Difference between revisions of "Team:Vilnius-Lithuania/test/Design"

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<div class="headingForIndex notvisible">Detection</div>
 
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<div class="headingForIndex notvisible">Overview</div>
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<div class="h3">Overview</div>
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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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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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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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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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<div class="h3">And other fine products you can try</div>
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<p class="content-paragraph">Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Amet nisl purus in mollis nunc. Accumsan tortor posuere ac ut consequat semper. Enim lobortis scelerisque fermentum dui faucibus in ornare. Sed lectus vestibulum mattis ullamcorper. Quis varius quam quisque id diam vel.</p>
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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 class="headingForIndex notvisible">HDA</div>
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<div class="headingForIndex notvisible">Helimerase</div>
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<div class="headingForIndex notvisible">Lateral Flow Assay</div>
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<p id="animLfaText" 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 class="h2 larger">TREATMENT</div>
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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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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">Endolysin & Exolysin System</div>
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<p id="anim2text" 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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<p id="anim2text" 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 id="anim2title" class="h3">In alginate beads</div>
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<p id="anim2text" 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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<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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 +
{ length: 0.14, title: "DNA", text: "Our lateral flow assay test is based on nucleic acid hybridization ŠALTINIAI for this reason sample added to the test must contain ssDNA. This test would work with fragments amplified during symmetric amplification which generates dsDNA also, but additional step of denaturation would be needed. For the ease of use we decided to perform asymmetric helicase dependent amplification after which a fraction of amplified sequences are double stranded but the majority of the DNA is single stranded and is immediately suitable for this test without the need of denaturation." },
 +
{ length: 0.03, title: "Conjugate pad", text: "Then the sample flows to a conjugate pad which has gold nanoparticles with conjugated detection probes. For nucleic acid hybridization based LFA 13 nm gold nanoparticles (AuNP) are most suitable SALTINIS. For these reasons we synthesised mentioned size gold nanoparticles using sodium citrate reduction method." },
 +
{ length: 0.07, title: "Detection probe", text: "Detekcijos zondo funkcija, dalelių funkcionalizavimo procesas, kaip tai vyksta, tiolio grupės modifikacija." },
 +
{ length: 0.09, title: "Gold nanoparticles", text: "Aukso nanodalelių sintezė, kaip tai vyksta, esminiai paaiškinimai viso proceso, (aukso redukcija naudojant citratą), galbūt paminėti tanino rūgštį." },
 +
{ length: 0.10, title: "Hybridization", text: "Paaiškinimas, jog vyksta pirminė hibridizacija, jei mėginyje buvo taikinio molekulė, nauji kompleksai juda toliau." },
 +
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 +
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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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{ 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." },
 +
{ 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." },
 +
{ 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." },
 +
{ 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." },
 +
{ length: 0.10, title: "", text: "After AI-2 is internalized thorough lsrABC type transporter into the cytoplasm, it has to be phosphorylated by LsrK." },
 +
{ length: 0.01, title: "", text: "Phosphorylated AI-2-P can bind to a repressor LsrR." },
 +
{ length: 0.01, title: "", text: "LsrR+AI-2-P (repressor and bound phosphorylated autoinducer-2) dissociates from repressed promoter." },
 +
{ length: 0.10, title: "", text: "This initiates AI-2 induced promoter activity and 2 protein synthesis." },
 +
{ 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." },
 +
{ 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: 0.52, bigTitle: "TREATMENT", 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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+
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+
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{ element: "#animLfa-5", length: 0.07, title: "Detection probe", text: "Detekcijos zondo funkcija, dalelių funkcionalizavimo procesas, kaip tai vyksta, tiolio grupės modifikacija." },
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+
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{ element: "#animLfa-7", length: 0.10, title: "Hybridization", text: "Paaiškinimas, jog vyksta pirminė hibridizacija, jei mėginyje buvo taikinio molekulė, nauji kompleksai juda toliau." },
+
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+
−
{ element: "#animLfa-9", 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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{ element: "#animLfa-10", length: 0.12, title: "Absorbent pad", text: "Absorbent pad funkcija, užtikrina kad netekėtų atgal ir surenka perteklių." },
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Revision as of 19:25, 12 October 2020

Detection
DETECTION

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.

Overview
Overview

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.

  1. A bioinformatic analysis of the marker gene sequences which does not match between Flavo species. Creation of LFA DNA probes and HDA primers.
  2. Helicase dependent asymmetric DNA amplification (HDA) of the marker gene fragments.
  3. Lateral-flow assay membrane test that just in a few minutes identifies an exact pathogen.

Bioinformatic Analysis
Bioinformatic Analysis

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 time2. 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).

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.

Helicase-dependent amplification
Helicase-dependent amplification

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)1.

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 test2. 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 development3.

HDA
Helimerase
Helimerase

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.

Lateral Flow Assay
Lateral Flow Assay

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.

Treatment
Choose treatment system
Endolysin & exolysin system
Toxin & antitoxin system
Endolysin & Exolysin System
Toxin & antitoxin system
Toxin & antitoxin system

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Prevention
PREVENTION

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.

Subunit vaccines
Subunit vaccines
In alginate beads

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,