Difference between revisions of "Team:XMU-China/Demonstrate"

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     <title>Team:XMU-China/Description - 2018.igem.org</title>
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     <title>Team:XMU-China/Demonstrate - 2018.igem.org</title>
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     <link rel="stylesheet" href="https://2018.igem.org/Team:XMU-China/css/font?action=raw&ctype=text/css">
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    <link rel="stylesheet" href="https://2018.igem.org/Team:XMU-China/css/nav_mobile?action=raw&ctype=text/css">
 
     <link rel="stylesheet" href="https://2018.igem.org/Team:XMU-China/css/material-scrolltop?action=raw&ctype=text/css">
 
     <link rel="stylesheet" href="https://2018.igem.org/Team:XMU-China/css/material-scrolltop?action=raw&ctype=text/css">
 
</head>
 
</head>
  
 
<body>
 
<body>
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    <header></header>
 
     <div id="container">
 
     <div id="container">
 
         <header>
 
         <header>
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                                 <li><a href="https://2018.igem.org/Team:XMU-China/Description">Description</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Description">Description</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Design">Design</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Design">Design</a></li>
                                <li><a href="https://2018.igem.org/Team:XMU-China/Demonstrate">Demonstrate</a></li>
 
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Results">Results</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Results">Results</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Demonstrate">Demonstrate</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Parts">Parts</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Parts">Parts</a></li>
 
                             </ul>
 
                             </ul>
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                             <ul>
 
                             <ul>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware">Overview</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware">Overview</a></li>
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware/Microfluidic_Chips">Microfluidic chips</a></li>
+
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Microfluidic_Chips">Microfluidic Chips</a></li>
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware/Fluorescenc_Detection">Fluorescence Detection</a></li>
+
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Fluorescence_Detection">Fluorescence Detection</a></li>
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware/Straberry_Pi">Straberry Pi</a></li>
+
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Raspberry_Pi">Raspberry Pi</a></li>
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Applied_Design">Applied Design</a></li>
+
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Application">Application</a></li>
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Software">APP</a></li>
+
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Software">Software</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Applied_Design">Product Design</a></li>
 
                             </ul>
 
                             </ul>
 
                         </li>
 
                         </li>
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                             <a href="#">Model</a>
 
                             <a href="#">Model</a>
 
                             <ul>
 
                             <ul>
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Model">Overview</a></li>
+
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Model#Summary">Summary</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Model#Thermodynamic_model">Thermodynamic Model</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Model#Fluid_dynamics_model">Fluid dynamics Model</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Model#Molecular_docking_model">Molecular Docking Model</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Model#The_dynamic_model">Derivation of Rate Equation</a></li>
 
                             </ul>
 
                             </ul>
 
                         </li>
 
                         </li>
 
                         <li class="Human_Practice">
 
                         <li class="Human_Practice">
                             <a href="#">Human Practice</a>
+
                             <a href="#">Social Works</a>
 
                             <ul>
 
                             <ul>
 
                                 <li><a href="https://2018.igem.org/Te
 
                                 <li><a href="https://2018.igem.org/Te
                                 am:XMU-China/Human_Practices">Overview</a></li>
+
                                 am:XMU-China/Human_Practices">Human Practice</a></li>
                                <li><a href="https://2018.igem.org/Team:XMU-China/HP/Silver">Silver</a></li>
+
                                <li><a href="https://2018.igem.org/Team:XMU-China/HP/Gold_Integrated">Gold</a></li>
+
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Public_Engagement">Engagement</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Public_Engagement">Engagement</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Collaborations">Collaborations</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Collaborations">Collaborations</a></li>
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                                 <li><a href="https://2018.igem.org/Team:XMU-China/Notebook">Notebook</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Notebook">Notebook</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Experiments">Experiments</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Experiments">Experiments</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/Engineering">Engineering</a></li>
 
                             </ul>
 
                             </ul>
 
                         </li>
 
                         </li>
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                                 <li><a href="https://2018.igem.org/Team:XMU-China/Attributions">Attributions</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Attributions">Attributions</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Judging">Judging</a></li>
 
                                 <li><a href="https://2018.igem.org/Team:XMU-China/Judging">Judging</a></li>
 +
                                <li><a href="https://2018.igem.org/Team:XMU-China/After_iGEM">After iGEM</a></li>
 
                             </ul>
 
                             </ul>
 
                         </li>
 
                         </li>
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     </div>
 
     </div>
 
     <script src="js/jquery-1.11.0.min.js"></script>
 
     <script src="js/jquery-1.11.0.min.js"></script>
    <!-- <script src="js/hc-mobile-nav.js"></script> -->
 
 
     <script src="https://2018.igem.org/Team:XMU-China/js/hc-mobile-nav?action=raw&ctype=text/javascript"></script>
 
     <script src="https://2018.igem.org/Team:XMU-China/js/hc-mobile-nav?action=raw&ctype=text/javascript"></script>
 
     <div class="header">
 
     <div class="header">
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                         <li><a href="https://2018.igem.org/Team:XMU-China/Attributions">Attributions</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Attributions">Attributions</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Judging">Judging</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Judging">Judging</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/After_iGEM">After iGEM</a></li>
 
                     </ul>
 
                     </ul>
 
                 </div>
 
                 </div>
 
                 <div id="Notebook">
 
                 <div id="Notebook">
                     <div>
+
                     <div class="nav-word">Notebook</div>
                        <div class="nav-word">Notebook</a></div>
+
                    </div>
+
 
                     <ul>
 
                     <ul>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Notebook">Notebook</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Notebook">Notebook</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Experiments">Experiments</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Experiments">Experiments</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Engineering">Engineering</a></li>
 
                     </ul>
 
                     </ul>
 
                 </div>
 
                 </div>
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                 </div>
 
                 </div>
 
                 <div id="Human_Practice">
 
                 <div id="Human_Practice">
                     <div class="nav-word">Human Practice</div>
+
                     <div class="nav-word">Social Works</div>
 
                     <ul>
 
                     <ul>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Human_Practices">Overview</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Human_Practices">Human Practice</a></li>
                        <li><a href="https://2018.igem.org/Team:XMU-China/HP/Silver">Silver</a></li>
+
                        <li><a href="https://2018.igem.org/Team:XMU-China/HP/Gold_Integrated">Gold</a></li>
+
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Public_Engagement">Engagement</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Public_Engagement">Engagement</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Collaborations">Collaborations</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Collaborations">Collaborations</a></li>
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                     <div class="nav-word">Model</div>
 
                     <div class="nav-word">Model</div>
 
                     <ul>
 
                     <ul>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Model">Overview</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Model">Summary</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Model#Thermodynamic_model">Thermodynamic Model</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Model#Fluid_dynamics_model">Fluid dynamics Model</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Model#Molecular_docking_model">Molecular Docking Model</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Model#The_dynamic_model">Derivation of Rate Equation</a></li>
 
                     </ul>
 
                     </ul>
 
                 </div>
 
                 </div>
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                     <ul>
 
                     <ul>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware">Overview</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware">Overview</a></li>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware/Microfluidic_Chips">Microfluidic chips</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Microfluidic_Chips">Microfluidic Chips</a></li>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware/Fluorescenc_Detection">Fluorescence Detection</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Fluorescence_Detection">Fluorescence Detection</a></li>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware/Straberry_Pi">Straberry Pi</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Raspberry_Pi">Raspberry Pi</a></li>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Applied_Design">Applied Design</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Hardware#Application">Application</a></li>
                         <li><a href="https://2018.igem.org/Team:XMU-China/Software">APP</a></li>
+
                         <li><a href="https://2018.igem.org/Team:XMU-China/Software">Software</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Applied_Design">Product Design</a></li>
 
                     </ul>
 
                     </ul>
 
                 </div>
 
                 </div>
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                         <li><a href="https://2018.igem.org/Team:XMU-China/Description">Description</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Description">Description</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Design">Design</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Design">Design</a></li>
                        <li><a href="https://2018.igem.org/Team:XMU-China/Demonstrate">Demonstrate</a></li>
 
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Results">Results</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Results">Results</a></li>
 +
                        <li><a href="https://2018.igem.org/Team:XMU-China/Demonstrate">Demonstrate</a></li>
 
                         <li><a href="https://2018.igem.org/Team:XMU-China/Parts">Parts</a></li>
 
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             <div class="word">Demonstrate</div>
 
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                     <a href="#Detection_Part"id="Quick_A">Detection Part</a></a>
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                     <a href="#Treatment_Part" id="Quick_B">Treatment Part</a></a>
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                 <div class="headline">Detection Part</div>
                    ABCDsystem
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                 <p>XMU-China set up a cell-free detection system platform for early screening of tumor biomarkers this year and named it <i>Fang</i>. The entire detection process is based on microfluidic chip technology. In order to link up all the experimental parts and show you the detection process clearly, we drew an operation instruction below.</p>
                </div>
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                 <p class="F2"><img src="https://static.igem.org/mediawiki/2018/1/18/T--XMU-China--Demonstrate1.jpg"></p>
                <h1>Background</h1>
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                     <p>Here are some possible factors that may influence the detection result in real life. Certainly these variates have already been controlled in ideal conditions. You can <a href="https://2018.igem.org/Team:XMU-China/Hardware">click here</a> to check our hardware's details.</p>
                 <p>Protein plays a significant role in performing physiological functions<sup>[1]</sup>. However, in diseased cells, protein carrying out a certain function may indicate the proceedings of disease. Such protein could be sorted to biomarkers, which have been regarded as the targets of disease detection and treatment in recent years.<sup>[2]-[4]</sup> Therefore, detecting those biomarkers of protein-type becomes more and more critical to biological and medical fields.
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                    <p class="reference">1. Type of testing sample (serum) <br>
                </p>
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                        2. Coating of complementary strands <br>
                <p>There are two main detecting approaches to detect a particular protein in a complex sample. One is direct determination of the content after purification, and the other is binding assays which include a target recognition probe and a signal transducer. The former approach includes gel filtration chromatography, ion exchange chromatography, nickel column and more. While on the down side, these methods involve high costs, strict equipment requirements and other drawbacks, which are not suitable for promotion and application. The enzyme-linked immunosorbent assay (ELISA) is a typical representative of the latter approach, nevertheless, such assays using antibodies as affinity ligands have cross-reactivity of antibodies compromising the specificity to the target of interest.<sup>[5]</sup> What’s worse, the premise of using ELISA is to find the corresponding antibodies, but the fact is that not all proteins can find their specific antibody protein. That is to say, the use of ELISA is also limited.
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                        3. Temperature of testing environment (Box) <br>
                </p>
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                        4. Establishment of standard curves <br>
                <p>In terms of binding assays, using aptamers as affinity ligands to recognize specific proteins are better than those using antibodies. Aptamers are short, synthetic single stranded oligonucleotides (DNA or RNA) that can bind to target molecules with high affinity and specificity.<sup>[6]-[9]</sup> They are commonly selected from random sequence libraries, using the systematic evolution of ligands by exponential enrichment (SELEX) techniques.<sup>[10]</sup> Advantages of aptamers over antibodies include longer shelflife, improved thermal stability and ease of modification and conjugation.<sup>[11]</sup>
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                        5. Stability of our hardware <br>
                </p>
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                        6. Biosecurity
                <p>An interesting binding assay is to use aptamers as the target recognition probes and CRISPR-Cas12a (Cpf1) as the signal amplifier, which is called Aptamer Based Cell-free Detection system(ABCD system, Figure 1). We developed this system to detect those biomarkers of protein-type for the purpose of disease detection or staging.
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                    </p>
                </p>
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                    <p>To address these issues, we propose following solutions.</p>
                 <p class="F1">
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                     <p>1. As we all know, there are many constituents in human’s whole blood, like red blood cells, white blood cells and platelets. So before adding sample to chips, we will pre-treat the sample by centrifuging that we would get pure serum without any colorful impurities to influence detection result.</p>
                    <img src="https://static.igem.org/mediawiki/2018/e/e2/T--XMU-China--ABCD_system.png">
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                     <p>2. To verify the effect of the coating, we disassemble the steps of <i>Ternary affinity coating</i> (TERACOAT) method. After each layer of coating is completed, we detect the fluorescence of washing solution to find whether ssDNA is tightly bonded to aptamer.</p>
                     <p class="Figure_word">Figure 1. <strong>A</strong>ptamer <strong>B</strong>ased <strong>C</strong>ell-free <strong>D</strong>etection system.</p>
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                     <p>3. The aptamer competition reaction needs to be carried out at room temperature. For now, the environment temperature when we do the verification experiment is above 25 ℃, which is basically conform to the requirements of experiment. If need in the later stage, we will put a temperature adjusting device in the box to keep the temperature constant.</p>
                </p>
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                     <p>4. Select two different pipeline on same microfluidic chips as the control group, add pure water into one pipeline and standard solution into another. You can compare the data of your water samples with that of the control group to reach your conclusion.</p>
                <h1>Abstract</h1>
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                    <p>The purpose of our project is definitely not to replace professional testing like pathological diagnosis. If our project can help people make preliminary judgments of samples and save their time and money, it will inspire us a lot.</p>
                <p>The core of the ABCD system is the specific binding of the aptamer and its target protein. We immobilize the aptamer-“complementary strand” complex on a solid phase, using a “competitive” approach to free the “complementary strand”; then the “complementary strand” was detected using the trans-cleavage property of the Cpf1 protein, which allows the fluorescence recovery of the static quenched complex whose fluorophore and quencher are linked by a ssDNA. In summary, we initially transform the protein signal to the acid signal, then transform the nucleic acid signal to the fluorescence signal. We use aptamer SYL3C<sup>[12]</sup> against EpCAM, an epithelial cell adhesion molecule that is highly expressed on the surface of adenocarcinoma cells, to test the feasibility of our system.</p>
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                     <p>5. Before the final version of hardware is mass produced, we will debug and improve the stability of hardware by simulating different experimental conditions.</p>
                <h1 class="reference">Reference</h1>
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                    <p>6. As we mentioned above, the biological reagents are in the state of freeze-dried powder. They have been placed in the microfluidic chips which are hermetic in the laboratory before you use them in practice. The microfluidic chips are disposable, so that they will not be exposed to biological safety problems if you collect them appropriately after using them. What’s more, we used purified EpCAM to replace serum to do verification experiment in our lab. As for clinic experiment, we will finish it under the guidance of professional doctors in clinic lab for the future plan, which meets the requirement of iGEM committee.</p>
                <p>
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                    <p>The operation instruction is following:</p>
                    [1] Janet Iwasa, Wallace Marshall. Karp's Cell and Molecular Biology: Concepts and Experiments (8th ed.). <i>Wiley: Hoboken, NJ.</i> <strong>2016</strong>, 48-49.
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                     <p class="reference">1) We get the peripheral blood to be tested from the user who maybe under high-risking or already sick, and pre-treat the sample by centrifugation. <br>
                    <br>[2] J. K. Aronson. Biomarkers and surrogate endpoints. <i>British Journal of Clinical Pharmacology.</i> <strong>2005</strong>, 59, 491-494.
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                        2) When the processed sample (serum) is added to the disc chip we designed, the chip would be placed in the detection hardware and remotely controlled by users’ app.<br>
                    <br>[3] Kyle Strimbu, Jorge A. Tavel. What are biomarkers? <i>Current Opinion in HIV and AIDS.</i> <strong>2010</strong>, 5, 463–466.
+
                        3) After a series of biological reactions, the protein signal is converted to fluorescent signals. <br>
                    <br>[4] Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: Preferred definitions and conceptual framework. <i>Clin. Pharmacol. Ther.</i> <strong>2001</strong>, 69, 89-95.
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                        4) App will convert the acquired image results into a visual and readability analysis report, which is based on its internal machine learning sample database. <br>
                     <br>[5] Hongquan Zhang, Feng Li, Brittany Dever, Xing-Fang Li, X. Chris Le. DNA-Mediated Homogeneous Binding Assays for Nucleic Acids and Proteins. <i>Chem. Rev.</i> <strong>2013</strong>, 113, 2812-2841.
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                        5) The professional doctor performs real-time diagnosis and treatment feedback through the communication platform built by the block chain, that is to achieve point-to-point information sharing between users and doctors.
                     <br>[6] Larry Gold, Barry Polisky, Olke Uhlenbeck, Michael Yarus. Diversity of Oligonucleotide Functions. <i>Annu. Rev. Biochem.</i> <strong>1995</strong>, 64, 763-797.
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                    </p>
                     <br>[7] Camille L.A. Hamula, Jeffrey W. Guthrie, Hongquan Zhang, Xing-Fang Li, X. Chris Le. Selection and analytical applications of aptamers. <i>Trends Anal. Chem.</i> <strong>2006</strong>, 25, 681-691.
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                     <br>[8] Renee K. Mosing, Shaun D. Mendonsa, Michael T. Bowser. Capillary Electrophoresis-SELEX Selection of Aptamers with Affinity for HIV-1 Reverse Transcriptase. <i>Anal. Chem.</i> <strong>2005</strong>, 77, 6107-6112.
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                     <br>[9] Maxim Berezovski, Andrei Drabovich, Svetlana M. Krylova, Michael Musheev, Victor Okhonin, Alexander Petrov, Sergey N. Krylov. Nonequilibrium Capillary Electrophoresis of Equilibrium Mixtures: A Universal Tool for Development of Aptamers. <i>J. Am. Chem. Soc.</i> <strong>2005</strong>, 127, 3165-3171.
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                    <br>[10] M Darmostuk, S Rimpelova, H Gbelcova, T Ruml. Current approaches in SELEX: an update to aptamer selection technology. <i>Biotechnology Advances.</i> <strong>2015</strong>, 33, 1141-1161.
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                     <br>[11] Sumedha D. Jayasena. Aptamers: an emerging class of molecules that rival antibodies in diagnostics. <i>Clin. Chem.</i> <strong>1999</strong>, 45, 1628-1650.
+
                    <br>[12] Yanling Song, Zhi Zhu, Yuan An, Weiting Zhang, Huimin Zhang, Dan Liu, Chundong Yu, Wei Duan, Chaoyong James Yang. Selection of DNA Aptamers against Epithelial Cell Adhesion Molecule for Cancer Cell Imaging and Circulating Tumor Cell Capture. <i>Anal Chem.</i> <strong>2013</strong>, 85, 4141-4149.
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                </p>
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             </section>
 
             </section>
             <section id="OMVs" class="js-scroll-step">
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             <section id="" class="js-scroll-step">
                 <div class="headline">
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                 <div class="headline">Treatment Part</div>
                    OMVs
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                 <p>For cell-free treatment, we were inclined to utilize outer-membrane vesicles (OMVs) to deliver nucleic acid. We designed three modules to anchor archaeal ribosomal protein L7Ae to the outer membrane protein A (OmpA) and then to encapsulate siRNA into OMVs (see our design page). Firstly, we identified our OMVs via transmission electron microscopy (TEM). Transmission electron microscopy (TEM) pictures identified what we extracted were exactly OMVs with membrane structure and 100-nm diameter (Figure 1). </p>
                </div>
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                <p class="F2"><img src="https://static.igem.org/mediawiki/2018/f/fb/T--XMU-China--Demonstrate2.png">
                <h1>Background</h1>
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                    <p class="Figure_word"><strong>Figure 1</strong>. TEM figures identify our OMVs.</p>
                 <p>Outer-membrane vesicles (OMVs) are lipid vesicles commonly produced by Gram-negative bacteria, which are filled with periplasmic content and are 20-250 nm in diameters (Figure 1). The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, and enabling bacterial delivery of nucleic acids and proteins. A recent paper by Kojima R et al. 2018, demonstrated an EXOtic device that can produce exosomes with specific nucleic acids cargo (Figure 2). We were impressed by the amazing OMVs and EXOtic device and came up with an idea to design a cell-free system to enable specific siRNA to be encapsulated into OMVs for cancer treatment.
+
 
                 </p>
 
                 </p>
                 <p class="F2">
+
                 <p>Secondly, we have successfully detected the OmpA-SpyTag (Figure 2) and the
                    <img src="https://static.igem.org/mediawiki/2018/4/43/T--XMU-China--OMVs11.png">
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                    SpyTag/SpyCatcher combination (Figure 3) inside OMVs via HSFCM <sup>[1-2]</sup>, indicating the presence of L7Ae.
                    <p class="Figure_word">Figure 2. The cell envelope of Gram-negative bacteria consists of two membranes, the outer membrane and the cytoplasmic membrane. Envelope stability comes from various crosslinks including the non-covalent interactions between the PG and the porin outer-membrane protein A (OmpA).</p>
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                 </p>
 
                 </p>
                 <p class="F2">
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                 <p class="F2"><img src="https://static.igem.org/mediawiki/2018/5/5b/T--XMU-China--Demonstrate3.png">
                    <img src="https://static.igem.org/mediawiki/2018/c/c0/T--XMU-China--OMVs12.png">
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                     <p class="Figure_word"><strong>Figure 2</strong>. Bivariate dot-plots of GFP green fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623021">BBa_K2623021</a> (Figure 2a) could secret more OMVs labeled with GFP than E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623022">BBa_K2623022</a> (Figure 2b). Similarly, E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623023">BBa_K2623023</a> (Figure 2c) would secret more OMVs labeled with GFP than E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623024">BBa_K2623024</a> (Figure 2d). Moreover, we could see E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623021">BBa_K2623021</a> (Figure 2a) would transport OmpA-ST to OMVs more efficiently than E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623023">BBa_K2623023</a> (Figure 2c). The remarkable difference of GFP-OMVs ratio between these two parts might account for the different inserted site of ST. SpyTag (ST) in <a href="http://parts.igem.org/Part:BBa_K2623021">BBa_K2623021</a> (Figure 2a) is inserted to N-termini of OmpA, while ST in <a href="http://parts.igem.org/Part:BBa_K2623023">BBa_K2623023</a> (Figure 2c) is inserted to C-termini of OmpA. Our HSFCM data shows that <a href="http://parts.igem.org/Part:BBa_K2623021">BBa_K2623021</a> is more efficient to transport ST-OmpA-GFP to OMVs and might be a good candidate for the following study.</p>
                     <p class="Figure_word">Figure 3. Schematic illustration of the EXOtic devices. Exosomes are nanoscale extracellular lipid bilayer vesicles of endocytic origin, and they are secreted by nearly all cell types in physiological and pathological conditions. Exosomes containing the RNA packaging device (CD63-L7Ae) and mRNA (e.g., nluc-C/Dbox) can efficiently deliver specific nucleic acids.</p>
+
                </p>
+
                <h1>Abstract</h1>
+
                <p>Not only eukaryotes but also prokaryotes can produce nanoscale bubbles to fulfill diverse functions, such as cellular communication, surface modifications and the elimination of undesired components. Additionally, because of this functional versatility, OMVs have been explored as a platform for bioengineering applications. This year, we XMU-China decide to utilize OMVs as a cell-free platform to deliver our nucleic acids agents to facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer.</p>
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                <p class="F3">
+
                    <img src="https://static.igem.org/mediawiki/2018/9/9d/T--XMU-China--OMVs13.png">
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                    <p class="Figure_word">Figure 4. We utilize a split protein SpyTag/SpyCatcher (ST/SC) bioconjugation system to create a synthetic linkage between protein OmpA and archaeal ribosomal protein L7Ae. We fuse SpyTag with OmpA at its C-termini and N-termini respectively.</p>
+
                </p>
+
                <p class="F3">
+
                    <img src="https://static.igem.org/mediawiki/2018/d/da/T--XMU-China--OMVs14.png">
+
                    <p class="Figure_word">Figure 5. After the induction of IPTG and Arabinose, we can get L7Ae-SpyCatcher and siRNA-C/Dbox. Archaeal ribosomal protein L7Ae owns the ability to bind with C/Dbox RNA structure.</p>
+
                </p>
+
                <p class="F4">
+
                    <img src="https://static.igem.org/mediawiki/2018/9/97/T--XMU-China--OMVs15.png">
+
                    <p class="Figure_word">Figure 6. With the interaction between SpyTag and SpyCatcher, and the ability of L7Ae to be bind with C/Dbox, we can produce customizable and cell-free OMVs containing specific siRNA to traget for oncogenic gene.</p>
+
                </p>
+
                <h1 class="reference">Reference</h1>
+
                <p>
+
                    [1] Kojima R, Bojar D, Rizzi G, et al. Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment[J]. <i>Nature Communications.</i> <strong>2018</strong>, 9(1):1305. <br>
+
                    [2] Alves N J, Turner K B, Medintz I L, et al. Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles: [J]. <i>Scientific Reports</i>, <strong>2016</strong>, 6:24866. <br>
+
                    [3] Schwechheimer C, Kuehn M J. Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. [J]. <i>Nature Reviews Microbiology</i>, <strong>2015</strong>, 13(10):605-19. <br>
+
                    [4] Vanaja S K, Russo A J, Behl B, et al. Bacterial Outer Membrane Vesicles Mediate Cytosolic Localization of LPS and Caspase-11 Activation. [J]. <i>Cell</i>, <strong>2016</strong>, 165(5):1106-1119. <br>
+
                    [5] Kamerkar S, Lebleu V S, Sugimoto H, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J]. <i>Nature</i>, <strong>2017</strong>, 546(7659):498-503. <br>
+
                    [6] https://en.wikipedia.org/wiki/Pancreatic_cancer<br>
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                 </p>
 
                 </p>
 +
                <p class="F2"><img src="https://static.igem.org/mediawiki/2018/8/83/T--XMU-China--Demonstrate4.png">
 +
                    <p class="Figure_word"><strong>Figure 3</strong>. Bivariate dot-plots of RFP red fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli BL21 transfected with <a href="http://parts.igem.org/Part:BBa_K2623028">BBa_K2623028</a>, <a href="http://parts.igem.org/Part:BBa_K2623029">BBa_K2623029</a>, <a href="http://parts.igem.org/Part:BBa_K2623030">BBa_K2623030</a>, <a href="http://parts.igem.org/Part:BBa_K2623031">BBa_K2623031</a> were incubated with OMVs-free LB culture for about 5 hours to get a 0.6-0.8 OD600 respectively, in which <a href="http://parts.igem.org/Part:BBa_K2623029">BBa_K2623029</a> and <a href="http://parts.igem.org/Part:BBa_K2623031">BBa_K2623031</a> were set as negative controls. Then IPTG was added to a final concentration at 0.5 mM and nurture the bacteria overnight. OMVs were isolated according to our protocols and then analyzed by HSFCM. It’s interesting to note that E. coli transfected with <a href="http://parts.igem.org/Part:BBa_K2623030">BBa_K2623030</a> (Figure 3c) could secret more RFP-OMVs than <a href="http://parts.igem.org/Part:BBa_K2623028">BBa_K2623028</a> (Figure 3a), indicating that ST/SC conjugation inside OMVs is more efficient with ST at the C-termini of OmpA. This result is inconsistent with the result shown in Figure 4, in which ST at the N-termini of OmpA (<a href="http://parts.igem.org/Part:BBa_K2623028">BBa_K2623028</a>) has a higher efficiency to be encapsulated inside OMVs. We propose that GFP inserted to OmpA might interfere the transport of OmpA-ST (<a href="http://parts.igem.org/Part:BBa_K2623021">BBa_K2623021</a> and <a href="http://parts.igem.org/Part:BBa_K2623023">BBa_K2623023</a>) to OMVs.
 +
                    </p>
 +
                    <p>Moreover, we have successfully detected the increase of RNA level inside OMVs between the positive group and the negative group, suggesting the presence of siRNA (Figure 4). </p>
 +
                    <p class="F2"><img src="https://static.igem.org/mediawiki/2018/7/7d/T--XMU-China--Demonstrate5.png">
 +
                        <p class="Figure_word"><strong>Figure 4</strong>. Bivariate dot-plots of green fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli BL21 transfected with <a href="http://parts.igem.org/Part:BBa_K2623029">BBa_K2623029</a> and <a href="http://parts.igem.org/Part:BBa_K2623032">BBa_K2623032</a> respectively were cultured in 100 mL OMVs-free LB culture for about 5 hours to get a OD600 at 0.6-0.8, in which <a href="http://parts.igem.org/Part:BBa_K2623029">BBa_K2623029</a> was set as negative control. Then Arabinose was added to a final concentration at 0.2% and after incubation for another 2 hours, IPTG was added to a final concentration at 0.5 mM and then nurtured the bacteria overnight. OMVs were isolated according to our protocols and stained with SYTOTM RNASelectTM. The stain OMVs were analyzed by HSFCM. As we expected, OMVs isolated from <a href="http://parts.igem.org/Part:BBa_K2623032">BBa_K2623032</a> could secret more OMVs containing with RNA stained positively by SYTOTM RNASelectTM (Figure 11b) than <a href="http://parts.igem.org/Part:BBa_K2623029">BBa_K2623029</a>, indicating that our L7Ae and C/Dbox could fulfill their function. It’s a pity that we didn’t have cell culture experiment to test our siOMVs to silence Kras in human pancreatic ductal adenocarcinoma (PDAC). In our future plans, we’ll finish our cell experiment and demonstrate the function of our siOMVs.
 +
                        </p>
 +
                        <p>However, we had no certification for cell culture experiments and it’s a pity that we haven’t incubated the OMVs with diseased cells to test the efficiency of our siOMVs (OMVs containing with siRNA). A paper published in 2017 demonstrated the potential to use extracellular vesicles (a similar thing to OMVs) for siRNA delivery and then treatment <sup>[3]</sup> . Hence we are confident with our OMVs for its ability to deliver siRNA as well. </p>
 
             </section>
 
             </section>
             <section id="Supporting" class="js-scroll-step">
+
             <section id="" class="js-scroll-step">
                 <div class="headline">
+
                 <div class="headline">References</div>
                    Supporting
+
                 <p class="reference">[1] Zhu S, Ma L, Wang S, et al. Light-Scattering Detection below the Level of Single Fluorescent Molecules for High-Resolution Characterization of Functional Nanoparticles. <i>Acs Nano</i>, <strong>2014</strong>, 8(10):10998-11006. <br>
                </div>
+
                     [2] Tian Y, Ma L, Gong M, et al. Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry[J]. <i>Acs Nano</i>, <strong>2018</strong>, 12(1). <br>
                <h1>Background</h1>
+
                    [3] Kamerkar S, Lebleu V S, Sugimoto H, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J]. <i>Nature</i>, <strong>2017</strong>, 546(7659):498-503.
                 <p>Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis.<sup>[1]</sup>Tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation olerance.<sup>[2]</sup>2012, Takekazu Kunieda and his team identified five abundant heat-soluble proteins in the tardigrades, which can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms.<sup>[1]</sup>
+
                </p>
+
                <p class="F4">
+
                    <img src="https://static.igem.org/mediawiki/2018/2/2d/T--XMU-China--TDP1.png">
+
                    <p class="Figure_word">Figure 7. Stage Photo of Tardigrades in Ant-Man 2.</p>
+
                </p>
+
                <p>In 2017, Thomas C. Boothby and his team segregated three TDP proteins in the water bears and explored their mechanism of action<sup>[3]</sup>. This is a schematic diagram of the mechanism they have done so far. At the same time, one of the 2017 iGEM teams <a href="https://2017.igem.org/Team:TUDelft/Design"><span class="click_here">TUDelft</span></a>, attempted to preserve the Cas13a protein using the TDP proteins, and they also tried to preserve the bacteria with the TDP proteins and obtained amazing outcome.
+
                     In our project, we are going to use TDPs to help preserve the protein Cas12a and OMVs.
+
                </p>
+
                <h1>Abstract</h1>
+
                <p>We have carried out research on TDP proteins this year. On the one hand, we plan to preserve the Cas12a required for protein detection and OMVs required for treatment with TDPs. On the other hand, as the wiki says, TDP is a new biological activity protector with great potential. So we are going to use TDP proteins to simplify existing methods of preserving proteins and bacteria.
+
                    There are two novel protein families with distinct subcellular localizations named Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. In our project, SAHS1 was used to preserve the proteins and CAHS1 was used for the preservation of the bacteria.
+
                </p>
+
                <p class="F4">
+
                    <img src="https://static.igem.org/mediawiki/2018/a/aa/T--XMU-China--TDP2.png">
+
                    <p class="Figure_word">Figure 8. The Expression of TDPs When The Tardigrades Suffer Form Fast Drying and Slow Drying.(Thomas C. Boothby et al. 2017).</p>
+
                </p>
+
                <h1>Reference</h1>
+
                <p class="reference">
+
                    [1]. Yamaguchi A. Two Novel Heat-Soluble Protein Families Abundantly Expressed in an Anhydrobiotic Tardigrade. <i>PLoS ONE</i>, <strong>2012</strong>;7(8):e44209. <br>
+
[2]. Boothby TC. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. <i>Mol Cell</i>. <strong>2017</strong> Mar16;65(6):975-984.e5.
+
 
+
 
                 </p>
 
                 </p>
 
             </section>
 
             </section>

Revision as of 20:22, 17 October 2018

Team:XMU-China/Demonstrate - 2018.igem.org

Demonstrate
Detection Part

XMU-China set up a cell-free detection system platform for early screening of tumor biomarkers this year and named it Fang. The entire detection process is based on microfluidic chip technology. In order to link up all the experimental parts and show you the detection process clearly, we drew an operation instruction below.

Here are some possible factors that may influence the detection result in real life. Certainly these variates have already been controlled in ideal conditions. You can click here to check our hardware's details.

1. Type of testing sample (serum)
2. Coating of complementary strands
3. Temperature of testing environment (Box)
4. Establishment of standard curves
5. Stability of our hardware
6. Biosecurity

To address these issues, we propose following solutions.

1. As we all know, there are many constituents in human’s whole blood, like red blood cells, white blood cells and platelets. So before adding sample to chips, we will pre-treat the sample by centrifuging that we would get pure serum without any colorful impurities to influence detection result.

2. To verify the effect of the coating, we disassemble the steps of Ternary affinity coating (TERACOAT) method. After each layer of coating is completed, we detect the fluorescence of washing solution to find whether ssDNA is tightly bonded to aptamer.

3. The aptamer competition reaction needs to be carried out at room temperature. For now, the environment temperature when we do the verification experiment is above 25 ℃, which is basically conform to the requirements of experiment. If need in the later stage, we will put a temperature adjusting device in the box to keep the temperature constant.

4. Select two different pipeline on same microfluidic chips as the control group, add pure water into one pipeline and standard solution into another. You can compare the data of your water samples with that of the control group to reach your conclusion.

The purpose of our project is definitely not to replace professional testing like pathological diagnosis. If our project can help people make preliminary judgments of samples and save their time and money, it will inspire us a lot.

5. Before the final version of hardware is mass produced, we will debug and improve the stability of hardware by simulating different experimental conditions.

6. As we mentioned above, the biological reagents are in the state of freeze-dried powder. They have been placed in the microfluidic chips which are hermetic in the laboratory before you use them in practice. The microfluidic chips are disposable, so that they will not be exposed to biological safety problems if you collect them appropriately after using them. What’s more, we used purified EpCAM to replace serum to do verification experiment in our lab. As for clinic experiment, we will finish it under the guidance of professional doctors in clinic lab for the future plan, which meets the requirement of iGEM committee.

The operation instruction is following:

1) We get the peripheral blood to be tested from the user who maybe under high-risking or already sick, and pre-treat the sample by centrifugation.
2) When the processed sample (serum) is added to the disc chip we designed, the chip would be placed in the detection hardware and remotely controlled by users’ app.
3) After a series of biological reactions, the protein signal is converted to fluorescent signals.
4) App will convert the acquired image results into a visual and readability analysis report, which is based on its internal machine learning sample database.
5) The professional doctor performs real-time diagnosis and treatment feedback through the communication platform built by the block chain, that is to achieve point-to-point information sharing between users and doctors.

Treatment Part

For cell-free treatment, we were inclined to utilize outer-membrane vesicles (OMVs) to deliver nucleic acid. We designed three modules to anchor archaeal ribosomal protein L7Ae to the outer membrane protein A (OmpA) and then to encapsulate siRNA into OMVs (see our design page). Firstly, we identified our OMVs via transmission electron microscopy (TEM). Transmission electron microscopy (TEM) pictures identified what we extracted were exactly OMVs with membrane structure and 100-nm diameter (Figure 1).

Figure 1. TEM figures identify our OMVs.

Secondly, we have successfully detected the OmpA-SpyTag (Figure 2) and the SpyTag/SpyCatcher combination (Figure 3) inside OMVs via HSFCM [1-2], indicating the presence of L7Ae.

Figure 2. Bivariate dot-plots of GFP green fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli transfected with BBa_K2623021 (Figure 2a) could secret more OMVs labeled with GFP than E. coli transfected with BBa_K2623022 (Figure 2b). Similarly, E. coli transfected with BBa_K2623023 (Figure 2c) would secret more OMVs labeled with GFP than E. coli transfected with BBa_K2623024 (Figure 2d). Moreover, we could see E. coli transfected with BBa_K2623021 (Figure 2a) would transport OmpA-ST to OMVs more efficiently than E. coli transfected with BBa_K2623023 (Figure 2c). The remarkable difference of GFP-OMVs ratio between these two parts might account for the different inserted site of ST. SpyTag (ST) in BBa_K2623021 (Figure 2a) is inserted to N-termini of OmpA, while ST in BBa_K2623023 (Figure 2c) is inserted to C-termini of OmpA. Our HSFCM data shows that BBa_K2623021 is more efficient to transport ST-OmpA-GFP to OMVs and might be a good candidate for the following study.

Figure 3. Bivariate dot-plots of RFP red fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli BL21 transfected with BBa_K2623028, BBa_K2623029, BBa_K2623030, BBa_K2623031 were incubated with OMVs-free LB culture for about 5 hours to get a 0.6-0.8 OD600 respectively, in which BBa_K2623029 and BBa_K2623031 were set as negative controls. Then IPTG was added to a final concentration at 0.5 mM and nurture the bacteria overnight. OMVs were isolated according to our protocols and then analyzed by HSFCM. It’s interesting to note that E. coli transfected with BBa_K2623030 (Figure 3c) could secret more RFP-OMVs than BBa_K2623028 (Figure 3a), indicating that ST/SC conjugation inside OMVs is more efficient with ST at the C-termini of OmpA. This result is inconsistent with the result shown in Figure 4, in which ST at the N-termini of OmpA (BBa_K2623028) has a higher efficiency to be encapsulated inside OMVs. We propose that GFP inserted to OmpA might interfere the transport of OmpA-ST (BBa_K2623021 and BBa_K2623023) to OMVs.

Moreover, we have successfully detected the increase of RNA level inside OMVs between the positive group and the negative group, suggesting the presence of siRNA (Figure 4).

Figure 4. Bivariate dot-plots of green fluorescence versus side scattering (SSC) for our OMVs isolates. E. coli BL21 transfected with BBa_K2623029 and BBa_K2623032 respectively were cultured in 100 mL OMVs-free LB culture for about 5 hours to get a OD600 at 0.6-0.8, in which BBa_K2623029 was set as negative control. Then Arabinose was added to a final concentration at 0.2% and after incubation for another 2 hours, IPTG was added to a final concentration at 0.5 mM and then nurtured the bacteria overnight. OMVs were isolated according to our protocols and stained with SYTOTM RNASelectTM. The stain OMVs were analyzed by HSFCM. As we expected, OMVs isolated from BBa_K2623032 could secret more OMVs containing with RNA stained positively by SYTOTM RNASelectTM (Figure 11b) than BBa_K2623029, indicating that our L7Ae and C/Dbox could fulfill their function. It’s a pity that we didn’t have cell culture experiment to test our siOMVs to silence Kras in human pancreatic ductal adenocarcinoma (PDAC). In our future plans, we’ll finish our cell experiment and demonstrate the function of our siOMVs.

However, we had no certification for cell culture experiments and it’s a pity that we haven’t incubated the OMVs with diseased cells to test the efficiency of our siOMVs (OMVs containing with siRNA). A paper published in 2017 demonstrated the potential to use extracellular vesicles (a similar thing to OMVs) for siRNA delivery and then treatment [3] . Hence we are confident with our OMVs for its ability to deliver siRNA as well.

References

[1] Zhu S, Ma L, Wang S, et al. Light-Scattering Detection below the Level of Single Fluorescent Molecules for High-Resolution Characterization of Functional Nanoparticles. Acs Nano, 2014, 8(10):10998-11006.
[2] Tian Y, Ma L, Gong M, et al. Protein Profiling and Sizing of Extracellular Vesicles from Colorectal Cancer Patients via Flow Cytometry[J]. Acs Nano, 2018, 12(1).
[3] Kamerkar S, Lebleu V S, Sugimoto H, et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer[J]. Nature, 2017, 546(7659):498-503.