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| − | <h1> Modular Tri-Display plasmid </h1>
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| − | <img src="https://static.igem.org/mediawiki/2017/2/2a/T--TECHNION-ISRAEL--co-plasmid.png" class="cover" alt="" style= "width:15% ; margin: auto;">
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| − | <br>
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| − | <br>
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| − | <div class = "col-md-offset-2 col-md-8" >
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| − | <p>A system capable of<strong> displaying various epitopes </strong> on the surface of mammalian cells is crucial for inducing <strong>immune tolerance</strong>. To that end, we have designed a modular display vector capable of displaying up to three proteins on the membrane of mammalian cells in equimolar ratios.
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| − | </p>
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| − | <p>Our display mechanism is composed of the following parts:
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| − | <br>
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| − | <div id= "wrapush">
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| − | <p class="text">click on the different parts in the image</p>
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| − | <img class="map" id="pic" src ="https://static.igem.org/mediawiki/2017/3/36/T--TECHNION-ISRAEL--co-design.png" width="700" style= "display: block; margin: auto;" alt="" usemap="#map" hidefocus="true"/>
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| − | <map name="map">
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| − | <area shape="poly" id="area1" coords="140,283,154,262,264,263,273,287,264,307,151,306" class="tooltip"
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| − | title="Ig κ-chain leader sequence fused to the N-termini of the proteins being expressed. This directs the proteins to the secretory pathway.
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| − | " alt="igk">
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| − | <area shape="poly" id="area2" coords="199,102,206,74,252,72,269,95,259,119,215,120,207,110" class="tooltip"
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| − | title="“Self-Cleaving” peptides that connect between each display segment. These peptides are believed to cause the ribosome to “skip” and therefore sever the connection between the protein upstream of the P2A sequence and the protein downstream of it. The complete sequence is translated each time, but due to the ribosome “skipping,” three different proteins are expressed, and in equimolar ratios." alt="2a">
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| − | <area shape="poly" id="area3" coords="272,290,281,265,314,263,334,285,325,303,288,308" class="tooltip"
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| − | title="Three tags that allow detection and quantification of each protein on the membrane. Hemagglutinin A tag, Myc tag and HIS tag. In our experiments we used flow cytometry to indirectly measure protein expression on the cellular membrane." alt="tag">
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| − | <area shape="poly" id="area4" coords="453,289,462,264,544,265,555,289,544,305,469,307" class="tooltip"
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| − | title="Platelet derived growth factor receptor (PDGFR) transmembrane domain, fused to the C-termini of the proteins being expressed. This anchors the proteins to the plasma membrane, allowing display on the extracellular side." alt="pdgfr">
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| − | <area shape="poly" id="area5" coords="331,291,338,263,443,263,459,286,440,308,347,309" class="tooltip"
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| − | title="" alt="epitope">
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| − | <area shape="poly" id="area6" coords="424,100,431,72,477,70,494,93,484,117,440,118,432,108" class="tooltip"
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| − | title="“Self-Cleaving” peptides that connect between each display segment. These peptides are believed to cause the ribosome to “skip” and therefore sever the connection between the protein upstream of the P2A sequence and the protein downstream of it. The complete sequence is translated each time, but due to the ribosome “skipping,” three different proteins are expressed, and in equimolar ratios." alt="2a">
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| − | <area shape="poly" id="area7" coords="41,97,48,69,195,64,206,98,192,123,56,119,44,116" class="tooltip"
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| − | title="" alt="epitope1">
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| − | <area shape="poly" id="area8" coords="266,95,274,62,420,62,431,96,417,121,280,123,269,114" class="tooltip"
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| − | title="" alt="epitope2">
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| − | <area shape="poly" id="area9" coords="488,95,496,62,642,62,653,96,639,121,502,123,491,114" class="tooltip"
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| − | title="" alt="epitope3">
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| − | </map>
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| − | </div>
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| − | <br>
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| − | <p style="text-align:center;"><strong>Figure 1:</strong> original Tri-Display
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| − | </p>
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| − | <br>
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| − | <br>
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| − |
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| − | <p>
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| − | To induce immune tolerance and prevent different allergies and autoimmune diseases we found the specific sequences which correspond to the epitopes that have been shown to <a href="" >trigger improper immune responses </a>. After we identified the desired epitopes, we ordered them as short single stranded DNA oligomers, combined them to create double stranded DNA, and integrated them into our modular plasmid.
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| − | </p>
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| − | <p>
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| − | We first tested our construct using <a href=""> HEK-293 cells </a>. The <a href="" >results of this experiment </a> were less than spectacular (link to results). We successfully expressed all three proteins on the membrane, but unfortunately only one protein was expressed significantly. This was both disparaging and confusing. The P2A system is often hailed as having
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| − | close to 100% cleavage efficiency,
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| − | <sup id = "cite ref-2 " class ="reference">
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| − | <a href="#ref2" original-title>[2] </a>
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| − | </sup>as such, we expected to see equal expression of all the proteins, or no expression at all. After extensive research, we discovered the staggering complexity and variability of 2A systems. Most importantly we discovered that 2A signals followed by signal sequences, such as the Ig-κ we were using, often had very low cleavage efficiencies. As time was running out, we used the most recent papers published on 2A systems and display mechanisms.
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| − | <sup id = "cite ref-4 " class ="reference">
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| − | <a href="#ref4" original-title>[4] </a>
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| − | </sup>
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| − | <sup id = "cite ref-5 " class ="reference">
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| − | <a href="#ref5" original-title>[5] </a>
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| − | </sup>
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| − | <sup id = "cite ref-6 " class ="reference">
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| − | <a href="#ref6" original-title>[6] </a>
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| − | </sup>
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| − | <sup id = "cite ref-2 " class ="reference">
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| − | <a href="#ref7" original-title>[7] </a>
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| − | </sup>
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| − | </p>
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| − |
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| − |
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| − | <p>
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| − | For guidance in crafting four new, and optimized, Tri-Display systems. Each of these constructs was carefully designed in accordance with a different strategy. Additionally, the protein order within each construct was chosen randomly to insure the issue was not caused by specific proteins.
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| − | </p>
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| − | <br>
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| − | <img src="https://static.igem.org/mediawiki/2017/3/3d/T--TECHNION-ISRAEL--plas-2.png" class="cover" alt="" style= "width:100% ; margin: auto;">
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| − | <br>
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| − | <p style="text-align:center;"><strong>Figure 2:</strong> Optimized Tri-Display constructs
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| − | </p>
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| − | <br>
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| − | <p>
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| − | We primarily focused on replacing the original P2A with the reportedly more efficient T2A, and on creating spacers between our signal sequence and the cleavage points. Additionally, we attempted to use the Secrecon signal sequence, a synthetic sequence with far greater secretion capacity. We <a href=""> tested these four constructs </a> in HEK-293 cells and discovered that the T2A sequence had far lower cleavage efficiency than our original P2A . Fortunately, combining both signal sequences (P2A-T2A construct) lead to <a href="" >significant expression </a> of all three proteins (Link to results). Expression was not perfectly equimolar, and there is certainly room for further optimization, but for the purpose of inducing immune tolerance, we believe this expression rate is
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| − | sufficient
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| − | <sup id = "cite ref-8 " class ="reference">
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| − | <a href="#ref8" original-title>[8] </a>
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| − | </sup>.
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| − | </p>
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| − | <h4> Future plans:</h4>
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| − | <p>
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| − | We believe the modular Tri-Display plasmid we created is a versatile and valuable tool. The usage of mammalian membrane display in research and therapeutics is on the rise. The need for multiple protein expression is a recurring
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| − | problem <sup id = "cite ref-9 " class ="reference">
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| − | <a href="#ref9" original-title>[9] </a>
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| − | </sup>
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| − | that has heretofore not been addressed in mammalian cells. To the best of our knowledge, this construct is the first of its kind, allowing simultaneous and nearly equimolar display of up to three unrelated proteins all within a single, easily modified, plasmid. In the future we would like to optimize this plasmid further, attempting to achieve greater levels, and more uniform expression, of all three proteins.
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| − | </p>
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| − | <br>
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| − | <br>
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| − | <br>
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| − | <div class= "references">
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| − | <ol>
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| − | <li id="ref1"> ThermoFisher scientific <i> sitehttps://www.thermofisher.com/order/catalog/product/V66020</i></li>
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| − | <li id="ref2"> Kim, Jin Hee, et al. "High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice." <i>PloS one </i>6.4 (2011): e18556. </li>
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| − | <li id="ref3"> de Felipe, Pablo, et al. "Inhibition of 2A-mediated ‘cleavage’of certain artificial polyproteins bearing N‐terminal signal sequences." <i>Biotechnology Journal </i> 5.2 (2010): 213-223.</li>
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| − | <li id="ref4"> Yan, Jun, et al. "Signal sequence is still required in genes downstream of “autocleaving” 2A peptide for secretary or membrane-anchored expression." <i>Analytical biochemistry</i> 399.1 (2010): 144-146. </li>
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| − | <li id="ref5"> Szymczak-Workman, Andrea L., Kate M. Vignali, and Dario AA Vignali. "Design and construction of 2A peptide-linked multicistronic vectors." <i>Cold Spring Harbor Protocols </i> 2012.2 (2012): pdb-ip067876.</li>
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| − | <li id="ref6"> Barash, Steve, Wei Wang, and Yanggu Shi. "Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression." <i>Biochemical and biophysical research communications </i>294.4 (2002): 835-842. </li>
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| − | <li id="ref7"> Minskaia, Ekaterina, and Martin D. Ryan. "Protein coexpression using FMDV 2A: effect of “linker” residues." <i>BioMed research international </i>2013 (2013). </li>
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| − | <li id="ref8"> Baranyi, U., et al. "Persistent molecular microchimerism induces long‐term tolerance towards a clinically relevant respiratory allergen." <i>Clinical & Experimental Allergy </i> 42.8 (2012): 1282-1292.</li>
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| − | <li id="ref9"> Korepanova, Alla, et al. "Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli." <i>Protein Science </i> 14.1 (2005): 148-158.</li>
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| − | </ol>
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