B. bigelowii, we obtained a culture of B. bigelowii FR-21 <a href="#cite1" style="color: #185A4F;">[1]</a>. This species is known to be difficult to work with, however, we were able to find the optimal conditions and grow it in our lab in Delft, establishing, to our knowledge, the first B. bigelowii culture in Europe. We followed Kyoko’s advice when deciding on our culture conditions, which can be found in our Materials and Methods section.</p>
<div class="img-pagestyle">
<img src="https://static.igem.wiki/teams/5054/bbigelowii.png" alt="Fig 1: Graphical overview of the experiment plan.">
- <figcaption>Figure 10: <em>Braarudosphaera bigelowii</em>, imaged at 1000X magnification on xenic culture medium.
+ <figcaption>Figure 11: <em>Braarudosphaera bigelowii</em>, imaged at 1000X magnification on xenic culture medium.
</figcaption>
</div>
@@ -136,11 +150,11 @@
</p>
<div class="img-pagestyle">
<img src="https://static.igem.wiki/teams/5054/bbigelowii.png" alt="Fig 1: Graphical overview of the experiment plan.">
- <figcaption>Figure 11: Flow cytometry plot and PCR result for the three populations we identified on B.bigelowii lysate.
+ <figcaption>Figure 12: Flow cytometry plot and PCR result for the three populations we identified on B.bigelowii lysate.
</figcaption>
</div>
- <p>The isolates are not completely pure, as each of the three fractures contained UCYN-A. However, after comparing our cytometry plots with those from the literature, we hypothesize the third fracture to contain the densest sample of UCYN-A. This is supported by the relative intensity of the bands in the PCR gel, with the third lane being the strongest. This suggests the third population had the highest concentration of UCYN-A, since an equal amount of cells were collected in each sample and all PCR conditions were identical, meaning the intensity is approximately proportional to the amount of DNA in the starting sample.
+ <p>The isolated populations are not completely pure, as each of the three fractions contained UCYN-A. However, after comparing our cytometry plots with those from <a href="#cite1" style="color: #185A4F;">[1]</a>, we hypothesize the third fraction to contain the densest sample of UCYN-A. This is supported by the relative intensity of the bands in the PCR gel, with the third lane being the strongest. This suggests the third population had the highest concentration of UCYN-A, since an equal amount of cells were collected in each sample and all PCR conditions were identical, meaning the intensity is approximately proportional to the amount of DNA in the starting sample.
</p>
<p>For future investigation we would recommend the usage of qPCR in order to more precisely quantify the presence of UCYN-A DNA in different isolates.</p>
@@ -153,7 +167,7 @@
<div class="img-pagestyle">
<img src="https://static.igem.wiki/teams/5054/peg.png" alt="Fig 1: Graphical overview of the experiment plan.">
- <figcaption>Figure 12: Scanning confocal images of a sample PEG fusion procedure (left) as well as a negative control containing regular yeast cells (right)
+ <figcaption>Figure 13: Scanning confocal images of a sample PEG fusion procedure (left) as well as a negative control containing regular yeast cells (right)
</figcaption>
</div>