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<ul>
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            <li><a class="active" data-scroll-nav='0'>Core of APOllO E.Cotector: Customization</a></li>
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            <li><a data-scroll-nav="1">Single Chain Variable Fragment</a>
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                <ul>
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                <li><a data-scroll-nav="2">Probe: scFv from targeted drugs</a>
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                <li><a data-scroll-nav="3">Transmembrane Protein of scFv</a></li>
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                <li><a data-scroll-nav="4">Color Signal</a></li>
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                </ul>
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            </li>
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            <li><a data-scroll-nav="5">Gold Binding Polypeptide</a>
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                <ul>
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                <li><a data-scroll-nav="6">Transmembrane Protein of GBP</a>
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                <li><a data-scroll-nav="7">Application: Immobilize on Gold</a></li>
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                </ul>
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            </li>
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</ul>
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    </div>
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<div class="p01">
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<div class="background1"></div>
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<div class="title">Design</div>
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<div class="p02">
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<div class="content" data-scroll-index="0"><h1>Core of APOllO E.Cotector: Customization</h1>
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<p>By utilizing the concept of <font color="#AC1F4A">co-transformation</font>, APOllO can offer various E.Cotectors with scFv, color signal or GBP and even any desired combination. Therefore, APOllO could <font color="#AC1F4A">customize the E.Cotector to satisfy the need of various detection platforms.</font></p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/0/07/Nctu_formosa_design_newfig1.png" height="400px"><br><br>
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Figure 1. With the co-transform technique, we can insert any scFv or signal-related genetic sequences into the <i>E.coli</i>, and create a customized platform ─ the APOllO E.Cotector.
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</div>
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</div>
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<div class="content" data-scroll-index="1"><h1>Single Chain Variable Fragment</h1>
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  <h2 data-scroll-index="2">Probe: scFv from targeted drugs</h2>
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<p>
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In order to provide doctors with new, direct, and innovative methods in determining the usage of <font color="#AC1F4A">monoclonal-antibody-targeted drugs</font>, we redesigned the <font color="#AC1F4A">FDA approved</font> monoclonal antibody targeted drugs, such as <font color="#AC1F4A">Bevacizumab (Avastin<sup>®</sup> anti-VEGF)<sup>[1]</sup></font>, <font color="#AC1F4A">Cetuximab (Erbitux<sup>®</sup> anti-EGFR)<sup>[2]</sup></font> and <font color="#AC1F4A">Trastuzumab (Herceptin<sup>®</sup> anti-HER2)<sup>[3]</sup></font> into scFv as probes.
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</p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/e/ef/Nctu_Formosa_design_newfig2.png" height="300px"> <br><br>
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Figure 2. We design the platform to detect multimarkers as the reference for applying the combination therapy.
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</div>
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<p>Each distinct scFv of targeted drugs is displayed on the surfaces of <i>E.coli</i> allowing it to specifically detect target molecules. </p>
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<p>Furthermore, since APOllO E.Cotector expresses scFv and <font color="#AC1F4A">color signals</font> at the same time, the specific molecules that correspond to the scFv can be identified by different colors, which allows an accurate prescription of monoclonal antibody targeted drugs.</p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/thumb/a/a6/Figure_2_E.Cotector.png/800px-Figure_2_E.Cotector.png" height="250px"> <br><br>
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Figure 3. E.Cotector expresses scFv from targeted drugs.
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</div>
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<h2 data-scroll-index="3">Transmembrane Protein of scFv</h2>
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  <p>To display scFv on the surface of <i>E.coli</i>, we use a <font color="#AC1F4A">transmembrane protein</font>. The transmembrane protein is composed of <font color="#AC1F4A">lipoprotein (Lpp)</font> and <font color="#AC1F4A">outer membrane protein A (OmpA)</font>.</p>
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<div class="image">
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  <img src="https://static.igem.org/mediawiki/2015/0/0b/Nctu_formosa_design_Lpp-Ompa123.png" height="200px"><br><br>
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  Figure 4. Transmembrane protein: Lpp-OmpA is composed of lipoprotein (Lpp) and outer membrane protein A (OmpA).
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  </div>
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  <p>Lpp-OmpA was designed as a fusion protein consisting of the signal sequence, first 9 amino acids of Lpp, and residue 46~159 amino acids of OmpA. The Lpp of the N-terminal of this fusion protein targets the protein on the membrane while the transmembrane domain of OmpA serves as an anchor. Owing to the fact that it is on the external exposed loops of the C-terminal of OmpA, scFv can be easily anchored to the outer membrane. Between the OmpA and scFv, there is <font color="#AC1F4A">a cut site of restriction enzyme</font> called <font color="#AC1F4A"><i>NcoI</i></font> allowing the linked scFv to be easily changed (like a cassette)<sup>[4]</sup>.</p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/b/b4/Figure_4_NocI_scFv.png" height="200px">
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<img src="https://static.igem.org/mediawiki/2015/8/84/Nctu_formosa_design_figure4-2.png" height="250px"><br> <br> <br> <br>
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<img src="https://static.igem.org/mediawiki/2015/b/b4/Lpp-OmpA_NcoI.png" height="70px">
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<img src="https://static.igem.org/mediawiki/2015/a/ab/Ncol_scFv.png" height="85px"> <br><br>
 +
  Figure 5. To change the scFv sequence easily, we designed the <I>Nco</I>I restriction site between Lpp-OmpA and scFv. When designing <i>XbaI</i>-<i>Spe</i>I restriction site between Lpp-OmpA and scFv, it can cause a mixed site. Therefore, the <i>NcoI</i> restriction site rather than the EX-SP restriction site was designed.
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  </div>
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 +
  <h2 data-scroll-index="4">Color Signal</h2>
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<p>The color signals that we have selected are <font color="#AC1F4A">fluorescent proteins</font> and <font color="#AC1F4A">chromoproteins</font>. Cooperating with iGEM, all of the resources that were from the giant registry of iGEM is accessible to every iGEMer.
 +
APOllO utilized red flourescent protein <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>, green fluorescent protein <a href="http://parts.igem.org/Part:BBa_E0040">BBa_E0040</a>, and blue chromoprotein <a href="http://parts.igem.org/Part:BBa_K592009">BBa_K592009</a>.</p><br>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/4/48/Design_Figure_5-1.png" height="100px"> 
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<img src="https://static.igem.org/mediawiki/2015/2/26/Design_Figure_5.png" height="100px">
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<img src="https://static.igem.org/mediawiki/2015/d/d3/Design_Figure_5-3.png" height="100px"> <br><br>
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    Figure 6. Different Color Signal
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  </div>
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<h2><a href="https://2015.igem.org/Team:NCTU_Formosa/Results">Jump to results to check the scFv function.</a></h2>
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</div>
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<div class="content" data-scroll-index="5"><h1>Gold Binding Polypeptide</h1>
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<p>Another plasmid is <font color="#AC1F4A">gold binding polypeptide</font>, abbreviated as GBP. APOllO may display GBP on the surface of <i>E.coli</i> for <font color="#AC1F4A">binding on gold surface</font>.</p>
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 +
<p>GBP was designed with the three-repeated following 14 amino acids sequences: [MHGKTQATSGTIQS]. The binding sequence of GBP does not contain cysteine which can form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs)<sup>[5]</sup>.</P>
 +
<p>The mechanism of the connection between GBP and gold metal plane remains unknown. By using Molecular Dynamics (MD), it indicates that GBP, with an antiparallel β-sheet structure, can recognize gold surface via OH-binding. It is likely that the hydroxyl, together with amine, ligands on GBP recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface<sup>[6]</sup>.</p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/thumb/3/36/Design_Figure_6.png/800px-Design_Figure_6.png" height="200px">  <br><br>
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  Figure 7. GBP can recognize and bind on the gold surface.
 +
  </div>
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</div>
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<div class="content" data-scroll-index="6"><h2>Transmembrane Protein of GBP</h2>
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<p>To display the GBP to the outer membrane of E.Cotector, we use a transmembrane protein called <font color="#AC1F4A">Long-chain fatty acid</font> short as FadL. We selected the first 384 amino acids sequence from the FadL for the transmembrane protein of GBP<sup>[7]</sup>.</p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/thumb/1/1a/Design_Figure_7.png/800px-Design_Figure_7.png" height="200px">  <br><br>
 +
  Figure 8. Transmembrane Protein FadL transports GBP out of the surface of <i>E.coli</i>.
 +
  </div>   
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 +
<h2 data-scroll-index="7">Application: Immobilize on Gold</h2>
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<p> According to the concept of biosensor, APOllO’s customers may deem the E.Cotector to play the role of biological recognition part and the gold chip to <font color="#AC1F4A">act as the signal transducer part</font>. Combining with multiple physicochemical nanoscale detectors in various fields, such as optical, electrochemistry, microbalance and etcetera, greater precision and accuracy will be achieved.</p>
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<div class="image">
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<img src="https://static.igem.org/mediawiki/2015/7/79/Nctu_formosa_design_figure12.png" height="300px"> <br><br>
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Figure 9. Concept of Biosensor via E.Cotector
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</div>
  
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<h2><a href="https://2015.igem.org/Team:NCTU_Formosa/Results">Jump to results to check GBP function.</a></h2>
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<h2>APOllO E.Cotector</h2>
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<b>Reference<br></b>
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<P>To help doctors with innovated methods to judge whether to use <font color="#AC1F4A";>monoclonal antibody targeted drugs</font> more directly ,we redesigned the FDA approved monoclonal antibody targeted drugs,
+
[1] <a href="http://www.ncbi.nlm.nih.gov/pubmed/15961063">Bevacimab(Avastin)</a><br>
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such as <font color="#AC1F4A";>Bevacizumab (Avastin<sup>®</sup> anti-VEGF)<sup>[1]</sup></font>, <font color="#AC1F4A";>Cetuximab (Erbitux<sup>®</sup> anti-EGFR)<sup>[2]</font></sup> and Trastuzumab (Herceptin<sup>®</sup> anti-HER2)<sup>[3]</sup></font> into recombinant antibodies : scFv (single chain variable fragment).
+
[2] <a href="http://www.drugbank.ca/drugs/DB00002">Cetuximab(Erbitux)</a><br>
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    For detecting the specific molecules ("targeted molecules"), which are specific to these <font color="#AC1F4A";>scFv of targeted drugs</font>, scFv are displayed on the surfaces of E.coli by using a transmembrane fusion protein, <font color="#AC1F4A";>Lpp-OmpA<sup>[5]</sup></font> respectively;
+
[3] <a href="http://www.drugbank.ca/drugs/DB00072">Trastuzumab(Herceptin)</a><br>
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    that is one APOllO E.Cotector displays one kind of targeted drugs of scFv each time. Take an example, while the anti-EGFR scFv (originate from Cetuximab) is displayed on the surface of E.coli, we can detect EGFR by this APOllO E.Cotector.
+
[4] <a href="http://www.uniprot.org/uniprot/P0A910">UniProtKB - P0A910 (OMPA_ECOLI)</a><br>
−
    Furthermore, as APOllO E.Cotector expressed fluorescence protein at the same time, the specific molecules ("targeted molecules") that correspond to those scFv of targeted drugs can be identified individually <font color="#AC1F4A";>in direct</font> by those different colors APOllO E.Cotector.  
+
[5] <a href="http://onlinelibrary.wiley.com/doi/10.1002/sia.2536/abstract">Adsorption of genetically engineered proteins studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Part A: data acquisition and principal component analysis (PCA), Noriaki Suzuki,1 Lara Gamble,2 Candan Tamerler,3 Mehmet Sarikaya,1 David G. Castner2,4 (2007)</a><br>
−
    In a simple word, because of the APOllO E.Cotector that display scFv of targeted drugs can we identify the corresponding specific molecules ("targeted molecules") ,then achieve to offer a prescription of monoclonal antibody targeted drug in direct . </P>
+
[6] <a href="http://austinpublishinggroup.com/biosensors-bioelectronics/fulltext/ajbb-v1-id1005.php">Assembly of Gold-Binding Proteins for Biomolecular Recognition, Zareie HM1,2* and Sarikaya M3, Austin Journal of Biosensors & Bioelectronics (2015)</a><br>
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<div style="text-align:center; float:left; width:80%; font-size:8pt"><img style="margin:0 auto; " src="https://static.igem.org/mediawiki/2015/3/32/NCTU_Formosa_APOllO_E.Cotecter_1.jpg" height="250vh" >
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[7] <a href="http://www.ncbi.nlm.nih.gov/pubmed/19228193">Tae Jung Park et al. (2009) Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface </a><br>
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<p>Figure 1. Concept of APOllO E.Cotecter:Displaying scFv of antibody drugs on the surface of E.coli</p></div>
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<div style="text-align:center; width:80%; font-size:8pt"> <img style="margin:0 auto; " src="https://static.igem.org/mediawiki/2015/9/90/NCTU_Formosa_APOllO_E.Cotecter_2.jpg" height="250vh">
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    <p>Figure 2. Concept of our Application: Cell Staining</p></div>
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<a href="https://2015.igem.org/Team:NCTU_Formosa/Project"><img src="https://static.igem.org/mediawiki/2015/3/3c/%E7%AE%AD%E9%A0%AD1.png"; width=50vw;><br><br>Back to Navigation</a>
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<h3><strong>The introduction of scFv</strong></h3>
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</div>
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<p>scFv is a fusion protein of the variable regions of the heavy chains(<b>VH</b>) and light chains (<b>VL</b>) of antibody connected by a flexible linker peptide.
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scFv still reserve <font color="#AC1F4A";>completely functional antigen-binding fragment </font> and specificity of the original immunoglobulin; that is ,
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<a href="https://2015.igem.org/Team:NCTU_Formosa/Results"><img src="https://static.igem.org/mediawiki/2015/c/c2/%E7%AE%AD%E9%A0%AD2.png"; width=50vw;><br><br>Go to Results</a>
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the property of specificity of antibody to antigen has being maintained. Moreover, scFv is only 20 percent the size as antibody <sup>[4]</sup>,
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therefore it will not cause stress to E.coli for displaying it</p>
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Latest revision as of 03:28, 19 September 2015

Design

Core of APOllO E.Cotector: Customization

By utilizing the concept of co-transformation, APOllO can offer various E.Cotectors with scFv, color signal or GBP and even any desired combination. Therefore, APOllO could customize the E.Cotector to satisfy the need of various detection platforms.



Figure 1. With the co-transform technique, we can insert any scFv or signal-related genetic sequences into the E.coli, and create a customized platform ─ the APOllO E.Cotector.

Single Chain Variable Fragment

Probe: scFv from targeted drugs

In order to provide doctors with new, direct, and innovative methods in determining the usage of monoclonal-antibody-targeted drugs, we redesigned the FDA approved monoclonal antibody targeted drugs, such as Bevacizumab (Avastin® anti-VEGF)[1], Cetuximab (Erbitux® anti-EGFR)[2] and Trastuzumab (Herceptin® anti-HER2)[3] into scFv as probes.



Figure 2. We design the platform to detect multimarkers as the reference for applying the combination therapy.

Each distinct scFv of targeted drugs is displayed on the surfaces of E.coli allowing it to specifically detect target molecules.

Furthermore, since APOllO E.Cotector expresses scFv and color signals at the same time, the specific molecules that correspond to the scFv can be identified by different colors, which allows an accurate prescription of monoclonal antibody targeted drugs.



Figure 3. E.Cotector expresses scFv from targeted drugs.

Transmembrane Protein of scFv

To display scFv on the surface of E.coli, we use a transmembrane protein. The transmembrane protein is composed of lipoprotein (Lpp) and outer membrane protein A (OmpA).



Figure 4. Transmembrane protein: Lpp-OmpA is composed of lipoprotein (Lpp) and outer membrane protein A (OmpA).

Lpp-OmpA was designed as a fusion protein consisting of the signal sequence, first 9 amino acids of Lpp, and residue 46~159 amino acids of OmpA. The Lpp of the N-terminal of this fusion protein targets the protein on the membrane while the transmembrane domain of OmpA serves as an anchor. Owing to the fact that it is on the external exposed loops of the C-terminal of OmpA, scFv can be easily anchored to the outer membrane. Between the OmpA and scFv, there is a cut site of restriction enzyme called NcoI allowing the linked scFv to be easily changed (like a cassette)[4].







Figure 5. To change the scFv sequence easily, we designed the NcoI restriction site between Lpp-OmpA and scFv. When designing XbaI-SpeI restriction site between Lpp-OmpA and scFv, it can cause a mixed site. Therefore, the NcoI restriction site rather than the EX-SP restriction site was designed.

Color Signal

The color signals that we have selected are fluorescent proteins and chromoproteins. Cooperating with iGEM, all of the resources that were from the giant registry of iGEM is accessible to every iGEMer. APOllO utilized red flourescent protein BBa_E1010, green fluorescent protein BBa_E0040, and blue chromoprotein BBa_K592009.




Figure 6. Different Color Signal

Jump to results to check the scFv function.

Gold Binding Polypeptide

Another plasmid is gold binding polypeptide, abbreviated as GBP. APOllO may display GBP on the surface of E.coli for binding on gold surface.

GBP was designed with the three-repeated following 14 amino acids sequences: [MHGKTQATSGTIQS]. The binding sequence of GBP does not contain cysteine which can form a covalent thiol linkage with gold, the linkage to the gold surface in Self-Assembled Monolayers (SAMs)[5].

The mechanism of the connection between GBP and gold metal plane remains unknown. By using Molecular Dynamics (MD), it indicates that GBP, with an antiparallel β-sheet structure, can recognize gold surface via OH-binding. It is likely that the hydroxyl, together with amine, ligands on GBP recognize the atomic lattice of gold, aligning the molecule along the variants of a six-fold axis on the Au (111) surface[6].



Figure 7. GBP can recognize and bind on the gold surface.

Transmembrane Protein of GBP

To display the GBP to the outer membrane of E.Cotector, we use a transmembrane protein called Long-chain fatty acid short as FadL. We selected the first 384 amino acids sequence from the FadL for the transmembrane protein of GBP[7].



Figure 8. Transmembrane Protein FadL transports GBP out of the surface of E.coli.

Application: Immobilize on Gold

According to the concept of biosensor, APOllO’s customers may deem the E.Cotector to play the role of biological recognition part and the gold chip to act as the signal transducer part. Combining with multiple physicochemical nanoscale detectors in various fields, such as optical, electrochemistry, microbalance and etcetera, greater precision and accuracy will be achieved.



Figure 9. Concept of Biosensor via E.Cotector

Jump to results to check GBP function.

Reference
 [1] Bevacimab(Avastin)
[2] Cetuximab(Erbitux)
[3] Trastuzumab(Herceptin)
[4] UniProtKB - P0A910 (OMPA_ECOLI)
[5] Adsorption of genetically engineered proteins studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS). Part A: data acquisition and principal component analysis (PCA), Noriaki Suzuki,1 Lara Gamble,2 Candan Tamerler,3 Mehmet Sarikaya,1 David G. Castner2,4 (2007)
[6] Assembly of Gold-Binding Proteins for Biomolecular Recognition, Zareie HM1,2* and Sarikaya M3, Austin Journal of Biosensors & Bioelectronics (2015)
[7] Tae Jung Park et al. (2009) Development of a whole-cell biosensor by cell surface display of a gold-binding polypeptide on the gold surface


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