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.
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<imgsrc="https://static.igem.wiki/teams/5054/experiment-abstract.jpeg.png"alt="Fig 1: Graphical overview of the experiment plan.">
<imgsrc="https://static.igem.wiki/teams/5054/experiment-abstract.jpeg"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 8: Overview of experiment
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<p>
We used Gibson assembly to construct the uTP-FP and transit-peptide-FP plasmids,
We used Gibson assembly to construct the uTP-FP and transit-peptide-FP plasmids, transformed <em>E. coli</em>
For our transformation, selection and culturing protocols, see our Materials and Methods page.
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<divclass="img-pagestyle">
<imgsrc="https://static.igem.wiki/teams/5054/experiment-abstract.jpeg"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 9: Example of a diagnostic colony PCR gel: Here,
<imgsrc="https://static.igem.wiki/teams/5054/diagnosis.jpg"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 9: Example of a diagnostic colony PCR gel: Here, we are checking for the presence of the uTP sequence on <em>E. coli</em> colonies that have grown on a selective plate. Note the false positive colonies - we hypothesize these grew after being transformed with leftover pUDE1311 used as a template to linearize for Gibson assembly.
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Due to technical difficulties, we were not able to send over our assembled constructs for sequencing until very late in our project - only when our sequencing results came back we noticed that our pUDE1311-uTP2 construct was in fact not
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This unfortunate turn of events so late on our timeline prevented us from progressing further with our experiments.
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<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>
<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 <em>S. cerevisiae</em> cultures into <em>B. bigelowii</em> 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>
