Difference between revisions of "Team:NCTU Formosa/Proof"

 
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           <div class="topic"><p class="text_color">Insect Test Result</p></div>
 
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<!----------cloning results----------->
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<div>
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<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>
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<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>
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    <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>
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<div>
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<p class="title">Expression Result</p>
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<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>
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<p class="content">The gel of SDS-PAGE result is correct, and here is our result.(Figure 2a, 2b, 2c)</p>
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<p class="content-image"style="text-align:justify !important;">Figure 2. SDS-PAGE result of the six Pantide-expressed sonicated products compared with the unexpressed sonicated one as a control and with Marker labeled length on the left hand. A: add β-mercaptoethanol and sample buffer, B: add sample buffer. (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) </p>
 +
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 +
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<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>
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<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>
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    <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, and with Marker labeled length on the left hand. A is the sonication product. B is the elution product of purification. (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>
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<!------------Feeding Assay-------------->
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   <div>
 
   <div>
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     <p class="title">Overview<p>
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     <p class="title">Feeding Assays</p>
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     <p class="content"><i> “We are witnessing the dawn of a new era of Internet of Things. IoT will increase the ubiquity of the Internet by integrating every object for interaction via embedded systems, which leads to a highly distributed network of devices communicating with human beings as well as other devices.”</i></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>
 +
**Note: The word “Pantide” in the following paragraph refers to the collection of all the toxin design in our project.
 +
</p>
  
−
     <p class="content" style="text-align:right">Feng Xia<br>
+
 
−
Head, Department of Cyber School of Software, Dalian University of <br>
+
<ul style="list-style-image:none;list-style-type:disc;">
−
Technology, China
+
    <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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 +
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>
 
</p>
  
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     <p class="content">In modern times, technology has advanced at a surprising speed because of the internet. Since the Internet came into existence, its role has become indispensable in multiple fields. Therefore, integrating the device with the internet that can be used in the farmland is what we aim to achieve. </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>
  
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    <p class="content">We integrate the state-of-the-art technology into the farmland and subvert the public stereotype of the conventional agriculture. The device is an automatic system that has multiple functions including detection and elimination of the pests. In the device, it consists of three parts—the detector, the controller, and the clientele.</p>
 
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    <p class="title">Detector Design</p>
 
  
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<div>
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     <img src="##" class="picture">Detector 的圖
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     <p class="title">Insect Test Result</p>
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     <p class="content-image" style="text-align:center">Figure 1.</p>
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    <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 <i>E. coli</i> 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>
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<div>
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<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;">Figur4. Remained leaf disks in the pre-test with the Hv1a and Hv1a-lectin.</p>
 
     </div>
 
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     <p class="content">First, inside the detector(Figure 1.), we utilize Pheromone Biosynthesis Activation Neuropeptide(PBAN) to attract specific species of pests into our device that will count the number of the species respectively. With the detector, we can know the situation remotely in the app we created. Second, the controller will then analyze the data from the detector to decide whether to spray the Pantide or not. Lastly, the client will receive the data of the real-time condition in the farmland.</p>
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<div style="margin-top:20px;">
 +
    <img src="https://static.igem.org/mediawiki/2016/f/fb/NCTU_result_dos_H.jpg" class="picture">
 +
     <p class="content-image" style="text-align:center !important;">Figure5. The dose response analysis of Hv1a/Hv1a-lectin.</p>
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 +
    <img src="https://static.igem.org/mediawiki/2016/b/be/Fig3-HHL.jpeg" class="picture">
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    <p class="content-image" style="text-align:center !important;">Figure6. The T-test analysis in different dose of Hv1a/Hv1a-lectin.</p>
 +
    </div>
  
−
    <p class="content">Our detector contains several parts. The main part is to catch different kinds of pests and calculate the number of various pests, and a double layered box with an inverted pyramid inside it, and we used a 3D printer to make the entry channels. Inside the inverted pyramid, we separate the volume into four parts. Each part has one kind of PBAN to attract a specific species of pest. When an insect comes into the channel of the device, the infrared light will be cut off, and the signal will be sent to the Arduino chip which is in the IoT board linkit smart 7688 duo.  The above process allows the device to count the bugs inside each channel. The function helps the users know the population growth of pests in the farmland. Apart from getting hold of the pest population in the farmland, the users can also grasp various conditions in farmland with the detector. The detector is equipped with a hygrometer, a thermometer, a rain gauge, a UV sensor, a CO2 sensor, a soil moisture sensor, an illuminance sensor and a barometric pressure sensor to know whether the land is too dry or not. We optimize the value of the data above into the database, hoping that the users can take good care of their plants by utilizing the information in the database in the future. Moreover, we will also record the weather condition as a reference for the users.</p>
 
  
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    <p class="title">Controller Design</p>
 
  
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     <p class="content" style="padding-top:40px !important;padding-bottom:30px; !important">Figure4 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. Figure5 shows the percentage of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. Figure6 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>
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        <img src="https://static.igem.org/mediawiki/2016/d/dc/NCTU_Controller_Design.png" class="picture">
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        <p class="content-image" style="text-align:center !important;">Figure 2. </p>
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 +
 
 +
<ul style="list-style-image:none;list-style-type:disc;">
 +
 
 +
    <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 figure5.</li>
 +
 
 +
    <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 figure5.</li>
 +
    <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 figure6.</li>
 +
 
 +
    <p class="content">To sum up, the Hv1a and Hv1a-lectin work well <i>in vivo</i>. The effect can be roughly ranked as:<br>Hv1a-lectin > Hv1a > <i>E. coli</i> > Negative control</p>
 +
 
 +
    <p class="content">We got the similar result in the Sf1a/Sf1a-lectin and OAIP/OAIP-lectin.</p>
 +
 
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<div>
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<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;">Figure7. Remained leaf disks in the pre-test with the Sf1a and Sf1a-lectin.</p>
 
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<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;">Figure8. The dose response analysis of Sf1a/Sf1a-lectin.</p>
 +
    </div>
  
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    <p class="content">The controller contains IoT board linkit smart 7688 duos, released by MediaTek, which has an MPU and an MCU ( in other words, a MediaTek chip and an Arduino chip). The MCU gets the data from the detector and sensors and sends to the MPU; then the MPU does a few computation and translates the data into the information we want. Subsequently, the Arduino sensors communicate with the server through WiFi, receiving the command from it and sending the information to the server. Also, the MPU will send the command to the MCU to tell it what it should do.</p>
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<div style="margin-top:20px;">
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     <p class="content">The server is the most important part of the IoT system. It is the central to the device. Most of the computational work occurs on the server. Also, the server has an SQLite database that is used to save the information and the result of computation. In the past, building an IoT system required large knowledge and skills in programming, and once a device is connected to the system, it was difficult to change the slaver. This year, we used IoT talk, a system developed by the lab of Dr.Lin in National Chiao Tung University Taiwan. It is a system that makes the IoT contact easier and more convenient, even for the users not familiar with programming knowledge and background can easily utilize it; as it has simplified to the connected device below.</p>
+
    <img src="https://static.igem.org/mediawiki/2016/2/29/Fig6-S.jpeg" class="picture">
 +
     <p class="content-image" style="text-align:center !important;">Figure9. The T-test analysis in different dose of Sf1a/Sf1a-lectin.</p>
 +
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<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>
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     <p class="content-image" style="text-align:center !important;">Figure 3. </p>
+
     <p class="content-image" style="text-align:center!important;">Figure10. Remained leaf disks in the pre-test with the OAIP and OAIP-lectin.</p>
 
     </div>
 
     </div>
−
   
 
−
</div>
 
−
 
 
−
  <div>
 
−
    <p class="title">Clientele Design</p>
 
−
    <p class="content">As for the clientele, we use the open source app released by MediaTek. Inside the app, we will have a panel to show us the various conditions of farm. By using the app, we can turn on or turn off the sprinklers at any time and anywhere as long as we have access to the internet.</p>
 
  
−
    <div>
+
<div style="margin-top:20px;">
−
     <img src="" class="picture">
+
     <img src="https://static.igem.org/mediawiki/2016/2/2f/NCTU_result_dos_O.jpg" class="picture">
−
     <p class="content-image"style="text-align:center !important;">Figure 4.</p>
+
     <p class="content-image" style="text-align:center !important;">Figure11. The dose response analysis of OAIP/OAIP-lectin.</p>
 
     </div>
 
     </div>
  
−
    <p class="content">With the detector, we can know the situation without going to the farm. With the server, we can grow the plant without labors. With the clientele, we can still manage our farm remotely. With the combination of all the parts above, we make up our device. </p>
+
<div style="margin-top:20px;">
 +
    <img src="https://static.igem.org/mediawiki/2016/0/03/Fig9-O.jpeg" class="picture">
 +
    <p class="content-image" style="text-align:center !important;">Figure12. The T-test analysis in different dose of OAIP/OAIP-lectin.</p>
 +
    </div>
 +
 
 +
 
 +
 
 +
 
 +
<div style="width:1px;height:60px;"id="GS_Linker">
 
   </div>
 
   </div>
 +
 +
    <p class="content-1">The Fusion Protein Improvement Test</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_K1974033" style="color:#44E287;">link</a>)</p>
  
 
<div>
 
<div>
−
    <p class="title">Hardware</p>
+
    <img src="https://static.igem.org/mediawiki/2016/c/c8/Fig10-pro.jpeg" class="picture" style="width:100%;left:0.5vw !important;">
−
    <p class="content">In the hardware, we designed an insect box, which had separated into four sections—the outside shell, the pest channels, the blue light LEDs and a PBAN container.</p>
+
    <p class="content-image" style="text-align:justify !important;">Figure13. Remained leaves in the GS-linker improvement test with the Hv1a, Hv1a-lectin, and Hv1a-lectin GS-linker improved.</p>
 +
    </div>
  
−
    <ul style="list-style-image:none;list-style-type:decimal;">
+
<div>
−
            <li class="list">Outside Shell:
+
    <img src="https://static.igem.org/mediawiki/2016/b/b8/Fig11-pro.jpeg" class="picture">
−
<br>&nbsp;&nbsp;&nbsp;&nbsp;The outside shell is a cube cover of our hardware which has four holes for moths entry. It also protects the whole hardware from destroying.</li>
+
    <p class="content-image" style="text-align:center !important;">Figure14. The T-test analysis in different dose of GS-linker improvement test.</p>
−
            <li class="list">Pest Channels:
+
    </div>
−
<br>&nbsp;&nbsp;&nbsp;&nbsp;There are four pest channels printed by 3D printer inside the device; We set the infrared LEDs and infrared receivers in them to count the number of moths. There are two layers of infrared receivers let moth cut off two of the infrared light to make sure that the count of moths enter the insect box is accurate. </li>
+
−
            <li class="list">Blue Light LEDs:
+
−
<br>&nbsp;&nbsp;&nbsp;&nbsp;According to the result of 2014 NCTU_Formosa, blue light has more attraction to <i>Spodoptera Litura</i> than other color light. Thus we use the blue light in another device to attraction moths.</li>
+
−
            <li class="list">PBAN container:
+
−
<br>&nbsp;&nbsp;&nbsp;&nbsp;This year, our project adopts the concept of 2014 NCTU_Formosa, using PBAN to attract moth. In our hardware, there is an inverted pyramid that had two layers that contain PBAN. Moreover, we have improved the design of container into the shape of an inverted pyramid to trap the pests, so that the pests can fly into the pest collector easily but hard to exit.</li>
+
  
−
   
+
    <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. Figure13 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>
−
    </ul>
+
 
 +
 
 +
    <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>
 +
 +
 +
 +
 +
 +
 
</section>
 
</section>
  

Latest revision as of 03:09, 4 November 2016

Cloning Result

Gene Constructions of Pantide—PCR

To ensure the genes are cloned into E. coli 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)

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)

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).

Expression Result

Expression of Pantide

Our goal is to express Pantide successfully. We used E. coli BL21 rosetta-gami strain to cultivate genes. Afterward, we sonicated E. coli 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)

The gel of SDS-PAGE result is correct, and here is our result.(Figure 2a, 2b, 2c)

Figure 2. SDS-PAGE result of the six Pantide-expressed sonicated products compared with the unexpressed sonicated one as a control and with Marker labeled length on the left hand. A: add β-mercaptoethanol and sample buffer, B: add sample buffer. (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)

Purification of Pantide

After sonicated E. coli, we further purified Pantide to check whether it is well-expressed. The purified products’ length can be seen in the table below.(Table 3)

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.

Figure 3. SDS-PAGE result of the seven purified Pantide products compared with the unpurified one, and with Marker labeled length on the left hand. A is the sonication product. B is the elution product of purification. (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)

Feeding Assays

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.
**Note: The word “Pantide” in the following paragraph refers to the collection of all the toxin design in our project.

  • Testee Selection

    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 in vivo. 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.

  • Experiment Design

    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.
           In the following experiments, we used E. coli 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 Protocols.

  • Hypothesis

    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.

Insect Test Result

Feeding Assay Pre-test

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.

Feeding Assay Pre-test of Hv1a and Hv1a-lectin

Figur4. Remained leaf disks in the pre-test with the Hv1a and Hv1a-lectin.

Figure5. The dose response analysis of Hv1a/Hv1a-lectin.

Figure6. The T-test analysis in different dose of Hv1a/Hv1a-lectin.

Figure4 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. Figure5 shows the percentage of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. Figure6 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.

  • With the dilution of Hv1a/Hv1a-lectin, the remained leaves area decreased, which indicates the dose response. The dose response is shown in figure5.
  • 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 figure5.
  • 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 figure6.

    • To sum up, the Hv1a and Hv1a-lectin work well in vivo. The effect can be roughly ranked as:
    Hv1a-lectin > Hv1a > E. coli > Negative control

    We got the similar result in the Sf1a/Sf1a-lectin and OAIP/OAIP-lectin.

    Feeding Assay Pre-test of Sf1a and Sf1a-lectin

    Figure7. Remained leaf disks in the pre-test with the Sf1a and Sf1a-lectin.

    Figure8. The dose response analysis of Sf1a/Sf1a-lectin.

    Figure9. The T-test analysis in different dose of Sf1a/Sf1a-lectin.

    Feeding Assay Pre-test of OAIP and OAIP-lectin

    Figure10. Remained leaf disks in the pre-test with the OAIP and OAIP-lectin.

    Figure11. The dose response analysis of OAIP/OAIP-lectin.

    Figure12. The T-test analysis in different dose of OAIP/OAIP-lectin.

    The Fusion Protein Improvement Test

    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 link)

    Figure13. Remained leaves in the GS-linker improvement test with the Hv1a, Hv1a-lectin, and Hv1a-lectin GS-linker improved.

    Figure14. The T-test analysis in different dose of GS-linker improvement test.

    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. Figure13 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.

  • 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.
  • 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.

    • Preference Test

    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.

    Conclusion

    After the three experiments, we have three conclusions.

  • 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.
  • The toxicity efficacy of Pantide is equal to Bacillus thuringiensis.
  • With the elongation of the linker, the improved fusion protein has the best repellent effect.
  • The tobacco cutworms have a preference on the leave disks without applying Pantide.

    • 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.