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

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<h1>Design</h1>
 
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Design is the first step in the design-build-test cycle in engineering and synthetic biology. Use this page to describe the process that you used in the design of your project. You should clearly explain the engineering principles used to design your project.
 
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<div class="grandHeading" style="margin-top:32px" id="section-text">DETECTION</div>
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<div class="headingForIndex notvisible">Detection</div>
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Our detection system is based on identification of an exact <i>Flavobacterium</i> species marker gene fragments. This detection design is made up of these three main steps.
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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 we wanted to differentiate between Flavobacterium species, instead of antibody-antigen interaction, our detection test is based on nucleic acid hybridization. We found out that for identification purposes, 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 marker genes, which allows us to identify an exact bacteria species. 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 highly specific, we made a multiple sequence alignment with 16S rRNA genes from other species within the same genus using Clustal Omega tool (1. 2. 4.). Unique target sequences for <i>F. columnare</i> and <i>F. psychrophilum</i> were selected based on the absence of matching alignments between sequences.
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<p>Picture 1</p>
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Also we found that rpoC gene can be used as marker from <i>F. psychrophilum</i>.
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We decided to create probes for this gene sequence as well.
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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 <b>rapid, specific and cost-effective</b> 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 <b>as minimal pipetting steps</b> 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 <b>cheap</b> and <b>fully-portable</b> equipment. By leading these main requirements, we have separated some isothermal amplification methods such as helicase dependent amplification (<b class="semi">HDA</b>), loop-mediated isothermal amplification (<b class="semi">LAMP</b>), strand-displacement amplification (<b class="semi">SDA</b>) and rolling circle amplification (<b class="semi">RCA</b>)<sup>1</sup>.
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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, <b>helicase-dependent amplification</b> 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">Helimerase</div>
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<div class="content">
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<div class="h3">Helimerase</div>
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<p>
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However, regarding the WHO guidelines for point-of-care testing, our detection tool should be not only sensitive, specific and user-friendly, but also it should be <b>affordable for target customers</b><sup>1</sup>. Keeping in mind that HDA amplification, performed using a commercial kit, is still too <b>expensive</b>, we have decided to search for new <b>alternatives</b> to reduce the cost of the test as much as possible.
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Our solution to this problem - <b>helimerase</b>. This protein complex contains <b>two</b> enzymes – <i>Thermoanaerobacter tengcongensis</i> </b>UvrD helicase</b> (<b class="semi">TteUvrD</b>) and <i>Bacillus stearothermophilus</i> <b>DNA polymerase I large fragment</b> (<b class="semi">BstPol</b>). TteUvrD is fused with one part of coiled-coil structure <b>WinZip-A2</b> (<b class="semi">WZA2</b>) via linker L1 and possesses a maltose-binding protein (<b class="semi">MBP</b>) and <b>10xHis Tag</b> in the N terminal end. BstPol is fused with the second coiled-coil part <b>WinZip-B1</b> (<b class="semi">WZB1</b>) through linker L1 and possesses <b>StrepII tag</b> in the N terminal end<sup>2</sup>. It was presumed that this <b>non-covalent fusion</b> strategy through coiled-coil structures should  improve HDA reaction by letting to amplify longer DNA fragments as well as it allows to perform amplification reaction without using additional proteins such as MutL or others SSB proteins, which are used in this type of reactions<sup>2,3</sup>.
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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>
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Lateral flow assay (<b class="semi">LFA</b>) 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.
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<div class="grandHeading">TREATMENT</div>
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<div class="headingForIndex notvisible">Treatment</div>
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After detection of an exact flavobacterium species, farmers need to start an exact treatment process as soon as possible. Currently, fish infected with flavobacterial diseases are treated with different types of antibiotics<sup>13</sup>. Scientific data shows<sup>14</sup> that the most abundant antibiotics used for salmon cultivation was quinolone, which consumption (by mass) in 2007 reached 821,997 tonnes. That is only in Norway! Other commonly used antibiotics in farms are oxolinic acid and florfenicol, which consumption reached 681 kg in 2008 and 166 kg in 2010 respectively. This enormous usage of wide variety antibiotics forces the evolving of antibiotic-resistant bacterial fish pathogens. These numbers raise huge concerns and questions on how we can reduce the usage of antibiotics?
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<p>
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Scientific data already shows that some <i>F. psychrophilum</i> isolates already have reduced susceptibility to quinolones, oxolinic acid, and enrofloxacin<sup>1</sup>. To reduce the amount of antibiotics used in aquaculture farms, our second goal is the development of two exogenous fish infection treatment systems, which are based on quorum sensing mechanisms.
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</p>
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<p>
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Flavobacterial diseases are caused when <i>Flavobacterium</i> forms the same biofilm on fish fins or gills<sup>15</sup>. Our target bacteria - <i>Flavobacterium</i> uses signaling molecules for cell-cell communication. 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>. We used the circumstances in our advantage: build a genetic circuit that is enhanced by quorum sensing signaling molecules.
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<div class="buttonHeading">Choose treatment system</div>
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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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<div class="headingForIndex notvisible">Endolysin & Exolysin System</div>
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<div class="grandHeading">PREVENTION</div>
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Usually, in order to prevent fish infections, vaccines are injected intraperitoneally. Because of manual handling, which is unavoidable in such a type of vaccination, fish experience a lot of stress and it paradoxically weakens their immune system. With the aim to avoid such consequences, we have set a third goal - to create a prevention system based on subunit vaccines against bacterial and viral fish infections.
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<div class="headingForIndex notvisible">Subunit vaccines</div>
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<div class="headingForIndex notvisible">References</div>
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<div class="h3">Big Mega Super Duper List of References</div>
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<p>R. Meškys et al. iGEM Vilnius-Lithuania 2020</p>
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<script type="text/javascript" src="https://2020.igem.org/wiki/index.php?title=Template:Vilnius-Lithuania/JSON/HDA&action=raw&ctype=text/javascript"></script>
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const animations = [
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animationData: animLfa,
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{ length: 0.245, title: "Sample Pad", text: "The first step in lateral flow assay is sample, which contains ssDNA molecules obtained during an asymmetric HDA reaction, addition to the sample application pad, which is made from glass fibre. Then the running buffer is added 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." },
 +
{ length: 0.03, title: "Conjugate Pad", text: "Fluid absorbed into sample pad flows to the subsequent membrane called a <b>conjugate pad</b>. On this membrane, labelled biorecognition molecules - <b>gold nanoparticles conjugated with detection probes</b> are dispersed. Upon contact with a moving liquid sample, the pad easily releases biorecognition molecules. According to literature research, <b>13 nm gold nanoparticles</b> are the most suitable for LFA based on nucleic acid hybridization<sup>1</sup>. For this reason, we chose to synthesize 13 nm gold nanoparticles using sodium citrate reduction method otherwise called Turkevich method." },
 +
{ length: 0.065, 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. For LFA test development we also decided to use tannic acid in the gold nanoparticles synthesis process since it helps to maintain the uniformity of gold nanoparticles size." },
 +
{ length: 0.065, title: "Detection Probe", text: "Synthesized gold nanoparticles must be functionalized with <b>detection probes</b>. Detection probe is a ssDNA molecule with <b>5’ thiol group modification</b> created to hybridize to HDA produced specific to species ssDNA amplicon. To functionalize gold nanoparticles a method, which includes the step of “aging” with NaCl is used. Salt helps to overcome the long-ranged electrostatic repulsion between the DNA and gold nanoparticles since they are both negatively charged<sup>2</sup>. Our team created probes with <b>poly-A</b> spacer, so an alternative and quicker, low pH assisted method can be used for functionalization<sup>3</sup>. In pH 3, adenine is protonated, and this reduces the repulsion between DNA and gold nanoparticle." },
 +
{ 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>." },
 +
{ length: 0.10, title: "Hybridization", text: "<b>Conjugate pad</b> filled with biorecognition molecules is essential for nucleic acid hybridization.<br>If specific ssDNA from the HDA reaction is present in the sample, then the first hybridization reaction occurs between the <b>detection probe and ssDNA amplicon</b>. Newly formed complex immediately flows to the subsequent nitrocellulose membrane.<br>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." },
 +
{ length: 0.05, title: "Test Line", text: "Mentioned nitrocellulose membrane has two lines with dispersed DNA probes. The first line is called <b>the test line</b>, and the further line is <b>the control line</b>. On the test line, capture probes are dispersed. The capture probe is a ssDNA molecule with 3’ biotin modification which is necessary so that the probe could be immobilized <b>via a biotin-streptavidin non-covalent bond<b> on the membrane." },
 +
{ length: 0.06, text: "When the fluid reaches the test line, another hybridization reaction occurs between the <b>capture probe and ssDNA amplicon-gold nanoparticle complex</b>. Since capture probes are immobilized on the membrane, the resulting complex does not move any further and stays on the test line. Eventually, due to hybridization gold nanoparticles start to accumulate and the red line becomes visible to the naked eye. If there is no specific ssDNA amplicon in the sample, then no hybridization takes place and labeled gold nanoparticles move further the membrane." },
 +
{ length: 0.05, title: "Control Line", text: "In the conjugate pad, not all labelled gold nanoparticles hybridize to ssDNA amplicon, meaning that there are gold nanoparticles with free detection probes. This is important for <b>the control line</b> where control probes are immobilized via the same biotin-streptavidin bond. The only difference is that the control probe has a 5’ biotin modification and is complementary to the detection probe. For this reason, hybridization between <b>free detection probe on gold nanoparticle and control probe</b> occurs resulting in the red line formation. Mentioned hybridization reaction has to occur during each test because it shows that the test operated correctly." },
 +
{ length: 0.10, text: "We chose to use Hi-Flow plus HF180 nitrocellulose membrane, which has a nominal capillary <b>flow time of 180 seconds/4cm</b>, which is relatively slow. The migration speed of the fluid is critical because it affects the response of the test. Slower flow membrane results in a longer time assay, but response signal is higher, and lines are better defined<sup>1</sup>. To get the best results optimization is needed, for this reason we decided to create a mathematical model which helps to find out the best place for capture and control probes." },
 +
{ length: 0.12, title: "Absorbent Pad", text: "Finally, the last membrane is <b>the absorbent pad</b> which helps in maintaining the flow rate of the liquid over the membrane and stops backflow of the sample. Also, it absorbs excess fluid. Different kinds of membranes are assembled with overlapping ends (2mm) to ensure that continuous flow is achieved and mounted over a backing card which helps as support and makes it easy to handle the test." },
 +
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 +
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 +
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 +
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 +
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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 <b><i>F. columnare</i></b>, and <b><i>F. psychrophilum</i> 16S rRNA</b> DNA fragments and rpoC gene, obtained from <i>F. psychrophilum</i>." },
 +
{ length: 0.15, title: "Asymmetric HDA", text: "These genomic DNA marker sequences for further analysis are being amplified during <b>asymmetric helicase dependent amplification</b>. This system is based on a natural mechanism where two complementary DNA strands are separated by thermostable <b>DNA helicase</b> and coated with single-stranded DNA-binding proteins (<b class=\"semi\">SSB</b>). 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 hybridization, 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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 +
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 +
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 +
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 +
{ length: 0.10, bigTitle: "Endolysin & Exolysin System", title: "Contact", text: "<i>E. coli</i> bacteria make contact with exogenous AI-2 in the environment. The <i>E. coli</i> - transporter cell - senses the AI-2 that is released from <i>Flavobacterium</i> biofilms. The <i>E. coli</i> strain DH5α can not produce AI-2 due to frameshift mutation in the <i>luxS</i> gene. It is 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 binds 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 and 2 protein coding genes transcription." },
 +
{ length: 0.20, title: "Exolysin", text: "Our desirable protein - exolysin. The main protein of this genetic circuit is 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). The idea is to synthesize <i>Flavobacterium</i> bacteriophage’s depolymerase and use it as a drug that destroys the biofilm." },
 +
{ length: 0.38, title: "", text: "Finally, the concentration of our toxin increases until it reaches the critical point where our <i>E. coli</i> bursts and releases a high concentration of exolysin to the bacterial infection site." },
 +
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 +
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 +
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 +
{ length: 0.52, title: "Biofilm", text: "Flavobacterial diseases occur when <i>Flavobacterium</i> biofilm forms on fish gills or fins. Like other pathogenic bacteria, <i>Flavobacterium spp.</i> forms biofilm for increasing the resistance of antibiotics. These biofilms aggravate the breathing of fish and cause internal organs' failure<sup>7</sup>. Due to this, infected fish die in 24-72 hours after first physical changes are being seen." },
 +
{ length: 0.31, title: "AI-2", text: "<i>Flavobacterium</i> uses the same signaling molecule as <i>E. coli</i> - AI-2. Flavobacterium is a Gram-negative bacteria that uses a <i>universal</i> interspecies signaling molecule so-called autoinducer-2 for cell-cell communication. This action is often being called a quorum sensing. 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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{ length: 0.017, noanim: true, bigTitle: "Subunit Vaccines", text: "Subunit vaccines are based on proteins that induce a specific immune response against an exact pathogen. These immunogenic proteins cannot replicate in the host, thus there is no risk of infection to the host or non-target species<sup>1</sup>. Additionally, the chances of spontaneous reversions or denaturing of antigenic peptides, which occur by using attenuated or inactivated virus vaccines, are reduced. Considering that these subunit vaccines will be used in aquaculture farms, a key aspect of the prevention system is its easy transportation and storage conditions. Subunit vaccines can be freeze-dried, allowing  non-refrigerated transport and storage<sup>2</sup>." },
 +
{ length: 0.03, title: "Bacterial Pathogens", text: "In the beginning, our primary goal was to immunise fish against one of the chosen <i>Flavobacterium</i> - <i>F. columnare</i>. Firstly, we considered picking <i>F. psychrophilum</i> or <i>F. branchiophilum</i>. However, after our research, we decided to focus on <i>F. columnare</i>, similar to other strains by its severity, yet having a well studied and effectively immunogenic outer membrane protein <b>GldJ</b>, which has well-exposed epitopes on its surface, is recognized by fish immune cells<sup>11</sup>. (link to parts)" },
 +
{ length: 0.03, title: "Viral Pathogens", text: "However, after a virtual meeting with <b>Darius Nienius, deputy director of National Food and Veterinary Risk Assessment Institute of Lithuania</b>, we were informed that <b>viruses</b> are as equally dangerous to aquaculture farms as bacteria. It was explained that viral infections are tough to deal with because while bacterial infections can be treated with antibiotics, there are barely any commercial drugs for viral infections. Especially problematic of them - <b>viral hemorrhagic septicemia virus (VHSV)</b> - does not have a commercially available vaccine yet, so it was added to our focus pathogens. VHSV has <b>Glycoprotein G</b> on its surface, that induces an immune response against the virus in fish. (link to parts)" },
 +
{ length: 0.03, title: "GFP", text: "To ensure that the encapsulation process does not damage the proteins, we had to test it on easily examinable protein. By using purified a green fluorescent protein with 6xHis Tag and by treating it with the most common digestive enzymes in their appropriate buffer, we can tell whether green fluorescent protein is intact and or when it was degraded in a plate reader assay. By adding the same amount of alginate beads of the same size into the plate reader and by reading emission we can determine whether alginate beads would pass through the digestive tract of the fish and how much of the protein would be lost during that process<sup>23</sup>." },
 +
{ length: 0.03, title: "Optimization", text: "Immunogenic recombinant proteins had to be expressed and purified, to be used in vaccine production. By cloning GldJ coding sequence into pET28-a(+) and VHSV into pfX-7 vector we tried to optimize protein induction conditions. By changing them we tried to get the most soluble protein and purify it with the ÄKTA chromatography system." },
 +
{ length: 0.09, title: "Why Alginate", text: "We decided to encapsulate the immunogenic proteins in <b>calcium alginate beads</b>. Using brown algae (kelp) polysaccharide - <b>alginate</b>, the same protective effect against digestive enzymes and pH differences occurring in the fish gut as with live algae could be achieved. Alginate is <b>cheaper</b> and <b>safer</b> for the environment as it does not rely on living genetically modified algae species, reducing the chance of spreading organisms in the ecosystem. <b>The calcium alginate layer</b> is biocompatible and highly resistant to acidic or digestive environments<sup>18</sup>. Alginate cannot be broken down in the fish stomach, and pyloric caeca and calcium make protein absorption more rapid<sup>19</sup>." },
 +
{ length: 0.20, title: "Protein Encapsulation", text: "The protein must be protected from partial or full digestion in the stomach of the fish. We found that scientists have already tried feeding fish with green fluorescent protein which was bio encapsulated in lyophilised Chlamydomonas reinhardtii. Non-digested green fluorescent protein was then found in fish blood and plasma, as well as in intestinal tissues<sup>17</sup>." },
 +
{ length: 0.06, title: "Digestive Tract Environment", text: "The biggest issue with oral subunit vaccines is that the protein needs to be absorbed into the bloodstream to induce an immune response, and some of it is lost in the process while going through the digestive tract. As mentioned above, due to the gastrointestinal tract’s severe environment, a significant amount of the vaccine is lost before reaching the target environment." },
 +
{ length: 0.21, title: "Delivery Route", text: "Therefore, an oral vaccine administration route can be used. In this way, the bioactive substances are given together with the fish food. All things considered, we concluded that <b>subunit vaccination with GldJ and VHSV G proteins</b> should be administered orally as it is considered relatively non-invasive. (info box Why oral?)" },
 +
{ length: 0.16, title: "Alginate Lysis", text: "Alginates can only be enzymatically depolymerised by <b>alginate lyases (algLs)</b>. Lyases catalyse β-elimination reaction of glycoside bonds to yield a 4,5-unsaturated sugar, 4-deoxy-L-erythro-hex-4-enopyranosyluronic acid at the non-reducing end<sup>20</sup>. Some bacteria can produce algLs and in that way can degrade alginate into oligosaccharides or monosaccharides. Some examples of such bacteria are <i>Bacillus circulans, Azotobacter vinelandii, Klebsiella aerogenes, K. pneumonia, Pseudomonas maltophilia, P. putida, and P. aeruginosa</i><sup>21</sup>. (Info box How was it tested?)" },
 +
{ length: 0.07, title: "Decomposition of Alginate Envelope", text: "<b>Calcium alginate envelopes</b> can only be decomposed in the midgut - the exact place where <i>Pseudomonas</i> bacteria live<sup>22</sup>. In addition, mineral and biomolecule absorption is most intensive in the fish midgut. It is an ideal environment for our protein to be stripped out of calcium alginate beads and pass through the gut wall barrier into the bloodstream. Immune cells would then start producing antibodies against this specific pathogen protein, forming the protective immunity." },
 +
{ length: 0.03, title: "Validation", text: "To test that alginate beads can protect from environmental damage and fish digestive system, we continued to examine the beads in vitro. With more time the immunogenic properties of the proteins could be tested in vivo by feeding the fish with oral subunit vaccines in a two step immunization program and challenging them with a bath containing pathogens of interest." },
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Revision as of 10:46, 21 October 2020
DETECTION
Detection
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

Since we wanted to differentiate between Flavobacterium species, instead of antibody-antigen interaction, our detection test is based on nucleic acid hybridization. We found out that for identification purposes, 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 marker genes, which allows us to identify an exact bacteria species. 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 highly specific, we made a multiple sequence alignment with 16S rRNA genes from other species within the same genus using Clustal Omega tool (1. 2. 4.). Unique target sequences for F. columnare and F. psychrophilum were selected based on the absence of matching alignments between sequences.

Picture 1

Also we found that rpoC gene can be used as marker from F. psychrophilum. We decided to create probes for this gene sequence as well.

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.

Helimerase
WZB1WZA2TteUvrDBstPolStrepII Tag10xHis TagMBP TagL1L1
Helimerase

However, regarding the WHO guidelines for point-of-care testing, our detection tool should be not only sensitive, specific and user-friendly, but also it should be affordable for target customers1. Keeping in mind that HDA amplification, performed using a commercial kit, is still too expensive, we have decided to search for new alternatives to reduce the cost of the test as much as possible.

Our solution to this problem - helimerase. This protein complex contains two enzymes – Thermoanaerobacter tengcongensis UvrD helicase (TteUvrD) and Bacillus stearothermophilus DNA polymerase I large fragment (BstPol). TteUvrD is fused with one part of coiled-coil structure WinZip-A2 (WZA2) via linker L1 and possesses a maltose-binding protein (MBP) and 10xHis Tag in the N terminal end. BstPol is fused with the second coiled-coil part WinZip-B1 (WZB1) through linker L1 and possesses StrepII tag in the N terminal end2. It was presumed that this non-covalent fusion strategy through coiled-coil structures should improve HDA reaction by letting to amplify longer DNA fragments as well as it allows to perform amplification reaction without using additional proteins such as MutL or others SSB proteins, which are used in this type of reactions2,3.

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.

TREATMENT
Treatment

After detection of an exact flavobacterium species, farmers need to start an exact treatment process as soon as possible. Currently, fish infected with flavobacterial diseases are treated with different types of antibiotics13. Scientific data shows14 that the most abundant antibiotics used for salmon cultivation was quinolone, which consumption (by mass) in 2007 reached 821,997 tonnes. That is only in Norway! Other commonly used antibiotics in farms are oxolinic acid and florfenicol, which consumption reached 681 kg in 2008 and 166 kg in 2010 respectively. This enormous usage of wide variety antibiotics forces the evolving of antibiotic-resistant bacterial fish pathogens. These numbers raise huge concerns and questions on how we can reduce the usage of antibiotics?

Scientific data already shows that some F. psychrophilum isolates already have reduced susceptibility to quinolones, oxolinic acid, and enrofloxacin1. To reduce the amount of antibiotics used in aquaculture farms, our second goal is the development of two exogenous fish infection treatment systems, which are based on quorum sensing mechanisms.

Flavobacterial diseases are caused when Flavobacterium forms the same biofilm on fish fins or gills15. Our target bacteria - Flavobacterium uses signaling molecules for cell-cell communication. Quorum sensing is a bacterial communication process that leads to the regulation of genes and response to changes2–4. The quorum sensing has two distinguished systems - AHL and AI-2 for Gram-negative bacteria for now4–6. We used the circumstances in our advantage: build a genetic circuit that is enhanced by quorum sensing signaling molecules.

Choose treatment system
Endolysin & Exolysin System
Toxin & Antitoxin System
Endolysin & Exolysin System
Toxin & Antitoxin System
PREVENTION
Prevention

Usually, in order to prevent fish infections, vaccines are injected intraperitoneally. Because of manual handling, which is unavoidable in such a type of vaccination, fish experience a lot of stress and it paradoxically weakens their immune system. With the aim to avoid such consequences, we have set a third goal - to create a prevention system based on subunit vaccines against bacterial and viral fish infections.

Subunit vaccines
References
Big Mega Super Duper List of References

R. Meškys et al. iGEM Vilnius-Lithuania 2020