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                     ABCDsystem
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                     Xiamen Science and Technology Museum
 
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                <h1>Background</h1>
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                 <p>This year our team XMU-China came to Xiamen Science and Technology Museum to advocate the synthetic biology and our project .The whole project about 3 parts, game, Poster and Wish Wall. First, the game part, children play the roles of antibody and antigen respectively, they battled to be the winner by RPS.After the game they all left a deep impression of the general method that our body fight with cancer. Second,Poster,we had some talkative teammates to take charge of this part, so many student from high school and primary school were attracted ,they crowded in front of the poster and listened to our announcers absorbedly. Finally, the Wish Wall part, after our diverse introductions about the synthetic biology, they had learn a lot about it ,and some of them even inspire to give their contributions to synthetic biology, and want to attend the innovative, fantastic competition ——iGEM. It’s also a cheerful Day for our team to propagate the synthetic biology to so many children.</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="F3"><img src="https://static.igem.org/mediawiki/2018/d/d6/T--XMU-China--HP-part3-1.jpg"><img src="https://static.igem.org/mediawiki/2018/0/00/T--XMU-China--HP-part3-2.jpg"><img src="https://static.igem.org/mediawiki/2018/a/a3/T--XMU-China--HP-part3-3.jpg"></p>
                </p>
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                <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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                </p>
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                <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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                </p>
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                <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>
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                 <p class="F1">
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                    <img src="https://static.igem.org/mediawiki/2018/e/e2/T--XMU-China--ABCD_system.png">
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                    <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>
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                <h1>Abstract</h1>
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                <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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                <h1 class="reference">Reference</h1>
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                <p>
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                    [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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                    <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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                    <br>[3] Kyle Strimbu, Jorge A. Tavel. What are biomarkers? <i>Current Opinion in HIV and AIDS.</i> <strong>2010</strong>, 5, 463–466.
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                    <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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                    <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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                    <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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                    <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.
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                    <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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                     OMVs
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                     Fighting for Mooncake —— A Special Activity in Mid-Autumn Festival
 
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                <h1>Background</h1>
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                 <p>Fighting for Mooncake is a traditional activity spent with relatives or close friends in Xiamen area in order to celebrate the Mid-Autumn Festival. Throughout the game, we advertised the iGEM to freshmen, and introduced our project this year. It was worth mentioning that we also collected solicited opinions from all the students about the feasibility of our disease detective device and treatment device with cell-free system this year. 86.7% of participants showed positive attitudes towards our detective device, with the common sense of this device could performed pre-diagnosis quickly to guide patients. 66.8% students thought that it was difficult to treat tumors with vesicles, but it was very promising.</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.
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                 <p class="F3"><img src="https://static.igem.org/mediawiki/2018/f/f8/T--XMU-China--HP-part3-4.jpg"><img src="https://static.igem.org/mediawiki/2018/7/7c/T--XMU-China--HP-part3-5.jpg"><img src="https://static.igem.org/mediawiki/2018/0/0f/T--XMU-China--HP-part3-6.jpg"><img src="https://static.igem.org/mediawiki/2018/3/38/T--XMU-China--HP-part3-7.jpg"></p>
                </p>
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                <p class="F2">
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                    <img src="https://static.igem.org/mediawiki/2018/4/43/T--XMU-China--OMVs11.png">
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                    <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>
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                 <p class="F2">
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                    <img src="https://static.igem.org/mediawiki/2018/c/c0/T--XMU-China--OMVs12.png">
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                    <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>
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                </p>
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                <h1>Abstract</h1>
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                <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">
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                    <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>
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                </p>
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                <p class="F3">
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                    <img src="https://static.igem.org/mediawiki/2018/d/da/T--XMU-China--OMVs14.png">
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                    <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>
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                </p>
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                <p class="F4">
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                    <img src="https://static.igem.org/mediawiki/2018/9/97/T--XMU-China--OMVs15.png">
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                    <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>
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                </p>
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                <h1 class="reference">Reference</h1>
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                <p>
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                    [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>
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                    [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>
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                    [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>
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                    [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>
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                    [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>
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                    [6] https://en.wikipedia.org/wiki/Pancreatic_cancer<br>
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                     Supporting
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                     Visiting Yanwu Elementary School
 
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                 <h1>Background</h1>
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                 <p>In order to popularize the common knowledge of biology to the public and meanwhile to draw the public attention to synthetic biology, this year we xmu-china team visited to Yanwu Elementary School, volunteering to teach elementary students biology, introducing our team and work, and directing them to do some simple biological experiments. For instance, we let them extract DNA from bananas. </p>
                 <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>
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                 <p>In the course of our introduction, the children of Yanwu Primary school expressed their great enthusiasm, responded positively to our interaction, and moreover they showed terrific zest for our introduction. In these next simple experiments, the children were thrilled to be involved in one of the experiments.</p>
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                <p>In the end, through our teaching and guidance, most of the children have achieved remarkable results during the experiments. After the event, Yanwu Elementary school students said they learned a lot of useful knowledge, and very happy to do these interesting experiments by themselves within our guidance.</p>
                 <p class="F4">
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                 <p>Also, the teachers feed back that they think this activity is so meaningful, extremely hoping to hold more such events in the primary school campus.</p>
                    <img src="https://static.igem.org/mediawiki/2018/2/2d/T--XMU-China--TDP1.png">
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                 <p class="F3"><img src="https://static.igem.org/mediawiki/2018/8/8e/T--XMU-China--HP-part3-8.jpg"><img src="https://static.igem.org/mediawiki/2018/7/73/T--XMU-China--HP-part3-9.jpg"></p>
                    <p class="Figure_word">Figure 7. Stage Photo of Tardigrades in Ant-Man 2.</p>
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            </section>
                 </p>
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            <section id="Provenance" class="js-scroll-step">
                 <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.
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                <div class="headline">
                    In our project, we are going to use TDPs to help preserve the protein Cas12a and OMVs.
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                    Consulting the Chief Physician Weiwei Tang in Medical Oncology
                </p>
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                 </div>
                 <h1>Abstract</h1>
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                 <p>This year,team XMU-China developed new methods named ABCD System and OMVs Treatment for detecting and treating diseases. In order to prove the feasibility of our project, we consulted the Chief Physician Weiwei Tang in Medical Oncology, The First Affiliated Hospital of Xiamen University, on August 23th. We introduced briefly the structure and function of our self-designed hardware Eye of Agamotto(EA) to Dr.Tang. During the talk, Dr.Tang appreciated it that our ideas are innovative and helpful. What’s more, Dr.Tang also told us those frequently-used and classical clinical detection methods, like ELISA, CLIA and so on. Most of which are time-consuming , expensive and complex, compared with EA. As for EA, Dr.Tang agreed with our  proposals that we are going to make EA widely used among basic medical institutions, especially in remote, and poverty-stricken areas because of its convenient and practicability. But it’s worth noting that today there is an authoritative method for disease-diagnose, i.e., pathological diagnosis. So our detection method would just serves as a kind of screening tool rather than diagnosis method, according to Tang’s suggestion. </p>
                 <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.
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                <p class="F3"><img src="https://static.igem.org/mediawiki/2018/9/9f/T--XMU-China--Entrepreneurship-6.png"><img src="https://static.igem.org/mediawiki/2018/6/64/T--XMU-China--hp-%3Di.png"></p>
                    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.
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            </section>
                </p>
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            <section id="Reference" class="js-scroll-step">
                 <p class="F4">
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                 <div class="headline ">
                    <img src="https://static.igem.org/mediawiki/2018/a/aa/T--XMU-China--TDP2.png">
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                    Visiting Health Service Center of Datong Street
                    <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>
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                </div>
                </p>
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                 <p>We visited Health Service Center of Datong Street, Tong’an District, Xiamen city. We learned about the development of tumor markers detection work in primary hospitals from director Zhan. We found that primary hospitals were mostly able to carry out screening of basic tumor markers such as alpha fetoprotein. If people want to carry out more systematic and specific tumor screening, they had to be referred to higher-level hospitals. In addition, due to the implementation effect of China's medical security system and Xiamen's health policy, the screening of tumor markers at the grassroots-level health center could save about 50% of the cost compared with the large hospitals. Therefore, to sum up, president Zhan affirmed the future prospects of our instruments, believing it would solve problems such as "difficult access to medical services" in real life, to better provide more convenient medical and health services.</p>
                <h1>Reference</h1>
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                 <p class="F25"><img src="https://static.igem.org/mediawiki/2018/c/cf/T--XMU-China--Entrepreneurship-10.png"></p>
                 <p class="reference">
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            </section>
                     [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>
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            <section id="Reference" class="js-scroll-step">
[2]. Boothby TC. Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation. <i>Mol Cell</i>. <strong>2017</strong> Mar16;65(6):975-984.e5.  
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                 <div class="headline ">
 
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                     Market analysis
                </p>
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                </div>
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                <p>This year, XMU-China designed a device that could be applied in cancer screening field by detecting biomarkers. We wondered if it would have a bright future in the IVD market,so we launched many Human Practices, including visiting specialists. Those specialists gave us some beneficial advice about the product-sale strategies, which was beneficial for our project’s further development. What’s more, we also visited the Community Health Service Center where our distributed devices could play an important role in testing the lab. See more in 商业策划书链接。</p>
 
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Team:XMU-China/Engagement - 2018.igem.org

Engagement