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| − | <div class="introContainer">
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| − | <style>
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| − | <main style="padding-top:32px" id="section-text">
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| − | <div class="headingForIndex notvisible">Detection</div>
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| − | <div class="h2 larger">DETECTION</div>
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| − | <div class="h3">Overview</div>
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| − | <p class="content-paragraph" style="margin-bottom: 8px;">
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| − | Our detection system is based on identification of an exact Flavobacterium species marker gene fragments. This detection design is made up of these three main steps.
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| − | <ol>
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| − | <li>A bioinformatic analysis of the marker gene sequences which does not match between Flavo species. Creation of LFA DNA probes and HDA primers.</li>
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| − | <li>Helicase dependent asymmetric DNA amplification (HDA) of the marker gene fragments.</li>
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| − | <li>Lateral-flow assay membrane test that just in a few minutes identifies an exact pathogen.</li>
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| − | <div class="headingForIndex notvisible">Bioinformatic Analysis</div>
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| − | <div class="h3">Bioinformatic Analysis</div>
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| − | <p class="content-paragraph">
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| − | Our test is based on nucleic acid hybridization instead of antibody-antigen recognition since we wanted to differentiate between Flavobacterium species. We found out that for this purpose, nucleic acids are a more reliable and specific source than antibodies SALTINIS. The first step in developing a lateral flow assay test based on nucleic acid hybridization is choosing genes for species identification. According to our literature research 16S rRNA gene is a suitable candidate for this purpose because it is present in almost all bacteria and its function did not change over time<sup>2</sup>. To make sure that flavotest is specific we made a multiple sequence alignment with 16S rRNA genes from other species within the same Flavobacterium columnare, F. branchiophilum and F. psychrophilum species using Clustal Omega tool. Unique target sequences for F. columnare and F. psychrophilum were selected based on the absence of matching alignments between them (pic. 1).
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| − | We used Flavobacterium columnare and Flavobacterium psychrophilum 16S rRNA gene as a unique marker to each of the species. After bioinformatics analysis, a specific region in the sequences where no matches between the bases were found was chosen. For the short fragment, we created detection and capture probes.
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| − | <div class="headingForIndex notvisible">Helicase-dependent amplification</div>
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| − | <div class="h3">Helicase-dependent amplification</div>
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| − | <p class="content-paragraph">
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| − | With the aim to create a rapid, specific and cost-effective point-of-care detection system, at first, we needed to find the most suitable isothermal DNA amplification method. This method should be usable for farmers who have no scientific background. This factor pinpoints a huge need to be able to perform these isothermal reactions with as minimal pipetting steps as possible by means of avoiding errors and false-positive results. Although, amplification of marker sequences should be done in constant temperature by the needs of cheap and fully-portable equipment. By leading these main requirements, we have separated some isothermal amplification methods such as helicase dependent amplification (HDA), loop-mediated isothermal amplification (LAMP), strand-displacement amplification (SDA) and rolling circle amplification (RCA)<sup>1</sup>.
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| − | <p class="content-paragraph">
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| − | However, LAMP, SDA or RCA amplification methods have their own limitations such as complicated reaction schemes or multiplex sets of primers. Also, it should be mentioned that each of these methods are incapable of amplifying DNA targets of sufficient length required for lateral flow assay test<sup>2</sup>. After further analysis, we found out that in order to fulfil these goals, helicase dependent amplification would be a perfect solution. This method allows us to make our detection test as specific as possible by using an exact length of target sequences. Thus, it provides a simple reaction scheme and enables the generation of single-stranded DNA fragments, which are essential for lateral flow assay test development<sup>3</sup>.
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| − | <div id="animHdaBlock" class="scrollBlock"></div>
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| − | <div class="headingForIndex notvisible">Helimerase</div>
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| − | <div class="h3">Helimerase</div>
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| − | <p class="content-paragraph">
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| − | Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create.
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </main>
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| − | <main>
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| − | <div class="headingForIndex notvisible">Lateral Flow Assay</div>
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| − | <div class="h3">Lateral Flow Assay</div>
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| − | <p class="content-paragraph">
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| − | Lateral flow assay (LFA) is a simple method that can be used for isothermal amplification results visualisation. The use of the test is very intuitive and requires no prior training. Also, this LFA based test method is cost-effective and portable. Because of this, LFA is commonly used in remote locations where access to scientific laboratories is limited. For these reasons, we have decided that the best strategy for rapid flavobacterium caused infections detection tool development is the combination of HDA and LFA methods. since the users of this detection kit would be people without a scientific background.
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| − | <div class="headingForIndex notvisible">Treatment</div>
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| − | <div class="h2 larger">TREATMENT</div>
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| − | <p>
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| − | After detection of flavobacteriosis or any other infection, there should be an immediate treatment process. Currently, fish infected with flavobacterial diseases are treated with antibiotics. The biggest downside with it is that the farmers need to either pour gallons of antibiotics in the water tanks. Consequently, because the water tanks volume is so huge the antibiotic becomes highly diluted. Moreover, there is a huge risk that diluted antibiotics will only provide sustainable media for bacteria to develop resistance.
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| − | </p>
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| − | <p>
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| − | Scientific data already shows that some F. psychrophilum isolates already have reduced susceptibility to quinolones, oxolinic acid, and enrofloxacin<sup>1</sup>. To reduce the amount of antibiotics used in aquaculture farms, we are suggesting two systems intended for treatment and based on quorum sensing.
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| − | </p>
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| − | <p>
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| − | Quorum sensing is a bacterial communication process that leads to the regulation of genes and response to changes<sup>2-4</sup>. The quorum sensing has two distinguished systems - AHL and AI-2 for Gram-negative bacteria for now<sup>4-6</sup>.
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| − | <div class="buttonHeading">Choose treatment system</div>
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| − | <div class="buttonContainer">
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| − | <div id="buttonLysin">Endolysin & exolysin system</div>
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| − | <div id="buttonToxin">Toxin & antitoxin system</div>
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| − | <main id="sectionLysin">
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| − | <div class="headingForIndex notvisible">Endolysin & Exolysin System</div>
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| − | </main>
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| − | <main id="sectionToxin">
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| − | <div class="headingForIndex notvisible">Toxin & antitoxin system</div>
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| − | <div class="h3">Toxin & antitoxin system</div>
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| − | <p class="content-paragraph">Lorem ipsum, dolor sit amet sit con...</p>
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| − | <main>
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| − | <div class="headingForIndex notvisible">Prevention</div>
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| − | <div class="content">
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| − | <div class="h2 larger">PREVENTION</div>
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| − | <p class="content-paragraph">
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| − | Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create.
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| − | </p>
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| − | </div>
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| − | </div>
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| − | </main>
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| − | <main>
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| − | <div class="headingForIndex notvisible">Subunit vaccines</div>
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| − | <div class="scrollBlock">
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| − | <div class="scrollItem">
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| − | <div></div>
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| − | </div>
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| − | <div class="content">
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| − | <div class="h2 larger">Subunit vaccines</div>
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| − | <div class="h3">In alginate beads</div>
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| − | <p class="content-paragraph">Since it's inception iGEM has worked to ensure that excellence in synthetic biology goes beyond what happens in the lab. Decisions in science and engineering shape, and are shaped by, the societies we create. Social,</p>
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| − | </div>
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| − | <script>
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| − | <script type="text/javascript" src="https://2020.igem.org/wiki/index.php?title=Template:Vilnius-Lithuania/JSON/LFA&action=raw&ctype=text/javascript"></script>
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| − | { length: 0.105, title: "Sample pad", text: "The first step in lateral flow assay is HDA sample addition to the sample application pad, which is made from glass fibre. Then the test is placed in a running buffer which would minimize the nonspecific adsorption and increase the sensitivity and reproducibility of the test. Mentioned sample pad can also be pretreated with different kinds of buffers which help in processing the added fluid." },
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| − | { length: 0.14, text: "Keeping in mind that our Flavotest is based on nucleic acid hybridization<sup>1</sup>, the sample, which would be added onto this membrane must contain ssDNA molecules obtained during an asymmetric HDA assay. This test would work with fragments amplified during symmetric amplification which generates dsDNA also, but an additional step of denaturation would be needed." },
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| − | { length: 0.03, title: "Conjugate pad", text: "Fluid absorbed into sample pad flows to the subsequent membrane called a conjugate pad. On this membrane, labelled biorecognition molecules - gold nanoparticles conjugated with detection probes are dispersed. Upon contact with a moving liquid sample, the pad easily releases biorecognition molecules. According to literature research for LFA based on nucleic acid hybridisation, 13 nm gold nanoparticles are the most suitable1. For this reason, we synthesised 13 nm gold nanoparticles using sodium citrate reduction method otherwise called Turkevich method." },
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| − | { length: 0.07, title: "Detection probe", text: "Detection probe." },
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| − | { length: 0.06, title: "Gold nanoparticles", text: "Gold nanoparticles can be bought premade or synthesized in the laboratory. Usually, the Turkevich method with slight modifications is used for synthesis in which HAuCl<sub>4</sub> is reduced by trisodium citrate under reflux conditions. In this technique, citrate ions act as both stabilizing and reducing agents. The size of gold nanoparticles is determined by the amount of sodium citrate added. With a lower concentration of sodium citrate, larger gold nanoparticles are produced." },
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| − | { length: 0.03, text: "We also used tannic acid in the synthesis process since it helps to maintain the uniformity of gold nanoparticles size. It is crucial to remember that HAuCl<sub>4</sub> is corrosive so a glass or plastic spatula must be used also all glassware has to be cleaned with aqua regia to avoid gold nanoparticle aggregation during synthesis<sup>2</sup>." },
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| − | { length: 0.10, title: "Hybridization", text: "Conjugate pad filled with biorecognition molecules is essential for nucleic acid hybridization. If specific ssDNA from the HDA reaction is present in the sample, then the first hybridization reaction occurs between the detection probe and ssDNA amplicon. Newly formed complex immediately flows to the subsequent nitrocellulose membrane. If the added sample does not have a ssDNA amplicon, then no hybridization occurs in the conjugate pad. Further flows labelled gold nanoparticles unhybridized to ssDNA." },
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| − | { length: 0.11, title: "Test line", text: "Antrinė hibridizacijos reakcija, paaiškinimas, kodėl yra streptavidinas ir biotinas." },
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| − | { length: 0.15, title: "Control line", text: "Trečia/kontrolinė hibridizacijos reakcija, kodėl ji reikalinga ir kodėl turi kiekvieno testo metu atsirasti." },
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| − | { length: 0.12, title: "Absorbent pad", text: "Absorbent pad funkcija, užtikrina kad netekėtų atgal ir surenka perteklių." },
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| − | ],
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| − | { length: 0.15, title: "Sample processing", text: "The specimens obtained from the fish gills are lysed mechanically or using ionic liquids. For the exact species identification, we have selected F. columnare, and F. psychrophilum 16S rRNA DNA fragments and xx gene, obtained from F. psychrophilum." },
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| − | { length: 0.15, title: "Asymmetric HDA", text: "These genomic DNA marker sequences for further analysis are being amplified during asymmetric helicase dependent amplification. This system is based on a natural mechanism where two complementary DNA strands are separated by thermostable DNA helicase and coated with single-stranded DNA-binding proteins (SSB). After generation of ssDNA two sequence-specific primers hybridise to each border of ssDNA." },
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| − | { length: 0.15, text: "As it is an asymmetric HDA assay, the limiting primer is used up with the purpose to obtain ssDNA templates. After hybridisation, DNA polymerase extends these annealed to the template primers. Newly synthesised fragments are then used as substrates for DNA helicase, which enters the next round of isothermal amplification." },
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| − | { length: 0.42, title: "HDA products", text: "Finally, after asymmetric HDA assay, due to excess reverse primer concentration in the assay, ssDNA templates are prepared for the lateral flow assay test." },
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| − | { length: 0.10, bigTitle: "Endolysin & Exolysin system", title: "E.coli bacteria makes contact with exogenous AI-2 in the environment", text: "The E.Coli transporter cell senses the AI-2 that is released from Flavobacterium biofilms. The E.Coli strain DH5-Alpha can not produce AI-2 due to frameshift mutation in the luxS gene. It's a perfect control chassis for sensing and uptaking AI-2." },
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| − | { length: 0.10, title: "", text: "After AI-2 is internalized thorough lsrABC type transporter into the cytoplasm, it has to be phosphorylated by LsrK." },
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| − | { length: 0.01, title: "", text: "Phosphorylated AI-2-P can bind to a repressor LsrR." },
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| − | { length: 0.01, title: "", text: "LsrR+AI-2-P (repressor and bound phosphorylated autoinducer-2) dissociates from repressed promoter." },
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| − | { length: 0.10, title: "", text: "This initiates AI-2 induced promoter activity and 2 protein synthesis." },
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| − | { length: 0.20, title: "Our desirable protein - exolysin", text: "A specific exolytic virus protein which is naturally located in the tail fibers of a virus. Exolysins are highly specific and, thus, largely determine the host spectrum of the phage (mostly specific to single bacterial species but also are often specific to only a few strains within that species). Also, phage can have multiple different exolysin proteins which broadens the host spectrum." },
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| − | { length: 0.13, title: "", text: "Finally, the concentration of our toxin increases until it reaches the critical point where our E.coli bursts and releases a high concentration of exolysin." },
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| − | { length: 0.25, title: "", text: "The exolysin floats to the bacterial infection site." },
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| − | { length: 0.52, title: "Flavobacterial diseases occur when Flavobacterium biofilm forms on fish gills or fins.", text: "Like other pathogenic bacteria, Flavobacterium forms biofilm for increasing the resistance of antibiotics. This aggravates the breathing of fish and causes internal organs' failure<sup>7</sup>. The infected fish dies in only a few days." },
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| − | { length: 0.31, title: "Flavobacterium uses the same signaling molecule as E.coli - AI-2", text: "Flavobacterium is a Gram-negative bacteria and uses so-called autoinducer-2 for cell-cell communication, which is ascribed as a ‘universal’ interspecies signaling molecule<sup>8–10</sup>. Autoinducer-2 is a furanosyl borate diester or a borated DPD, which can be recognized as a signaling molecule as well<sup>8</sup>. During the biofilm formation the AI-2 concentration skyrockets. Therefore, we thought that using AI-2 induced promoter would be a perfect sensing system to start producing ‘killer-protein’." },
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