<imgsrc="https://static.igem.wiki/teams/5054/msa.png"alt="Fig 1: Graphical overview of the experiment plan.">
<imgsrc="https://static.igem.wiki/teams/5054/msa.png"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 5: Structural predictions. (a) Consensus structure of all uTP sequences extracted from UCYN-A imported proteins. (b) Consensus structure of all uTP sequences with charged residues shown (red=negative, blue=positive). (c) Consensus structure aligned onto uTP1 + mNeonGreen construct (RMSD=4.00Å). (d) Consensus structure aligned onto uTP2 + mNeonGreen construct (RMSD=3.77Å). </figcaption>
<figcaption>Figure 5: Structural predictions. (a) Consensus structure of all uTP sequences extracted from UCYN-A imported proteins. (b) Consensus structure of all uTP sequences with charged residues shown (red=negative, blue=positive). (c) Consensus structure aligned onto uTP1 + mNeonGreen construct (RMSD=4.00Å). (d) Consensus structure aligned onto uTP2 + mNeonGreen construct (RMSD=3.77Å). </figcaption>
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<p>In the case of mitochondrial and chloroplast imported proteins, there is a well-known overlap: numerous proteins have twin transit peptides or ambiguous targeting sequences targeting both organelles. To investigate whether there is a similar overlap between the potential UCYN-A import system and other known cellular transport systems, we used established protein localization prediction tools on the potential list of UCYN-A imported proteins. These predictions proved to be inconclusive. A large minority (28%) of them were classified as secreted (Fig 4). This suggests that the UCYN-A import system, similar to other protein transport mechanisms, might be related to the Sec system.
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<p>We also investigated both sequence and structural homologs of uTP in public databases (NCBI, PDB, AlphaFold/Proteome) and found no significant matches.</p>
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<imgsrc="https://static.igem.wiki/teams/5054/msa.png"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 6: Average predicted confidence for different localization categories. </figcaption>
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<p>The inconclusive homology search and localization prediction results, together with the fact that in most cases transport signals are found on the N-terminal of proteins [2, 3, 8] as opposed to the C-terminal in the case of uTP, suggest the protein import machinery associated with uTP is quite distinct from other known systems in the cell.</p>
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<imgsrc="https://static.igem.wiki/teams/5054/msa.png"alt="Fig 1: Graphical overview of the experiment plan.">
<figcaption>Figure 7: Overview of hypothesized UCYN-A protein import. </figcaption>
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<p>The successful fusion of E. coli into S. cerevisiae has been shown in literature before [5] using polyethylene glycol (PEG) to make the host cells permeable. 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 [5] using polyethylene glycol (PEG) to make the host cells permeable. 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>
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<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><em>Scanning confocal images of a sample PEG fusion procedure (left) as well as a negative control containing regular yeast cells (right)</em>
<figcaption><em>Scanning confocal images of a sample PEG fusion procedure (left) as well as a negative control containing regular yeast cells (right)</em>