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| − | font-size:15pt !important;
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| | text-align:justify; | | text-align:justify; |
| | color:#F3F7F7; | | color:#F3F7F7; |
| − | text-indent:2em;
| + | font-weight: bold !important; |
| − | padding-top:15px;
| + | padding-top:30px; |
| − | padding-bottom:15px;
| + | padding-bottom:10px |
| − | }
| + | |
| − | .reference-content{
| + | |
| − | font-size:13pt !important; | + | |
| − | text-align:justify;
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| − | color:#F3F7F7;
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| − | padding-top:15px; | + | |
| − | padding-bottom:15px; | + | |
| | } | | } |
| | + | |
| | .content-image{ | | .content-image{ |
| | color:#F3F7F7 !important; | | color:#F3F7F7 !important; |
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| | } | | } |
| | .quote{ | | .quote{ |
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| | | | |
| | | | |
| | + | /*image*/ |
| | + | .web{ |
| | + | position:fixed !important; |
| | + | left:0%; |
| | + | bottom:0%; |
| | + | display:block; |
| | + | margin: auto; |
| | + | } |
| | + | .right{ |
| | + | position:fixed !important; |
| | + | bottom:-15px; |
| | + | right:0vw; |
| | + | margin: auto; |
| | + | } |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | .img-position{ |
| | + | position:absolute; |
| | + | z-index:-1; |
| | + | } |
| | + | .box{ |
| | + | width:auto; |
| | + | padding-top:20px; |
| | + | padding-bottom:20px; |
| | + | } |
| | + | .picture{ |
| | + | position:relative; |
| | + | left:7.5vw; |
| | + | width:80%; |
| | + | padding-bottom:5px; |
| | + | } |
| | + | |
| | + | .picture-1{ |
| | + | position:relative; |
| | + | left:1vw; |
| | + | width:100%; |
| | + | padding-bottom:10px; |
| | + | } |
| | + | |
| | + | /*board game video*/ |
| | + | .animate-box{ |
| | + | margin-left:9vw !important; |
| | + | width:50%; |
| | + | } |
| | </style> | | </style> |
| | </head> | | </head> |
| | <body> | | <body> |
| − | <!--圖片-->
| |
| − | <div class="img-container">
| |
| − | <img src="https://static.igem.org/mediawiki/2016/0/01/NCTU_MODELING_BIG_PICTURE.png" class="main-img" width="100%">
| |
| − | </div>
| |
| − | <div>
| |
| − | <img src='https://static.igem.org/mediawiki/2016/f/f8/Web.png' class="web img-position" height="200px" id="web">
| |
| − | </div>
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| − | <div>
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| − | <img src="https://static.igem.org/mediawiki/2016/6/65/Right_%281%29.png" class="right img-position" id="right" height="600px">
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| − | </div>
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| | </div> | | </div> |
| | | | |
| − | <div class="node" id="node1"> | + | |
| − | <div class="topic"><p class="text_color"><a href="#title1" style="text-decoration:none;color:#F3F7F7;">Software</p></div> | + | |
| | + | <div class="node"> |
| | + | <div class="topic"><p class="text_color">Cloning Result</p></div> |
| | <div class="active-circle"> | | <div class="active-circle"> |
| | <div class="splash"></div> | | <div class="splash"></div> |
| − | <div class="cover"> | + | <div class="cover"> |
| | + | |
| | + | |
| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
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| − | <div class="node " id="node2"> | + | |
| − | <div class="topic"><p class="text_color"><a href="#title2" style="text-decoration:none;color:#F3F7F7;">Pest Prediction System</a></p></div> | + | <div class="node"> |
| | + | <div class="topic"><p class="text_color">Expression Results</p></div> |
| | <div class="active-circle"> | | <div class="active-circle"> |
| | <div class="splash"></div> | | <div class="splash"></div> |
| | <div class="cover"> | | <div class="cover"> |
| | + | </div> |
| | + | </div> |
| | + | <div class="inactive-circle"> |
| | + | <div class="small-cover"> |
| | + | </div> |
| | + | </div> |
| | + | </div> |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | <div class="node"> |
| | + | <div class="topic"><p class="text_color">Feeding Assays</p></div> |
| | + | <div class="active-circle"> |
| | + | <div class="splash"></div> |
| | + | <div class="cover"> |
| | </div> | | </div> |
| | </div> | | </div> |
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| | </div> | | </div> |
| | | | |
| − | <div class="node" id="node3"> | + | <div class="node"> |
| − | <div class="topic"><p class="text_color"><a href="#title3" style="text-decoration:none;color:#F3F7F7;">Degradation Rate</a></p></div> | + | <div class="topic"><p class="text_color">Insect Test Result</p></div> |
| | <div class="active-circle"> | | <div class="active-circle"> |
| | <div class="splash"></div> | | <div class="splash"></div> |
| | <div class="cover"> | | <div class="cover"> |
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| | </div> | | </div> |
| | </div> | | </div> |
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| | </nav> | | </nav> |
| | | | |
| − | <!--------------------wiki content--------------------------> | + | |
| | + | |
| | + | <!--圖片--> |
| | + | <div class="img-container"> |
| | + | <img src="https://static.igem.org/mediawiki/2016/f/f0/NCTU_RESULTS_BIG_PICTURE.png" class="main-img" width="100%"> |
| | + | </div> |
| | + | <div> |
| | + | <img src='https://static.igem.org/mediawiki/2016/f/f8/Web.png' class="web img-position" height="200px" id="web"> |
| | + | </div> |
| | + | <div> |
| | + | <img src="https://static.igem.org/mediawiki/2016/6/65/Right_%281%29.png" class="right img-position" id="right" height="600px"> |
| | + | </div> |
| | + | |
| | + | |
| | + | |
| | + | <!---------------------wiki content---------------------> |
| | + | |
| | + | |
| | <section class="content_container"> | | <section class="content_container"> |
| | + | |
| | + | <!----------cloning results-----------> |
| | <div> | | <div> |
| | + | <p class="title">Cloning Result</p> |
| | + | <p class="content-1">Gene Constructions of Pantide—PCR</p> |
| | + | <p class="content">To ensure the genes are cloned into <i>E. coli</i> BL21 Rosetta-gami strain, we did electrophoresis of PCR products for checking insert gene’s length after amplification with PCR. Each of the BioBrick construction includes the conservative promoter, RBS, linker, and His-tag. Here are the theoretical length of seven BioBricks we construct.(Table 1)</p> |
| | | | |
| − | <div id="modelingContainer"> | + | <div> |
| − | <p class="title" id="title1">Software—Toxin selection</p> | + | <img src="https://static.igem.org/mediawiki/2016/6/6b/NCTU_table1.png" class="picture" style="left:9vw;width:60%;"> |
| | + | </div> |
| | | | |
| − | <div id="modelingContent"> | + | <p class="content">After amplification with PCR, the PCE products of Hv1a, Sf1a, and OAIP have a length of around 500bp； and Hv1a-Lectin, Sf1a-Lectin, and OAIP-Lectin has a length of 750bp~1000bp； and Hv1a-Lectin with Gs linker has a length of 893bp. (Figure 1a, 1b, 1c, 1d, 1e, 1f, 1g)</p> |
| − |
| + | |
| − | <!--modeling part-->
| + | |
| − | <!--1.-->
| + | |
| − | <div class="modelingPart">
| + | |
| − | <h2 class="content-1" id="titleA" style="color:#33FFCC">I. Purpose</h2>
| + | |
| − | <div class="modelingPartContent" id="partA">
| + | |
| | | | |
| − | <p class="content">To prove the concept of Pantide, we wanted to select three existing distinct spider toxin peptides with probable oral toxicity against the testee－Spodoptera litura(Tobacco cutworms). For the actual application of Pantide, we needed some more knowledge base of peptides which have different molecular targets to promote Pantide applying to other orders of insects, and a different toxic mechanism to regularly alternate so as to avoid drug resistance.</p>
| |
| − | <p class="content">To date, about 1500 toxin peptides from 97 spider species have been studied, though the number of spider toxin peptides is conservatively estimated up to 10 million. <sup>[1]</sup> So, our purpose is to establish a database collecting the information of those peptides, such as molecular target, taxon, toxicity, sequence. According to the database, if we first choose a target insect, then we can easily find out groups of suitable peptides used as Pantide. Therefore, we also need to create a method to select peptides from the database.</p>
| |
| | | | |
| − | </div>
| + | <div> |
| − | </div>
| + | <img src="7張店永" class="picture"> |
| − |
| + | <p class="content -image" style=" text- align: justify !important;"> Figure 1. Polymerase chain reaction (PCR) amplification analysis of electrophoresis of seven PCR products with Marker labeled length on the left hand (a)Hv1a (BBa_K1974011, 495 base pairs). (b)Sf1a (BBa_K1974012, 522 base pairs). (c)OAIP (BBa_K1974013, 489 base pairs). (d)Hv1a- Lectin (BBa_K1974021, 819 base pairs). (e)Sf1a- Lectin (BBa_K1974022, 846 base pairs). (f)OAIP- Lectin (BBa_K1974023, 813 base pairs). ( g) Hv1a-Lectin with Gs linker(BBa_K1974033, 883 base pairs).</p> |
| − |
| + | </div> |
| − | <!--2.-->
| + | |
| − | <div class="modelingPart">
| + | |
| − | <h2 class="content-1" id="titleB" style="color:#44FCCE">II. Method</h2>
| + | |
| − | <div class="modelingPartContent" id="partB">
| + | |
| − | <p class="content" >The method of toxin selection can be separated into three part: crawler, filter, and selection.</p> | + | |
| − | <ul style="list-style-image:none;list-style-type:decimal;">
| + | |
| − | <li class="list">Toxin Collection—we planned to collect information of toxin peptides to establish our own database for Pantide from protein databases and some research results like taxon and toxicity from published papers. </li>
| + | |
| − | <li class="list">Toxin Filtering—based on background knowledge of toxin peptides, we set up some conditions to filter out those unsuitable to use as Pantide.</li>
| + | |
| − | <li class="list">Toxin Processing—we used online protein analytic tools to classify the remained peptides into groups by their similarity. Finally, we select out three distinct peptides from different groups to proof concept of Pantide.</li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − |
| + | |
| − |
| + | |
| − | <!--3.-->
| + | |
| − | <div class="modelingPart">
| + | |
| − |
| + | |
| − | <h2 class="content-1" id="titleC" style="color:#5BFCD4">III. Step 1: Crawler</h2>
| + | |
| − | <div class="modelingPartContent" id="partC">
| + | |
| − | <p class="content">In the beginning, we searched on UniProtKB/Swiss-Prot. It is a freely accessible database of protein sequence and functional information that is the manually annotated and reviewed section. (<a href="http://www.uniprot.org" style="color:#44E287;">http://www.uniprot.org/</a>) By searching the keyword “insecticidal NOT crystal” we wanted to find all the proteins that have insecticidal activity excluding those crystal proteins of Bacillus thuringiensis, and we got 216 proteins as results.</p>
| + | |
| − | <p class="content">Using the result, we established our Pantide database by crawling 11 entries of the protein information from UniProt. The entries are as follows.</p>
| + | |
| | | | |
| − | <ul style="list-style-image:none;list-style-type:disc;"> | + | <div> |
| − | <li class="list">The name of the protein</li>
| + | <img src="GS" class="picture"> |
| − | <li class="list">The description of protein function</li>
| + | </div> |
| − | <li class="list">The organisms/source of the protein sequence</li>
| + | |
| − | <li class="list">The length of amino acids</li>
| + | |
| − | <li class="list">The number of disulfides bonds</li>
| + | |
| − | <li class="list">Propeptide & signal peptide—If the proteins have an N-terminal signal peptide and propeptide, a part of protein will be cleaved during maturation or activation.</li>
| + | |
| − | <li class="list">Uniprot entry & Arachnoserver id—the accession number of protein in UniProtKB and ArachnoServer*.</li>
| + | |
| − | </ul>
| + | |
| − | <p class="content-2">*ArachnoServer is a manually curated database for protein toxins derived from spider venom.(<a href="http://www.arachnoserver.org/" style="color:#44E287;">http://www.arachnoserver.org/</a>).</p>
| + | |
| − | <p class="content">We also crawled other seven entries of protein toxicity recorded by Arachnoserver—molecular target, taxon, ED50, LD50, PD50, qualitative information, protein sequence from Arachnoserver. The term, Molecular target, is the effect site of toxin peptides, such as voltage-gated ion channels, GABA receptors and so on. Taxon, ED50, LD50, PD50, and the qualitative information are the toxicity against taxon that had been tested by experiments. The protein sequence from two databases is entirely the same.</p>
| + | |
| − | <p class="content">We utilized BeautifulSoup 4.4.0, sqlite3 and gevent modules in Python 3.5 to develop our crawler. Moreover, we have submitted the code to GitHub.<br>(Link:<a href="https://github.com/chengchingwen/iGEM/blob/master/crawler.py" style="color:#44E287;">https://github.com/chengchingwen/iGEM/blob/master/crawler.py</a>)</p>
| + | |
| | | | |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − |
| |
| − | <!--4.-->
| |
| − | <div class="modelingPart" >
| |
| − | <h2 class="content-1" id="titleD" style="color:#6EFFDB">IV. Step 2: Filter</h2>
| |
| − | <div class="modelingPartContent" id="partD">
| |
| − | <p class="content">After crawling the data, we used DB Browser for SQLite software to browse and used SQL to process our Pantide database. We tried to build a filter to find out peptides suitable to use as Pantide.</p>
| |
| − | <p class="content">According to the previous articles, we knew that around 90% of spider venom toxin peptides contain ICK structure which is the most important domain that reacts with the voltage-gated ion channels of insects and some other receptors specifically. <sup>[2]</sup></p>
| |
| − | <p class="content">Therefore, to find these spider venom toxin peptides from Pantide database, we could start from searching for ICK structure, whose mass is among 1-10 kDa containing at least three disulfide bonds. <sup>[2]</sup> So we set a filter with three conditions.</p>
| |
| | | | |
| | | | |
| − | <ul style="list-style-image:none;list-style-type:decimal;">
| |
| − | <li class="list">The organism we choose must be spiders or tarantulas.</li>
| |
| − | <li class="list">The length of the a.a. sequences are between 27 and 271 base pairs (1 kDa of protein has averagely nine amino acids, encoded by 27 base pairs)</li>
| |
| − | <li class="list">The number of disulfide bonds is greater or equal to 3.
| |
| − | After filtering with the three conditions, 113 peptides remained. Next, we set another filter to find out insecticidal peptides.</li>
| |
| − | <li class="list">Molecular target contains “invertebrate,” but we also remain peptides without data.
| |
| − | <br> The reason why we keep the peptides without data was that they have the probability to be effective. In this stage, we got 63 candidates.</li>
| |
| − | <p class="content-2">For efficacy experiment of Pantide, we choose our testee－Spodoptera litura as target insect. While there are 14 kinds of distinct Taxon in our database, including 4 Lepidoptera genus. Thus, we also set the other filter to find out peptides against Lepidoptera:</p>
| |
| − | <li class="list">Taxon contains at least one of <i>Spodoptera litura</i>, <i>Heliothis virescens</i>, <i>Manduca sexta</i> and <i>Spodoptera exigua</i>, but we also remain peptides without data
| |
| − | <br> On the other hand, because we designed to produce Pantide by <i>E.coli</i>, that is difficult to express proteins containing disulfide bonds. We had chosen <i>E.coli</i> Rosetta-gami strain for enhanced disulfide bond formation, but to express a protein with more than four disulfide bonds is still a heavy load. So we finally filtered out those peptides containing too much disulfide bonds.</li>
| |
| − | <li class="list">The number of disulfide bonds is less than or equal to four.
| |
| − | <br> The result was that we got 46 peptides which have the possibility to use as Pantide in proof concept experiment, and all of them is targeted to insects’ voltage-gated ion channels (excluding NULL).</li>
| |
| − | </ul>
| |
| | | | |
| − | </div>
| |
| − | </div>
| |
| − |
| |
| − | <div class="modelingPart">
| |
| − | <h2 class="content-1" id="titleE" style="color:#80FFDF">V. Step 3: Selection</h2>
| |
| − | <div class="modelingPartContent" id="partE">
| |
| − | <p class="content">In this step, we tried to find three peptides that have different molecular target or mechanism from filtering result to do the test experiment. The method we used was to classify the remained peptides into groups by their structure similarity.</p>
| |
| − | <p class="content">We used online analytic tools on NCBI to process those peptides.</p>
| |
| − | <p class="content">We started with using Protein BLAST (Basic Local Alignment Search Tool) to search from the whole protein database for the similar query protein sequences related to all the 46 peptides and put those related peptides into groups.</p>
| |
| − | <p class="content">The next was using COBALT (Constraint-based Multiple Alignment Tool) to align the sequence between groups to find out whether or not the two groups have the similar structure while they were not got together on the last step because of side chains and other factors. At last, we separated 46 peptides into four groups, containing 27, 12, 3, 2 peptides, and two alone.</p>
| |
| − | <p class="content">Then we chose the three larger groups and used Conserved Domains Search, and found out that they belonged to the three conserved protein domain family. There are Omega-toxin Superfamily (cl05707), Toxin_28 Superfamily (cl06928) and Toxin_20 Superfamily (cl06915). The strings in brackets are unique ID of superfamilies in the conserved protein domain family database. Finally, we selected the representative peptides from each superfamily and got these three peptides, ω-hexatoxin-Hv1a, μ-segestritoxin-Sf1a and Orally active insecticidal peptide (OAIP).</p>
| |
| − | </div>
| |
| | </div> | | </div> |
| | + | <div> |
| | + | <p class="title">Expression Result</p> |
| | + | <p class="content-1">Expression of Pantide</p> |
| | + | <p class="content">Our goal is to express Pantide successfully. We used <i>E. coli</i> BL21 rosetta-gami strain to cultivate genes. Afterward, we sonicated <i>E. coli</i> and then ran SDS-PAGE to make sure that Pantide fell into the correct ranges. There are seven kinds of BioBricks, and the mass of them are showed below.(Table 2)</p> |
| | | | |
| | + | <div> |
| | + | <img src="https://static.igem.org/mediawiki/2016/3/3d/NCTU_Table2.png" class="picture" style="left:9vw;width:60%;"> |
| | + | </div> |
| | | | |
| − | <div class="modelingPart">
| + | <p class="content">The gel of SDS-PAGE result is correct, and here is our result.(Figure 2a, 2b, 2c)</p> |
| − | <h2 class="content-1" id="titleF" style="color:#88FCDF">VI. Future</h2>
| + | |
| − | <div class="modelingPartContent" id="partF">
| + | |
| − | <p class="content"> To promote the applicability of Pantide, we still need to extend our database. The next step is to integrate with other toxin peptide databases, such as scorpions or cone snails, collect more peptides’ information from research results, and even combine with bioinformatics to build a new scoring system, and search for new potential peptides.</p > | + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| | | | |
| − | <div class=" modelingPart"> | + | <div> |
| − | <h2 class="content-1" id="titleG" style="color:#9CFFE6">Reference</h2>
| + | <img src="" class="picture"> |
| − | <div class="modelingPartContent" id="partG">
| + | <p class="content-image"style="text-align:justify !important;">Figure 2. SDS-PAGE result of the seven Pantide-expressed sonicated products compared with the unexpressed sonicated one and with Marker labeled length on the left hand (a)well 1: unexpressed sonicated <i>E. coli</i>, well 2: Hv1a (5.3 kDa), well 3: Sf1a (6.2 kDa), well 4: OAIP (5.3 kDa). (b)well 1: unexpressed sonicated <i>E. coli</i>, well 2: Hv1a-Lectin (17.1 kDa), well 3: Sf1a-Lectin (18.1 kDa), well 4: OAIP-Lectin (17.2 kDa). (c)well 1: unexpressed sonicated <i>E. coli</i>, well 2: Hv1a-Lectin with Gs linker ( 18.2kDa). </p> |
| − | <p class="reference-content">[1] King, G.F.; Gentz, M.C.; Escoubas, P.; Nicholson, G.M. A rational nomenclature for naming peptide toxins from spiders and other venomous animals. Toxicon 2008, 52, 264–276.</p>
| + | </div> |
| − | <p class="reference-content">[2] Monique J. Windley, Volker Herzig, Sławomir A. Dziemborowicz, Margaret C. Hardy, Glenn F. King and Graham M. Nicholson (2012). Spider-Venom Peptides as Bioinsecticides. Toxins, 4, 191-227.</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| | + | |
| | + | |
| | + | |
| | + | <p class="content-1">Purification of Pantide</p> |
| | + | <p class="content">After sonicated <i>E. coli</i>, we further purified Pantide to check whether it is well-expressed. The purified products’ length can be seen in the table below.(Table 3)</p> |
| | + | |
| | + | <div> |
| | + | <img src="https://static.igem.org/mediawiki/2016/8/8c/NCTU_table3.png" class="picture" style="left:9vw;width:60%;"> |
| | </div> | | </div> |
| | | | |
| | + | <p class="content">The gel of SDS-PAGE result of Hv1a, Sf1a, OAIP have a length of 5kDa~7 kDa；and Hv1a-Lectin, Sf1a-Lectin, OAIP-Lectin, and Hv1a-Gs linker-Lectin have a length of 17kDa~19kDa.(Figure 3a, 3b, 3c, 3d, 3e, 3f, 3g) As a result, the constructions of BioBrick were correct.</p> |
| | | | |
| − | <!--------------------------------The Pest Prediction System ------------------------------------------->
| |
| | <div> | | <div> |
| − | <div id="modelingContainer"> | + | <img src="" class="picture"> |
| − | <p class="title" id="title2">The Pest Prediction System</p> | + | <p class=" content-image" style=" text-align:justify !important;"> Figure 3. SDS-PAGE result of the seven purified Pantide products compared with the unpurified one, we called it Before, and with Marker labeled length on the left hand. well 1: unpurified solution(Before), well 2~4: Wash 1~Wash 3, well 5~14: Elution 1,~Elution 10, well 15: NaCl, well 16: ddH2O (a) Hv1a (5.3 kDa) (b) Sf1a (6.2 kDa) (c) OAIP (5.3 kDa) (d) Hv1a-Lectin (17.1 kDa) (e) Sf1a-Lectin (18.1 kDa) (f) OAIP-Lectin (17.2 kDa) (g) Hv1a-Gs linker-Lectin (18.2 kDa)</p > |
| | + | </div> |
| | | | |
| − | <!--1-->
| |
| − | <div class="modelingPart">
| |
| − | <h2 class="content-1" id="titleH" style="color:#FFFF82">I. The Factors of Prediction</h2>
| |
| − | <div class="modelingPartContent" id="partH">
| |
| − | <p class="content">We built a prediction model for our device to support our spraying system. The model will predict the number of the pest, so we can know when our farm will be in the pest threat in the future. Thus we can open our spraying system spraying PANTIDE to protect our farmland. To know the number of pests in the future, we used seven weather data in the past 20 days including air pressure, the highest temperature, the lowest temperature, average temperature, humidity, precipitation, wind velocity, and also the accumulated number of bugs from the two periods including twenty to ten days ago and ten to one days ago. Because Pantide only influences on the larvae, we cannot easily predict the number of moths tomorrow or two or three days later. We needed to know the number of larvae in the future.</p>
| |
| − | </div>
| |
| − | </div>
| |
| | | | |
| | | | |
| − | <!--2-->
| + | </div> |
| − | <div class="modelingPart">
| + | |
| − | <h2 class="content-1" id="titleI" style="color:#FFFF82">II. The Life History of Moth</h2>
| + | |
| − | <div class="modelingPartContent" id="partI">
| + | |
| − | <p class="content">After understanding the life history of the moths from Dr. Huang in Taiwan Agriculture Research Institute, we can know that the time of pupa becoming moth ranges from six days to 14 days. So the moths we caught in 20 days must be the larvae in our farm right now. Therefore, we utilized the data of the accumulated number of moth in the next 20 days as the target of our prediction system, and we can use the output to know the time that requires spraying Pantide for prevention.</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| − | <!--3--> | + | <!------------Feeding Assay--------------> |
| − | <div class="modelingPart">
| + | |
| − | <h2 class="content-1" id="titleJ" style="color:#FFFF82">III.The Software Design of Prediction Model</h2>
| + | |
| − | <div class="modelingPartContent" id="partJ">
| + | |
| − | <p class="content">The method we used was called neural network, one of the popular machine learning solutions, or another well-known name called deep learning. The model we used was a combination of two kinds of neural network, Recurrent Neural Network (RNN) and Artificial Neural Network (ANN). Becuase we got seven features of 20 days, equals 140 features in total, we cannot simply put it into ANN to train the model. Therefore, we used RNN which was good for this kind of time series data to compress the 140 features into seven compressed data and got the seven feature and the other two feature which we have previously mentioned. Therefore, we only had nine features in total and then put them into the ANN as the input to get the final prediction. Then, it will compute the errors between the actual answer and the output and use gradient descent and backpropagation to modify the weight in each network each neuron. After a bunch of train steps, we got our model with about 80% of accuracy.</p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| | | | |
| − | <!--4--->
| |
| | | | |
| − | <div class=" modelingPart"> | + | |
| − | <h2 class="content-1" id="titleK" style="color:#FFFF82">IV. The Quality of the Model</h2>
| + | |
| − | <div class="modelingPartContent" id="partK">
| + | <p class="title">Feeding Assays</p> |
| − | <p class="content"> The prediction model is also a part of our device system. After using the device to collect data in a farm, we can not only predict the number of moth with the model we already trained but also retrain our model with the data we collect in that farm. So, now we can modify the model according to each farm to get a specific model for it.</p> | + | <p class="content">To understand the insect test in an easy way, the testee selection, and experiment design would be briefly described. The results consist of three parts-feeding assay pre-test, the fusion protein improvement test, and preference test.<br> |
| − | <p class="content"> The programming language we use is python3. 5, and the deep learning framework is the one developed by Google called tendorflow. (See on<a href="https:// github. com/ chengchingwen/ moth_prediction"style="color:#44E287;"> GitHub</a> )</p> | + | **Note: The word “Pantide” in the following paragraph refers to the collection of all the toxin design in our project. |
| − | </div>
| + | </p> |
| − | </div> | + | |
| | + | |
| | + | <ul style="list-style-image:none;list-style-type:disc;"> |
| | + | <li class="list">Testee Selection</p> |
| | + | <p class="content"> In the test of Pantide toxicity, we chose tobacco cutworm as the testee for the insect appetite tests because all the Pantide targets lepidopterans. We hope that we can observe the difference of the three toxins <i>in vivo</i>. Tobacco cutworms, which are one of the major pests in vegetable farms, impact on Crucifers in farmland around the world. In the serious pest damage, the density of tobacco cutworms is approximately up to two hundred million per hectare. Therefore, we used five tobacco cutworms to mimic the much more severe situation in our insect experiment. </p> |
| | + | |
| | + | <li class="list">Experiment Design</p> |
| | + | <p class="content"> All the experiments were to check the functions of Pantide for leaf protection, so the observation of the results would focus on the remained area of the leaf disks.<br> |
| | + | In the following experiments, we used <i>E. coli</i> BL21 Rosetta-Gami strain to produce Pantide. We aimed to evaluate the remained leaf area applying Pantide. In this insect test, Pantide was diluted into three concentrations to observe the trends of dose response and to confirm the quality of PANTIDE. All the following experiments have three dilution ratios including 1/125 x, 1/25x and 1/5x. Three repeated tests were done in each experiment. See the procedure of insect experiment on <a href="https:// 2016. igem.org/ Team:NCTU_Formosa/ Protocol" style="color:#44E287;"> Protocols</a> . |
| | + | </p> |
| | + | |
| | + | <li class="list">Hypothesis</p> |
| | + | <p class="content">According to the mechanism of Pantide, Pantide is supposed to perform its toxicity in the nervous system of the larvae. We might see larvae paralyze and finally die.</p> |
| | </div> | | </div> |
| | + | |
| | + | |
| | + | |
| | | | |
| | <div> | | <div> |
| − | <div id="modelingContainer"> | + | <p class="title">Insect Test Result</p> |
| − | <p class="title" id="title3">Degradation Rate</p> | + | <p class=" content-1" >Feeding Assay Pre-test</p> |
| | + | <p class="content"> To know the qualitative toxicity effect of Pantide, we prepared the samples with the sonicated LB solution lysate containing Pantide-expressed E. coli Rosetta-Gami strain and diluted it with the three concentration. This experiment is the pre-test that shows us whether the amount of Pantide is sufficient enough to perform the toxicity against the larvae. We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply Bacillus thuringiensis on the leaf disks, which is nowadays the most widely-used bioinsecticide, and the negative control group in the test was DD water. We preserved all the result of the remained leaves sealing with the glass paper and calculated the percentage of the remained area on the leaves. The collected data were analyzed by t–test and calculated with the p-value. The p-value is a function that measures how extreme the observation is. The widespread use of “statistical significance” (interpreted as “p ≤ 0.05”) is a license for making a claim of a scientific finding that leads to considerable distortion of the scientific process. In the picture, it shows as a star. The more the stars are, the more significant difference is. Here are the feeding assay results.</p> |
| | | | |
| | + | <div> |
| | + | <p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of Hv1a and Hv1a-lectin</p> |
| | + | <img src="https://static.igem.org/mediawiki/2016/8/81/NCTU_result_H.jpg" class="picture"> |
| | + | <p class="content-image" style="text-align:center !important;">Figur1. Remained leaf disks in the pre-test with the Hv1a and Hv1a-lectin.</p> |
| | + | </div> |
| | | | |
| − | <!--1-->
| + | <div style="margin-top:20px;"> |
| − | <div class=" modelingPart"> | + | <img src="https://static.igem.org/mediawiki/2016/f/fb/NCTU_result_dos_H.jpg" class="picture"> |
| − | <h2 class="content-1" id="titleL" style="color:#FFFF82">I. Summary</h2>
| + | <p class="content -image" style=" text-align:center !important;"> Figure2. The dose response analysis of Hv1a/Hv1a-lectin.</p> |
| − | <div class="modelingPartContent" id="partL">
| + | |
| − | <p class="content" >The aim of this modeling was to predict and simulate the degradation rate of Pantide. Practically, the results would be integrated into our device to promote automatic control system. Once Pantide degraded below the effective level, it will spray the solution to replenish.</p> | + | |
| − | <p class="content">After discussion and experimental verification, we show that UV radiolytic oxidation contributes to the degradation of Pantide. We tried to build a model for simulating the relationship between applied UV light and degradation rate and then combined with UV intensity sensor to calculate the period of spraying Pantide.</p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | | | |
| | + | <div style="margin-top:20px;"> |
| | + | <img src="https://static.igem.org/mediawiki/2016/b/be/Fig3-HHL.jpeg" class="picture"> |
| | + | <p class="content-image" style="text-align:center !important;">Figure3. The T-test analysis in different dose of Hv1a/Hv1a-lectin.</p> |
| | + | </div> |
| | | | |
| − | <!--2-->
| + | |
| − | <div class="modelingPart"> | + | |
| − | <h2 class="content-1" id="titleM" style="color:#FFFF82">II. Pantide degradation process</h2>
| + | |
| − | <div class="modelingPartContent" id="partM">
| + | |
| − | <p class="content">For the proteins used as Pantide, the inhibitor cystine knot (ICK) is significant to their function. Pantide with several disulfide bonds is often more stable in solution. If “native protein” is denatured, it loses disulfide bonds and becomes a less stable form, normally called “linear protein,” which is easily degradable.</p>
| + | <p class="content" style="padding-top:40px !important;padding-bottom:30px; !important">Figure1 shows the pictures of the remained leaf disks after twelve hours of feeding assays. After we had done the feeding assays on tobacco cutworms with the three dilution ratios, we measured the area with the software imageJ. Figure2 shows the percentage of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. Figure3 shows the p-value with the star attached to indicate P < 0.05 and two stars to indicate P < 0.01. The p-value of the T-test can be used to determine if two sets of data are significantly different from each other. The smaller the p-value is, the more significant the differences are between the two groups. The observed phenomenon can be analyzed below.</p> |
| − | <p class="content">Recent research shows that the spider toxin proteins containing ICK structure, for example, ω-hexatoxin-Hv1a (Hv1a), have high stability against temperature, pH, solvents and protease. In contrast, when Hv1a is denatured to linear form, it loses its stability and then degrades rapidly. [1]</p>
| + | |
| − | <p class="content">There are many possible processes of Pantide degradation we discussed below. (Figure 1) Pantide may have a chance to be reduced to a linear form by reductants or reductases. For both native form and linear form proteins, it may suffer hydrolysis and proteolysis, resulting in denaturing or amino acid cleavage. Also, UV light of sun also leads to Pantide degradation. Though the energy of UV light may not be not enough to break the covalent bonds efficiently, proteins still could undergo radiolytic oxidation.</p>
| + | |
| − | <div>
| + | |
| − | <img src=”” class=”picture”>
| + | <ul style="list-style-image:none;list-style-type:disc;"> |
| − | <p class=”Figure 1. Pantide degradation process”></p>
| + | |
| − | </div>
| + | <li class="list">With the dilution of Hv1a/Hv1a-lectin, the remained leaves area decreased, which indicates the dose response. The dose response is shown in figure2.</li> |
| − | <p class="content"> According to this degradation process, we attempted to build up our model. The degradation rate of Pantide is contributed by hydrolysis, proteolysis, UV radiolytic oxidation and the reduction to linear form. We considered that the rate Pantide transformed to linear form is a part of degradation rate because linear form protein loses its function and it is also likely to be degraded.</p> | + | |
| − | <p class=" content"> However, since the whole process is too complex to verify, we divided the experiment into three parts, hydrolysis test , proteolysis test and UV radiolytic oxidation test, and further summarized the results to conclude. The purpose is to find out the degradation rate of Pantide and to verify the less stable linear form protein has.</p> | + | <li class="list">Compared Hv1a with Hv1a-lectin, the remained leaves area in the Hv1a-lectin is higher than that of Hv1a, which means the repellent efficiency of Hv1a-lectin is higher than that of Hv1a. It shows in figure2.</li> |
| − | <p class="content"> On the other hand, because ICK structure domain mainly contributes the stability of these proteins; we could assume that the degradation processes of three target proteins are roughly the same, for the reason that, we chose Hv1a and Hv1a with lectin (Hv1a-lectin ) to demonstrate.</p > | + | <li class="list">To use the p-value as an indicator of statistical significance, we compared Hv1a/Hv1a-lectin with the negative control; it shows that the remained leaf disk area of Hv1a and Hv1a-lectin are significantly different from negative control, shown in figure3.</li> |
| − | </div>
| + | |
| | + | <p class="content">To sum up, the Hv1a and Hv1a-lectin work well in vivo. The effect can be roughly ranked as:<br>Hv1a-lectin > Hv1a > E. coli > Negative control</p> |
| | + | |
| | + | <p class="content">We got the similar result in the Sf1a/Sf1a-lectin and OAIP/OAIP-lectin.</p> |
| | + | |
| | + | |
| | + | |
| | + | <div> |
| | + | <p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of Sf1a and Sf1a-lectin</p> |
| | + | <img src="https://static.igem.org/mediawiki/2016/c/c4/NCTU_result_S.jpg" class="picture"> |
| | + | <p class="content -image" style="text-align:center !important;"> Figure4. Remained leaf disks in the pre-test with the Sf1a and Sf1a-lectin.</p> |
| | </div> | | </div> |
| | | | |
| | + | <div style="margin-top:20px;"> |
| | + | <img src="https://static.igem.org/mediawiki/2016/a/a7/NCTU_result_dos_S.jpg" class="picture"> |
| | + | <p class="content-image" style="text-align:center !important;">Figure5. The dose response analysis of Sf1a/Sf1a-lectin.</p> |
| | + | </div> |
| | | | |
| − | <!--3-->
| + | <div style="margin-top:20px;"> |
| − | <div class=" modelingPart"> | + | <img src="https://static.igem.org/mediawiki/2016/2/29/Fig6-S.jpeg" class="picture"> |
| − | <h2 class="content-1" id="titleN" style="color:#FFFF82">III. Hydrolysis test</h2>
| + | <p class="content- image" style=" text-align:center !important;"> Figure6. The T-test analysis in different dose of Sf1a/ Sf1a-lectin.</p> |
| − | <div class="modelingPartContent" id="partN">
| + | |
| − | <p class="content- 1" > i. Theory</p> | + | |
| − | <p class="content">Like the reverse direction of polypeptide formation, proteins can be hydrolyzed into their constituent amino acids. The mechanism undergoes an E2 elimination since a nucleotide attach to sp2 hybridized acyl carbon (amide). [2] Therefore, the reaction rate depends on the concentration of both nucleotides and the protein: </p>
| + | |
| − | <p class="content"></p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| − | <!--4-->
| + | |
| − | <div class=" modelingPart"> | + | |
| − | <h2 class="content-1" id="titleO" style="color:#FFFF82">IV. Proteolysis test</h2>
| + | <div style="margin-top:60px;"> |
| − | <div class="modelingPartContent" id="partO">
| + | <p style="text-align:center;padding-top:50px;font-size:22pt;color:#F3F7F7;padding-left:2px;">Feeding Assay Pre-test of OAIP and OAIP-lectin</p> |
| − | | + | <img src="https://static.igem.org/mediawiki/2016/a/a0/NCTU_result_O.jpg" class="picture"> |
| − | </div>
| + | <p class="content -image" style="text-align:center!important;"> Figure7. Remained leaf disks in the pre-test with the OAIP and OAIP-lectin.</p> |
| | </div> | | </div> |
| − | <!-5-->
| + | |
| − | <div class=" modelingPart"> | + | <div style="margin-top:20px;"> |
| − | <h2 class="content-1" id="titleP" style="color:#FFFF82">V. UV radiolytic oxidation test</h2>
| + | <img src="https://static.igem.org/mediawiki/2016/2/2f/NCTU_result_dos_O.jpg" class="picture"> |
| − | <div class="modelingPartContent" id="partP">
| + | <p class="content -image" style="text-align:center !important;"> Figure8. The dose response analysis of OAIP/OAIP-lectin.</p> |
| − | | + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | | | |
| − | <!--6--> | + | <div style="margin-top:20px;"> |
| − | <div class="modelingPart"> | + | <img src="https://static.igem.org/mediawiki/2016/0/03/Fig9-O.jpeg" class="picture"> |
| − | <h2 class="content-1" id="titleQ" style="color:#FFFF82">VI. Summarization</h2>
| + | <p class="content-image" style="text-align:center !important;">Figure9. The T-test analysis in different dose of OAIP/OAIP-lectin.</p> |
| − | <div class="modelingPartContent" id="partQ">
| + | |
| − | <p class="content"></p>
| + | |
| − | </div>
| + | |
| | </div> | | </div> |
| | | | |
| | | | |
| − | <div class="modelingPart"> | + | |
| − | <h2 class="content-1" id="titleR" style="color:#FFFF82">Reference</h2>
| + | |
| − | <div class="modelingPartContent" id="partR">
| + | |
| − | <p class=" reference-content"> [1] Volker Herzig and Glenn F. King (2015). The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω- Hexatoxin- Hv1a. Toxins, 7, 4366-4380.< /p> | + | <p class="content-1">The Fusion Protein Improvement Test</p> |
| − | <p class="reference-content">[2] Anonymous. (n.d.). HYDROLYSIS. Retrieved October 16, 2016 , from https://zh.scribd.com/document/79207692/Hydrolysis-2006</p>
| + | <p class="content"> In this experiment, we compared the toxicity effect of the three toxin designs including Hv1a/Hv1a- lectin/Hv1a- lectin with GS linker to test the toxicity effect. (For more about the improved Pantide with GS linker, see the < a href=" http:// parts. igem. org/ wiki/ index.php?title=Part:BBa_K1974030" style="color:#44E287;">link</ a>)</p> |
| − | <p class="reference-content">[3] Hedstrom, L. (2002, May 14). Serine Protease Mechanism and Specificity. Chem. Rev. 2002, 102, 4501-4523.</p>
| + | |
| − | <p class="reference-content">[4] Guozhong Xu & Mark R. Chance (2005). Radiolytic Modification of Sulfur-Containing Amino Acid Residues in Model Peptides: Fundamental Studies for Protein Footprinting. Anal. Chem, 77, 2437-2449.</p>
| + | <div> |
| − | <p class=" reference-content"> [5] Mary E. Dzaugis, Arthur J. Spivack, Steven D'Hondt (2015, April 10). A quantitative model of water radiolysis and chemical production rates near radionuclide- containing solids. Radiation Physics and Chemistry, 115, 127- 134.</p > | + | <img src="https://static.igem.org/mediawiki/2016/6/6c/NCTU_result_pro.jpg" class="picture" style="width:100%;left:0.5vw !important;"> |
| − | <p class="reference-content">[6] Bachari, T. S. (n.d.). Theoretical Investigation on The Kinetics of Free Radical Reactions of Styrene Emulsion Polymerization . Retrieved from http://www.iasj.net/iasj?func=fulltext&aId=13996</p>
| + | <p class="content -image" style="text-align:justify !important;"> Figure10. Remained leaves in the GS-linker improvement test with the Hv1a, Hv1a-lectin, and Hv1a- lectin GS- linker improved.</p> |
| − | </div>
| + | |
| | </div> | | </div> |
| | | | |
| − | </div>
| + | <div> |
| | + | <img src="https://static.igem.org/mediawiki/2016/b/b8/Fig11-pro.jpeg" class="picture"> |
| | + | <p class="content-image" style="text-align:center !important;">Figure11. The T-test analysis in different dose of GS-linker improvement test.</p> |
| | + | </div> |
| | + | |
| | + | <p class="content">To know how the length of the linker affect the function of the fusion protein, we constructed Hv1a-lectin with the original three-alanine linker and a longer GS linker. Besides, there is also a Hv1a without the linker between the repellent peptide and the lectin served as a comparative sample. Figure10 shows the pictures of the remained leaf disks after twelve hours of feeding assays. After we had done the feeding assays on tobacco cutworms with the three dilution ratio, we measured the area with the software imageJ. Figure11 shows the ratio of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. The observed phenomenon can be analyzed below.</p> |
| | + | |
| | + | |
| | + | <li class="list">We purified Pantide into three quantitative concentration with 0.4μM, 2μM, and 10 μM. The remained leaves area decreased as the concentration of PANTIDE decreased, which shows the dose response of Pantide.</li> |
| | + | <li class="list">In comparison with the two other design, we could find out that the remained leaves area with improved Hv1a-lectin were all more than that of the original Hv1a-lectin and Hv1a. The result shows that by enhancing the length of the linker, the fusion protein works performs its toxicity better.</li> |
| | + | |
| | + | <p class="content-1">Preference Test</p> |
| | + | <p class="content"In the preference test, we wanted to test if the larvae will have any preference on the leaf disks. There was an interesting finding that tobacco cutworms prefer the negative control to the sonicated or purified Pantide. The results are shown in the following video.</p> |
| | + | |
| | + | <div class="animate-box"> |
| | + | <div> |
| | + | <video controls width="150%"> |
| | + | <source src="https://static.igem.org/mediawiki/2016/4/4b/NCTU_result_prefer_test.mp4" type="video/mp4"> |
| | + | </video> |
| | + | </div> |
| | </div> | | </div> |
| | | | |
| | | | |
| | + | |
| | + | <p class="content">In the two-hour test, we can see that most of the tobacco cutworms move to eat the negative control group, which proves the repellent effect of Pantide.</p> |
| | + | |
| | + | |
| | + | |
| | + | |
| | + | <p class="content-1">Conclusion</p> |
| | + | |
| | + | |
| | + | <p class="content">After the three experiments, we have three conclusions.</p> |
| | + | <li class="list">Pantide performs its toxicity as a repellent in vivo. It is a noticeable finding that goes beyond our original hypothesis. The mechanism of the repellent effect is so far unknown.</li> |
| | + | <li class="list">The toxicity efficacy of Pantide is equal to <i>Bacillus thuringiensis</i>. </li> |
| | + | <li class="list">With the elongation of the linker, the improved fusion protein has the best repellent effect. </li> |
| | + | <li class="list">The tobacco cutworms have a preference on the leave disks without applying Pantide.</li> |
| | + | |
| | + | |
| | + | <p class="content">Although Pantide does not act as a biological pesticide as our hypothesis had mentioned, we may conclude that it is a biological insect repellent, which corresponds to our ideology of lowering the artificial impacts on the environment and simultaneously achieve effective pest control.</p> |
| | </div> | | </div> |
| − | </section>
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