Difference between revisions of "Team:TCFSH Taiwan/Design"

 
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           <div class="topic"><p class="text_color">Applied Design</p></div>
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           <div class="topic"><p class="text_color">How we design our biobrick</p></div>
 
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          <div class="topic"><p class="text_color">Orally Active Insecticidal Peptide</p></div>
 
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    <p class="title">Applied Design—A Detecting System<p>
 
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    <p class="content">For the practical application in the farmland, the farm will be divided into several areas, in each area, several sets of devices and sensors will be installed. The devices in each area will collect the farmland conditions respectively, and the data will be transmitted to one host device in each area through Bluetooth, and then the host will upload the data of its area up to the cloud through WiFi. When the data are uploaded to the cloud, it will send into the app in real time; thus the user can know the conditions in their farm simultaneously. As the time goes by, a database of the environmental information cloud will be created, the farm conditions will become big databases, and according to it, we can use the statistics of the big data to predict the future conditions as the number of pests, and auto-control the spraying system to spray Pantide or water more efficiently and accurately.<br>(See more in the <a href="https://2016.igem.org/Team:NCTU_Formosa/Demonstrate" style="color:#44E287;">Device</a>) </p>
 
 
  
  
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    <p class="title">What is “Detecoli”?<p>
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    <p class="content">We intend to invent a STICKER called “Detecoli”—a word we coin by combining “detect” and “<span style="font-style:italic;">E. coli</span>”—which changes color in environments of excess sunlight or inadequate temperatures. It will be attached to the product during manufacture and thus is able to monitor the whole process of transportation. Detecoli alerts consumers to possible deterioration or contamination by changing color and serves as a guarantee of quality.</p>
 
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<div style="width:1px;height:60px;"id="pantide">
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   <p class="title">How we design our biobrick</p>
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     <p class="content">First, we tried to find a UV promoter, and we located <font style="color: orange">BBa_I765001</font>. However, it simply didn’t work in our experiment. So after searching on the Internet, we found a project that had been conducted by Rice University. They found that protein UirR (<font style="color:orange">K1725420</font>) and UirS (<font style="color: orange">K1725410</font>) can be used as a photoreceptor. The UirS protein is anchored in the bacterial membrane where it “sees” the color illuminating the bacterium. If the illumination is UV, UirS activates itself and releases the protein, UirR. UirR will then be phosphorylated, and become active. Active UirR is mobile, capable of binding a specific promoter called P<sub>CsiR1</sub>, and triggering the expression of the desired gene—RFP (<font style="color: orange">E1010</font>). However, we couldn’t find the promoter sequence of PcsiR1 at first, so we used Plsir (<font style="color: orange">K1725400</font>) instead. But when we eventually found the sequence, it was too late for us. So we designed this biobrick:</p>
 
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    <p class="title">What is “Detecoli”?<p>
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     <p class="content">Then, we needed to find a way to measure the temperature, which is using the temperature regulated RBS (<font style="color: orange">BBa_K115001</font>). This RBS only allows ribosomes to bind to it at the temperature of 37 degree Celsius or above. Originally, we decided to put GFP after it, and the GFP would be activated if it reaches the target temperature. But we then noticed that GFP would produce green light, and the green light would cause the protein UirS to reverse back into an inactive state. So we then chose BFP (<font style="color: orange">K592009</font>) instead and designed this biobrick:</p>
     <p class="content">Pantide, a portmanteau word, conveys two concept-Pan and peptide-in a single blended neologism. In ancient Greek mythology, Pan is a god of shepherds and nature, whereas peptide indicates the essential substance of Pantide, amino acids. Pantide derives its toxicity from the spider venom. The inspiration for Pantide originates from the food chain. Predation is a scene ubiquitously observed in nature. Through evolution, animals have evolved diverse ways of predatory strategy. In this light, we hope to avail the natural evolutionary phenomenon into our project. Spiders are one of the most successful terrestrial venomous creatures on earth. In 300 million years of evolution, spiders have evolved arrays of complex venomous toxins. <sup>[1]</sup> Therefore, we found its potential for integrating spider toxins as a new source of bioinsecticide.</p>
+
     <img src="https://static.igem.org/mediawiki/2017/9/9e/Brick3.jpeg" class="bigphoto" width=70%>
    <p class="quote" style="color:#FFAF60 !important;">“More than a hundred different components can be found in the same venom, and in this parameter spiders are leaders in living nature.</p>
+
     <p class="content">Nevertheless, we were afraid that the length of exposure time to 37 degrees Celsius or above is too short for the bacteria to produce enough amounts of BFP. So we decided to use an irreversible inhibitor, and then we came across the Rhl promoter. When the product of RhlI (K1541017) C4-HSR and protein RhlR (C0171) bind together, Prhl will continuously work without consuming the proteins and thus will have enough time to produce BFP. Late after, we found that the team iGEM14_ETH_Zurich had improved this gene to prevent the “leakiness”. But unfortunately, it’s again too late for us to change. Eventually, we designed this biobrick:</p>
     <p style="text-align:right;color:#FFAF60 !important;"class="quote">Professor Alexander Vassilevski et al<br>
+
     <img src="https://static.igem.org/mediawiki/2017/1/17/Brick4.jpeg" class="bigphoto" width=70%>
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry<br>
+
     <p class="content">For fear that our products might be damaged, causing the bacteria inside to die, we designed a mechanism to guarantee that our product will remain effective. We knew that if we put an LVA tag behind the chromoprotein, it will degrade much faster. So our concept is to make the bacteria produce chromoprotein constantly, and it will be colorful when it is working. Nonetheless, when the bacteria aren’t alive anymore, the color will degrade fast and eventually become colorless. In the end, we designed this biobrick:</p>
Russia</p>
+
    <img src="https://static.igem.org/mediawiki/2017/0/0c/Brick5.jpeg" class="bigphoto" width=70%>
     <p class="content">On account of the vast multicomponent mixture in spider toxins, the selection of spider toxin required evaluation in an organized methodology. In this case, first, we searched two online databases-AnachnoServer and UniProt (Universal Protein Resource)-for toxic candidates. AnachnoServer is an online database that contains nearly 800 peptide toxin information from 78 spider species<sup>[1]</sup>, and UniProt is a library of protein information. We selected the toxin peptides from the databases with the following several criteria.</p>
+
    <p class="content">Also, to avoid the color mixture and the overconsuming of the amino acid, we designed a negative control promoter. We use LacI at the end since it is the most popular one.</p>
 
+
     <p class="content">Lastly, since it would be difficult to transform more than three plasmids into the bacteria, we combined two of them with one in the reverse direction (we are afraid that the gene behind will express poorly), and try to make the sequence as short as possible. So the final biobrick is:</p>
<ul style="list-style-image:none;list-style-type:disc;margin-left:10px !important">
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    <img src="https://static.igem.org/mediawiki/2017/4/4c/Brick6.jpeg" class="bigphoto" width="70%">
    <li class="list">The toxin should have multiple references which back up the origin, structure, and mechanism.</li>
+
     <li class="list">The toxin should not be toxic to mammals with the authentication of mice experiment.</li>
+
     <li class="list">The toxin should not have more than four disulfide bonds because we plan to express the toxin gene in <i>E. coli</i>.</li>
+
     <li class="list">The toxin was done with some orally-active experiment on certain species.</li>
+
    <li class="list">The toxin has no antibiotic activity to bacteria.</li>
+
</ul>
+
     <p class="content">For the detailed toxin selection process, see <a href="https://2016.igem.org/Team:NCTU_Formosa/Model#title1" style="color:#44E287;">toxin selection model</a>.</p>
+
     <p class="content">After months of searching and winnowing, Pantide comes into existence. The three selected toxins are Omega-hexatoxin-Hv1a (Hv1a), μ-segestritoxin-Sf1a (Sf1a) and Orally active insecticidal peptide (OAIP).</p>
+
 
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     <p class="title">Operation Model</p>
 
     <p class="title">Operation Model</p>
 
+
     <p class="content">We designed a device to detect harmful UV lights and high temperature, and we are able to confirm if it is working. Our design of the device will prevent <span style="font-style:italic;">E. coli</span> from leaking out, and the <span style="font-style:italic;">E. coli</span> in it will be annihilated after use. In the end, it can be freely disposed of without causing any potential health concern.</p>
     <p class="content">We designed a device to sense UV lights and high temperature, and we can confirm if it is working. When the device is working, and has not been exposed to UV light nor high temperature, it will be in the color of light green. When the device is working, and it has been exposed to excess UV lights, it will turn red; and if it has been under high temperature for too long, it will turn blue. However, if the device is not working at all, it won’t be in color (=white). The design of the device can also prevent e-coli from getting out, and the e-coli in it will be Sterilized after use. At the end, it’s free for disposal. </p>
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     <p class="title">Color changing system</p>
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     <p class="title">Mechanism</p>
     <p class="content">In normal conditions, the color of the sticker will be light green, of which indicates “safe”; nonetheless, if the products were exposed under sunlight or temperature higher than 37 degrees Celsius, the color will change drastically into red or blue respectively, of which indicates “not safe”. The longer the sticker was exposed under bad condition, the darker will the colors blue or red will be. The cause of both color is the synthesis of chromoprotein. Chromoproteins are very stable, which means that the color will last for a long period of time. </p>
+
     <p class="content-1">UV receptor</p>
 +
    <p class="content">Rice University found that protein UirR and UirS can be used as a photo receptor. The UirS protein is anchored in the bacterial membrane where it “sees” the color illuminating the bacterium. If the illumination is UV, UirS activates itself and releases the protein, UirR. UirR will then be phosphorylated, and become active. Active UirR is mobile, capable of binding a specific promoter called (PcsiR1), and triggering the expression of the desired gene.</p>
 +
    <p class="content-1">Temperature thermometer</p>
 +
    <p class="content">The RBS<sub>Temp</sub> only allows ribosomes to bind to it at the temperature of 37 degree Celsius or above. The main feature of all RNA thermometers is that they function through conformational shifts in structure. These shifts cause conformational changes to expose the Shine-Dalgarno sequence, which acts as a binding site to allow translation.3 For translation to occur, the ribosome must have the aforementioned SD sequence. The structural differences are caused by the transcription regions, but the SD sequence is common.</p>
 +
    <p class="content-1">Color changing</p>
 +
    <p class="content">The chromoprotein cj-blue, and the fluorescent proteins BFP and RFP can perform different colors.</p>
 +
    <p class="content-1">Chromoprotein</p>
 +
    <p class="content">The LVA tag served as a degradation peptide sequence, is one of the most effective of them. If we put LVA tags on our desired gene, we can make them degrade faster.</p>
 
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    <p class="title">Orally Active Insecticidal Peptide (OAIP)</p>
 
    <p class="content">OAIP is a toxic peptide derived from Selenotypus plumipes (Australian featherleg tarantula). It targets the voltage-gated ion channel of insects including species from the orders Lepidoptera and Coleoptera. It causes paralysis and finally death. OAIP is lethal to several insect orders but is not toxic to mice. </p>
 
  
<div>
 
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    <p class="content-image" style="text-align:center;">Figure 3. The animation shows the 3D structure of OAIP,<br> created by a software called Cn3D with the peptide information from NCBI. </p>
 
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    <p class="content">The three toxins are belong to a major category in spider venom-Short peptides that have disulfide bonds. Most of these toxin peptides have a structural motif that contains cysteine knottings and forms loops. The active site in the peptide that performs its toxicity are the amino acids located in loop regions. <sup>[4]</sup> The structure of these toxins are so-called “Inhibitor Cystine Knot (ICK)”. ICK has several features based on its disulfide-bond-rich structure-Stability. Take Hv1a as an example for proving the stability of ICK; Hv1a is highly stable in the temperature range of -20°C to 75°C and pH values of 1 to 8. Also, Hv1a is resistant to digestive enzyme-protease K. <sup>[5]</sup></p>
 
 
<div>
 
<img src="https://static.igem.org/mediawiki/2016/d/de/NCTU_ICK.png" class="picture" style="width:40%;padding-left:10vw !important;">
 
<p class="content-image">Figure 4. ICK structure.</p>
 
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    <p class="content">In nature, spiders inject venom into the haemolymph of insects’ that causes the death of the prey. However, Pantide is designed to be ingested by pests after application of Pantide onto the leaves. Therefore, there should be an amelioration done for the design of toxin.</p>
 
    <p class="quote" style="color:#FFAF60 !important;">“Many insecticidal venom peptides are typically ineffective, or at least much less potent, when delivered orally and this is thought to be due to the ineffective delivery of the toxins to their active sites of action in the central nervous system or peripheral nervous system.”</p>
 
    <p class="quote" style="text-align:right;color:#FFAF60 !important;">Doctor Elaine C. Fitches et al<br>
 
The Food and Environment Research Agency<br>
 
United Kingdom
 
</p>
 
    <p class="content">To promote the oral toxicity of toxin peptide, we designed a fusion protein with the addition of lectin. Lectins are glycoprotein-binding proteins. In this case, we chose snowdrop (Galanthus nivalis) lectin as a carrier of toxin peptides to create a fusion protein.<sup>[6]</sup> Snowdrop Lectin recognizes the glycoproteins on the epithelial cell in the insect gut and facilitates the fusion protein to cross the epithelial cell by transcytosis. Therefore, the fusion proteins are translocated into the haemolymph from the alimentary canal. Also, snowdrop lectin is proved to be resistant to proteolytic activity in the insect gut.<sup>[7]</sup></p>
 
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     <p class="title">Target</p>
 
     <p class="title">Target</p>
     <p class="content">The principal application for our sticker is to monitor the WHOLE transporting process. This is, from the second that the product was made by the factory, to the moment that the customer received the product (even until the customer finishes using the product), every moment was monitored. This not only guarantee the quality of the products, but helps to solve the conflict between the factory and the transport company. When the customers received the products with the stickers on it color changed, it indicates that the transporting condition is not acceptable. Likely, the transport company can also use this kind of stickers to prove to the factory that their employees do treat every cargo properly. This way, we can avoid consumer dispute and the problems of compensation, and add more goodwill for the company.  
+
     <p class="content">The principal application for our sticker is to monitor the WHOLE delivery process. That is, from the minute the manufacturing of the product is completed in the factory to the moment the customer receives the product, every moment will be monitored. In fact, the monitoring will continue until the customer finishes using the product. This not only guarantees the quality of the product, but helps to solve the possible legal disputes between the factory and the transport company, since you can add a new sticker in each stage of the delivery process. If the transport company or the customer receives the product with red or blue stickers on it, it indicates that the previous transporting condition is not acceptable. Likewise, the transportation company can also use this kind of stickers as evidence to prove that their employees do treat every cargo properly. This way, we can avoid consumer disputes and the ensuing problems of compensation, and the company will even get more goodwill.  
In addition, what we emphasize is to ensure EVERY product that have our stickers on and to maximize the accuracy of quality control. We extend the monitoring process from initially only in the factory to eventually the customers’ hands, making customers no longer buy, or use, the damaged product caused by inappropriately transporting or storing. The most considerable benefits of this bio-sticker are “long-term monitor” and “cumulative”. The factors of the harmful environments accumulate, and as soon as they surpass the limitation, it activates the color changing process. Still more, the basic material of this bio-sticker is e-coli. As the fact that e-coli replicates itself extremely rapidly, the cost of the sticker will substantially decrease.
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    <p class="content">As is shown in the above passage, our goal is to ensure the quality of EVERY product that has our stickers on it and to substantiate the reliability of quality control. Above all, we are able to extend the monitoring process from manufacturing, transportation, and eventually to the customer’s hands, preventing the customer from buying, or using, defective products which are damaged or contaminated due to inappropriate transporting or storing conditions. The most attractive features of this bio-sticker are “long-term monitoring” and “cumulative, layered supervision.The factors of harmful conditions accumulate, and as soon as they surpass the limits, they activate the color changing process in our stickers. The best part is, the basic material of this bio-sticker is <span style="font-style:italic;">E. coli</span>, and since <span style="font-style:italic;">E. coli</span> replicates itself extremely rapidly, the cost of the sticker will be considerably low. So, either replacing a sticker or adding a new one after each stage of the delivery process should still be cheap enough for the company or the consumer to afford, giving them extra incentive to use the product.</p>
 
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Latest revision as of 02:18, 2 November 2017

What is “Detecoli”?

We intend to invent a STICKER called “Detecoli”—a word we coin by combining “detect” and “E. coli”—which changes color in environments of excess sunlight or inadequate temperatures. It will be attached to the product during manufacture and thus is able to monitor the whole process of transportation. Detecoli alerts consumers to possible deterioration or contamination by changing color and serves as a guarantee of quality.

How we design our biobrick

First, we tried to find a UV promoter, and we located BBa_I765001. However, it simply didn’t work in our experiment. So after searching on the Internet, we found a project that had been conducted by Rice University. They found that protein UirR (K1725420) and UirS (K1725410) can be used as a photoreceptor. The UirS protein is anchored in the bacterial membrane where it “sees” the color illuminating the bacterium. If the illumination is UV, UirS activates itself and releases the protein, UirR. UirR will then be phosphorylated, and become active. Active UirR is mobile, capable of binding a specific promoter called PCsiR1, and triggering the expression of the desired gene—RFP (E1010). However, we couldn’t find the promoter sequence of PcsiR1 at first, so we used Plsir (K1725400) instead. But when we eventually found the sequence, it was too late for us. So we designed this biobrick:

Then, we needed to find a way to measure the temperature, which is using the temperature regulated RBS (BBa_K115001). This RBS only allows ribosomes to bind to it at the temperature of 37 degree Celsius or above. Originally, we decided to put GFP after it, and the GFP would be activated if it reaches the target temperature. But we then noticed that GFP would produce green light, and the green light would cause the protein UirS to reverse back into an inactive state. So we then chose BFP (K592009) instead and designed this biobrick:

Nevertheless, we were afraid that the length of exposure time to 37 degrees Celsius or above is too short for the bacteria to produce enough amounts of BFP. So we decided to use an irreversible inhibitor, and then we came across the Rhl promoter. When the product of RhlI (K1541017) C4-HSR and protein RhlR (C0171) bind together, Prhl will continuously work without consuming the proteins and thus will have enough time to produce BFP. Late after, we found that the team iGEM14_ETH_Zurich had improved this gene to prevent the “leakiness”. But unfortunately, it’s again too late for us to change. Eventually, we designed this biobrick:

For fear that our products might be damaged, causing the bacteria inside to die, we designed a mechanism to guarantee that our product will remain effective. We knew that if we put an LVA tag behind the chromoprotein, it will degrade much faster. So our concept is to make the bacteria produce chromoprotein constantly, and it will be colorful when it is working. Nonetheless, when the bacteria aren’t alive anymore, the color will degrade fast and eventually become colorless. In the end, we designed this biobrick:

Also, to avoid the color mixture and the overconsuming of the amino acid, we designed a negative control promoter. We use LacI at the end since it is the most popular one.

Lastly, since it would be difficult to transform more than three plasmids into the bacteria, we combined two of them with one in the reverse direction (we are afraid that the gene behind will express poorly), and try to make the sequence as short as possible. So the final biobrick is:

Operation Model

We designed a device to detect harmful UV lights and high temperature, and we are able to confirm if it is working. Our design of the device will prevent E. coli from leaking out, and the E. coli in it will be annihilated after use. In the end, it can be freely disposed of without causing any potential health concern.

Mechanism

UV receptor

Rice University found that protein UirR and UirS can be used as a photo receptor. The UirS protein is anchored in the bacterial membrane where it “sees” the color illuminating the bacterium. If the illumination is UV, UirS activates itself and releases the protein, UirR. UirR will then be phosphorylated, and become active. Active UirR is mobile, capable of binding a specific promoter called (PcsiR1), and triggering the expression of the desired gene.

Temperature thermometer

The RBSTemp only allows ribosomes to bind to it at the temperature of 37 degree Celsius or above. The main feature of all RNA thermometers is that they function through conformational shifts in structure. These shifts cause conformational changes to expose the Shine-Dalgarno sequence, which acts as a binding site to allow translation.3 For translation to occur, the ribosome must have the aforementioned SD sequence. The structural differences are caused by the transcription regions, but the SD sequence is common.

Color changing

The chromoprotein cj-blue, and the fluorescent proteins BFP and RFP can perform different colors.

Chromoprotein

The LVA tag served as a degradation peptide sequence, is one of the most effective of them. If we put LVA tags on our desired gene, we can make them degrade faster.

Target

The principal application for our sticker is to monitor the WHOLE delivery process. That is, from the minute the manufacturing of the product is completed in the factory to the moment the customer receives the product, every moment will be monitored. In fact, the monitoring will continue until the customer finishes using the product. This not only guarantees the quality of the product, but helps to solve the possible legal disputes between the factory and the transport company, since you can add a new sticker in each stage of the delivery process. If the transport company or the customer receives the product with red or blue stickers on it, it indicates that the previous transporting condition is not acceptable. Likewise, the transportation company can also use this kind of stickers as evidence to prove that their employees do treat every cargo properly. This way, we can avoid consumer disputes and the ensuing problems of compensation, and the company will even get more goodwill.

As is shown in the above passage, our goal is to ensure the quality of EVERY product that has our stickers on it and to substantiate the reliability of quality control. Above all, we are able to extend the monitoring process from manufacturing, transportation, and eventually to the customer’s hands, preventing the customer from buying, or using, defective products which are damaged or contaminated due to inappropriate transporting or storing conditions. The most attractive features of this bio-sticker are “long-term monitoring” and “cumulative, layered supervision.” The factors of harmful conditions accumulate, and as soon as they surpass the limits, they activate the color changing process in our stickers. The best part is, the basic material of this bio-sticker is E. coli, and since E. coli replicates itself extremely rapidly, the cost of the sticker will be considerably low. So, either replacing a sticker or adding a new one after each stage of the delivery process should still be cheap enough for the company or the consumer to afford, giving them extra incentive to use the product.