<divclass="h3"><strong>Expression of uTP tagged by Fluorescent proteins</strong></div>
<divclass="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>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
<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
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 <ahref="#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, <ahref="#cite3"style="color: #185A4F;">[3]</a>) and a mitochondrial transit peptide (mTP, <ahref="#cite11"style="color: #185A4F;">[11]</a>) respectively, both inserted in the N-terminus of mVenus.
</p>
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>
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<imgsrc="https://static.igem.wiki/teams/5054/mts1.png"alt="Fig 1: Graphical overview of the experiment plan.">
<imgsrc="https://static.igem.wiki/teams/5054/mts1.png"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>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 <ahref="#cite6"style="color: #185A4F;">[6]</a>.
<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 <ahref="#cite6"style="color: #185A4F;">[6]</a>.
</figcaption>
</figcaption>
</div>
</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>
<divclass="h3"><strong>Protein construct delivery into <em>B. bigelowii</em></strong></div>
<divclass="h3"><strong>Protein construct delivery into <em>B. bigelowii</em></strong></div>
<p>To verify the function of uTP in vivo, we aimed to deliver purified His-tagged uTP-mNeongreen from our transformed S. cerevisiae cultures into B. bigelowii cells via electroporation. This would be followed up by high resolution imaging to confirm localization of the fluorescent protein to the nitroplast. However, this experiment was prematurely terminated due to the failure of correct uTP assembly into the yeast backbone, as previously described</p>
<p>To verify the function of uTP in vivo, we aimed to deliver purified His-tagged uTP-mNeongreen from our transformed S. cerevisiae cultures into B. bigelowii cells via electroporation. This would be followed up by high resolution imaging to confirm localization of the fluorescent protein to the nitroplast. However, this experiment was prematurely terminated due to the failure of correct uTP assembly into the yeast backbone, as previously described</p>
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<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 <ahref="#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>
<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 <ahref="#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>
<divclass="img-pagestyle">
<divclass="img-pagestyle">
<imgsrc="https://static.igem.wiki/teams/5054/bbigelowii.png"alt="Fig 1: Graphical overview of the experiment plan.">
<imgsrc="https://static.igem.wiki/teams/5054/bbigelowii.png"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption><em>Braarudosphaera bigelowii</em>, imaged at 1000X magnification on xenic culture medium.
<figcaption>Figure 10: <em>Braarudosphaera bigelowii</em>, imaged at 1000X magnification on xenic culture medium.
</figcaption>
</figcaption>
</div>
</div>
<p>To allow the close study of UCYN-A by future iGEM teams and lay some groundwork for UCYN-A transplantation efforts, we developed a new, easier protocol for isolating it from a culture of B. bigelowii, compared to the known method reported by <ahref="#cite1"style="color: #185A4F;">[1]</a>. While Coale et al relied on a multistep procedure with a Percoll gradient and centrifugation, we used a sorting flow cytometer after lysing of the host cells. We confirmed UCYN-A’s presence in the isolate with PCR, with primers targeting part of the 16S rRNA gene exclusive to prokaryotes.
<p>To allow the close study of UCYN-A by future iGEM teams and lay some groundwork for UCYN-A transplantation efforts, we developed a new, easier protocol for isolating it from a culture of B. bigelowii, compared to the known method reported by <ahref="#cite1"style="color: #185A4F;">[1]</a>. While Coale et al relied on a multistep procedure with a Percoll gradient and centrifugation, we used a sorting flow cytometer after lysing of the host cells. We confirmed UCYN-A’s presence in the isolate with PCR, with primers targeting part of the 16S rRNA gene exclusive to prokaryotes.
</p>
</p>
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<imgsrc="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>
</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 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>
</p>
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<divclass="h3"><strong>PEG fusion</strong></div>
<divclass="h3"><strong>PEG fusion</strong></div>
<p>After identifying the proteins that must be supplied by a host to UCYN-A and the mechanism of their import, the next step to successfully transplanting it into a new host would be the actual insertion of UCYN-A into the host cells. We investigated a protocol to execute this insertion step using S. cerevisiae as a host and E. coli as a stand-in for UCYN-A.</p>
<p>After identifying the proteins that must be supplied by a host to UCYN-A and the mechanism of their import, the next step to successfully transplanting it into a new host would be the actual insertion of UCYN-A into the host cells. We investigated a protocol to execute this insertion step using S. cerevisiae as a host and E. coli as a stand-in for UCYN-A.</p>
<p>The successful fusion of E. coli into S. cerevisiae has been shown in literature before using polyethylene glycol (PEG) to make the host cells perme<ahref="#cite5"style="color: #185A4F;">[5]</a>able. As a first step we attempted to replicate these results. We used E. coli NCM3722 expressing PlsB-msGFP2 combined with fluorescent microscopy to validate the outcome of fusion. </p>
<p>The successful fusion of E. coli into S. cerevisiae has been shown in literature before using polyethylene glycol (PEG) to make the host cells permeable <ahref="#cite5"style="color: #185A4F;">[5]</a>. As a first step we attempted to replicate these results. We used E. coli NCM3722 expressing PlsB-msGFP2 combined with fluorescent microscopy to validate the outcome of fusion. </p>
<divclass="img-pagestyle">
<divclass="img-pagestyle">
<imgsrc="https://static.igem.wiki/teams/5054/peg.png"alt="Fig 1: Graphical overview of the experiment plan.">
<imgsrc="https://static.igem.wiki/teams/5054/peg.png"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Scanning confocal images of a sample PEG fusion procedure (left) as well as a negative control containing regular yeast cells (right)
<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>
</figcaption>
</div>
</div>
<p>Despite multiple attempts at the fusion procedure, we were unable to obtain conclusive results. The yeast cells' native autofluorescence interfered with measurements using the fluorescent microscope, since GFP expression in our E. coli strain was not very strong: we hypothesize this is due to the stress experienced by the cells during the fusion procedure. Since the intensity from autofluorescence was too similar to the <em>E. coli</em>'s, we could not draw conclusions from our images.
<p>Despite multiple attempts at the fusion procedure, we were unable to obtain conclusive results. The yeast cells' native autofluorescence interfered with measurements using the fluorescent microscope, since GFP expression in our E. coli strain was not very strong: we hypothesize this is due to the stress experienced by the cells during the fusion procedure. Since the intensity from autofluorescence was too similar to the <em>E. coli</em>'s, we could not draw conclusions from our images.
To circumvent these problems, selective staining of bacterial RNA or DAPI staining of E. coli prior to fusion were both proposed. Due to time constraints, we could not execute any of these changes.</p>
To circumvent these problems, selective staining of bacterial RNA or DAPI staining of E. coli prior to fusion were both proposed. Due to time constraints, we could not execute any of these changes.</p>
<p></p>
<p></p>
</div>
</div>
<ahref="#cite1"style="color: #185A4F;">[1]</a>
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<divclass="h1">References</div>
<divclass="h1">References</div>
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<liid="cite8"> Albiniak, A. M., Baglieri, J., & Robinson, C. (2012). Targeting of lumenal proteins across the thylakoid membrane. Journal of Experimental Botany, 63(4), 1689–1698. <ahref="https://doi.org/10.1093/JXB/ERR444"style="color:#185A4F;">https://doi.org/10.1093/JXB/ERR444</a></li>
<liid="cite8"> Albiniak, A. M., Baglieri, J., & Robinson, C. (2012). Targeting of lumenal proteins across the thylakoid membrane. Journal of Experimental Botany, 63(4), 1689–1698. <ahref="https://doi.org/10.1093/JXB/ERR444"style="color:#185A4F;">https://doi.org/10.1093/JXB/ERR444</a></li>
<liid="cite9"> Peeters, N., & Small, I. (2001). Dual targeting to mitochondria and chloroplasts. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1541(1–2), 54–63. <ahref="https://doi.org/10.1016/S0167-4889(01)00146-X"style="color:#185A4F;">https://doi.org/10.1016/S0167-4889(01)00146-X</a></li>
<liid="cite9"> Peeters, N., & Small, I. (2001). Dual targeting to mitochondria and chloroplasts. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1541(1–2), 54–63. <ahref="https://doi.org/10.1016/S0167-4889(01)00146-X"style="color:#185A4F;">https://doi.org/10.1016/S0167-4889(01)00146-X</a></li>
<liid="cite10"> Ojala and Garriga. <ahref="https://www.jmlr.org/papers/volume11/ojala10a/ojala10a.pdf"style="color:#185A4F;">Permutation Tests for Studying Classifier Performance.</a> J. Mach. Learn. Res. 2010</li>
<liid="cite10"> Ojala and Garriga. <ahref="https://www.jmlr.org/papers/volume11/ojala10a/ojala10a.pdf"style="color:#185A4F;">Permutation Tests for Studying Classifier Performance.</a> J. Mach. Learn. Res. 2010</li>
