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| | <h2 class="section-heading">Introduction </h2> | | <h2 class="section-heading">Introduction </h2> |
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| − | The third mechanism that we have investigated in our project is protein import into bacterial cells, as a stand in for a symbiont. In the two best known endosymbiotic organelles, mitochondria and chloroplasts, a majority of gene expression has moved from the symbiont to the host. Because this relationship seems to be an evolutionary foundation of the known endosymbiotic relationships, we will attempt to imitate this concept.
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| − | Besides our interest in the evolutionary aspect of protein import, we believe it is valuable when approaching modular endosymbiosis. The principle of protein import would enable additional modularity in an endosymbiotic system, as one host can be manipulated to produce proteins to be utilized by a range of symbionts.
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| − | The majority of the proteins, destined for mitochondria, are expressed in the cytosol and subsequently imported across the membranes via transport complexes taking up unfolded peptides with an N-terminal signal sequence targeting them to the mitochondria (Schmidt <i>et al</i> 2010). Similar transport complexes are found in chloroplast. We want a similar targeted uptake, but in a simpler system. Thus, we utilise a small peptide shown to penetrate many types of cells; a Cell Penetrating Peptide (CPP) (Chang <i>et al</i> 2005, and Chang<i>et al</i> 2014).
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| | <h2 class="section-heading">Final Design </h2> | | <h2 class="section-heading">Final Design </h2> |
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| − | <strong>Background</strong>: CPPs are small peptides, typically rich in arginines, which are able to facilitate transport of a wide variety of cargos across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain (Eudes and Chugh, 2008). In recent years, research on creating synthetics CPPs has been conducted and especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells. These experiments have shown that GFP connected to a CPP has entered the cells contained in vesicles (Chang <i>et al</i> 2005).
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| − | If CPP can be used as a protein tag for import into an endosymbiotic symbiont, the host proteins targeted to the symbiont would simply need the CPP added.
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| − | <strong>Goal</strong>: Evaluate the efficiency of protein uptake by our <i>Escherichia coli</i> chassis in presence and absence of the cell penetrating peptide (CPP).
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| − | <strong>Circuits and Biobricks</strong>: The parts in our circuit are fluorescent proteins and CPP. <br><br>
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| − | We have chosen the yellow and blue fluorescent proteins (<a href="http://parts.igem.org/Part:BBa_K864100">YFP</a> and <a href="http://parts.igem.org/Part:BBa_K592100">BFP</a>) from the Biobrick repository, and improved them by adding the CPP sequence to the C-terminal end. <br><br>
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| − | YFP: <a href="http://parts.igem.org/Part:BBa_K2455002">BBa_K2455002 </a><br><br>
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| − | BFP: <a href="http://parts.igem.org/Part:BBa_K2455005">BBa_K2455005 </a> <br><br>
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| − | Additionally, we created a biobrick of CPP with a USER casette, ready for insertion of any protein to be imported: <a href="http://parts.igem.org/Part:BBa_K2455003">BBa_K2455003</a>.
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| − | YFP and BFP were chosen to avoid overlapping colours with FM4-64, a red staining used for membranes, in case we wanted to look into the localization and potential vesicle breaking using confocal microscopy. As it turned out, we did not get far enough in the lab to do this.
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| | </p> | | </p> |
| | </div> | | </div> |
