Team:KU Leuven/Project/HoneydewSystem

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    <a href="https://2013.igem.org/Team:KU_Leuven/Project/Aphid_Background">
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    <i class="livicon activeicon" data-name="bug" data-color="white"></i></div>
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  <div class="span7 icon-text">
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    <h3>Aphid Background</h3>
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    </a>
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    <p>Crashcourse in aphid biology</p>
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  </div>
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  </div>
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</div>
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    <a href="https://2013.igem.org/Team:KU_Leuven/Project/HoneydewSystem">
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    <i class="livicon activeicon" data-name="drop" data-color="white"></i></div>
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    <h3>Honeydew System</h3> </a>
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    <p>You are here!</p>
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    <a href="https://2013.igem.org/Team:KU_Leuven/Project/StickerSystem">
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    <i class="livicon activeicon" data-name="refresh" data-color="white"></i>
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    </div>
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  <div class="span7 icon-text">
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    <h3>Sticker System</h3>
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    </a>
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    <p>BanAphid population oscillates!</p>
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    <a href="https://2013.igem.org/Team:KU_Leuven/Project/E.coligy">
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    <i class="livicon activeicon" data-name="leaf" data-color="white"></i>
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  <div class="span7 icon-text">
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    <h3><i>E. coligy</i></h3>
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    <p>Validating the BanAphids in vivo</p>
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    <i class="livicon activeicon" data-name="gears" data-color="white"></i></div>
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    <h3>Parts</h3> </a>
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    <p>BioBrick 'm all!</p>
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    <a href="https://2013.igem.org/Team:KU_Leuven/Project/DataPage">
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    <h3>Data Page</h3> </a>
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    <p>All our achievements on one small page!</p>
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   <h3 class="bg-green">The Glucose Model</h3>
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   <h3 class="bg-green">The Honeydew System</h3>
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   <p align = "justify">We aim to achieve a sustainable way to reduce the damage caused by aphid pests, and offer an effective alternative for insecticides. Our modified <I>E. coli</I> (‘BanAphids’, meaning ‘to ban aphids’ as well as with ‘benefits’) would imitate insecticides by using the aphid’s own alarm pheromone, E-β-farnesene, (EBF) to repel them off the plant. On top of that we want to attract aphid predators such as the ladybug by using methyl salicylate (MeS), a phytohormone. This way we make sure the aphids are thoroughly removed from the plant.<br/>
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<b>We aim to achieve a sustainable way to reduce the damage caused by aphid pests, and offer an effective alternative for insecticides</b>.
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We have established what might be possible hurdles in introducing this system in the agricultural industry. First we have to make sure that the plant cell’s metabolism is not over burdened. Besides that we have to take into account that aphids might habituate to constitutive expression of EBF (De Vos <I>et al.</I>, 2010, Kunert <I>et al.</I>, 2010). Finally, we do not want to attract the aphid’s natural predators when they are not needed. <br/>
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Our modified <i>E. coli</i>, the <b>BanAphid</b> (meaning ‘to ban aphids’ as well as with ‘benefits’), would imitate the effect of insecticides by using the aphid’s own alarm pheromone, <b>E-β-farnesene</b>, (EBF) to <b>repel them off the plant</b>. On top of that we want to <b>attract aphid predators</b> such as the ladybug by using <b>Methyl Salicylate</b>(MeS), a phytohormone. This way we make sure the aphids are thoroughly removed from the plant. MeS also activates plant defense mechanisms and EBF also attracts predators.</br></br>
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We have established possible hurdles in introducing this system in the agricultural industry and took steps to tackle them.</br> First we have to make sure that the <b>plant cell’s metabolism is not overburdened </b>.</br>
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We thought of two different methods to carry out our system. One method would be to spray our BanAphids onto the plants. Keeping into account the possible hurdles we mentioned before, BanAphids will produce MeS in response to an external signal that indicates the presence of aphids, in order to reduce the burden on the plant cell’s metabolism and attract predators only when needed. This external signal is honeydew, since aphids produce high amounts of this. Honeydew is a very glucose rich substance, which is the reason why ants ‘farm’ aphids, in order to milk their honeydew. <br/>
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Secondly we have to take into account that aphids might <b>habituate</b> to constitutive expression of EBF (De Vos <i>et al.</i>, 2010, Kunert <i>et al.</i>, 2010).</br>
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Finally, we do not want to attract the aphid’s natural predators when they are not needed. These factors along with input from potential end users all played a role in the design of the <b>Honeydew system</b>.
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<img src="https://static.igem.org/mediawiki/2013/8/86/TetRconstruct.png" width=500>
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    <p> <b>Tet repressor under low glucose promoter</b> </p>
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  <img src="https://static.igem.org/mediawiki/2013/6/6d/AroGconstruct.png" width=500>
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    <p> <b> AroG, LacI construct </b> </p>
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pCaiF is a low glucose promoter, so when aphids are present on the plant and thereby honeydew, TetR will not be transcribed. pTetR, a TetR repressible promoter, will be active in this case so that <I>lacI</I> and <I>aroG*</I> will be transcribed. <I>aroG</I> encodes for the enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, which will convert erythose-4-phosphate into 3-deoxy-arabine-heptulosonate-7-phosphate. We have mutated <I>aroG</I> into <I>aroG*</I> in order to inhibit the negative feedback mechanism of phenylalanine to increase the activity of DAHP synthase so that the chorismate concentration is increased.<br/>
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<img src="https://static.igem.org/mediawiki/2013/4/4f/Chorismatepathway.png" width=600>
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    <p><b>Chorismate pathway</b></p>
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The following construct will convert the chorismate produced into salicylic acid and then into MeS. The original BioBrick from the 2006 MIT team (Bba_J45700) contained a <I>lac</I> promoter in front of the <I>pchBA gene</I>. The <I>pchBA</I> gene encodes an enzyme that converts chorismate into salicylic acid. Since this would interfere with our system (we use LacI), we replaced this promoter with another pTetR promoter.<br/>
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<img src="https://static.igem.org/mediawiki/2013/2/2d/MITbiobrick.png" width=400>
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  <p><b>MIT BioBrick 2006 for salicylic acid to MeS conversion</b></p>
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<img src="https://static.igem.org/mediawiki/2013/5/54/MeSconstruct.png" width=400>
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    <p><b>MeS construct to convert salicylic acid into MeS</b></p>
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cpram is a constitutive promoter so EBF synthase will be constitutively transcribed and EBF constitutively expressed. However, there is a <I>lac</I> operator present and since LacI is transcribed when honeydew is present (see above), EBF synthase transcription is inhibited in the presence of honeydew.  In the absence of aphids, EBF is constitutively expressed and aphids are thus repelled. However, as mentioned before, EBF could lose its aphid repellent effect due to habituation.<br/>
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<img src="https://static.igem.org/mediawiki/2013/c/c4/EBFconstruct.png" width=500>
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    <p><b>EBF construct</b></p>
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So if certain aphids do happen to escape the EBF repellent signal, the MeS acts as a counter signal and attracts natural predators of the aphid such as ladybugs and green lacewings. Aphids will activate the MeS cycle due to the presence of honeydew.<br/>
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     <h3>E-β-Farnesene</h3> </a>
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     <h3>Designing the Honeydew System</h3> </a>
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       <p align="justify">EBF serves as the most universal aphid alarm pheromone. It is released from the cornicles of the aphids to warn others against upcoming danger, such as the natural predators of aphids. Because of the fact that EBF is highly susceptible to oxidation, we want to make our BanAphids produce EBF regularly.</p>
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       <p align="justify">In this Honeydew System we opt to spray our BanAphids onto the plants. If aphids do happen to escape the EBF repellent signal, they activate the MeS BioBrick due to the presence of honeydew. This direct contact with honeydew is why we call this our Honeydew System. MeS acts as a second line of defence and attracts natural predators of the aphids such as ladybugs and green lacewings.</p>
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     <h3>Methyl Salicylate - Wetlab</h3> </a>
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     <h3>E-β-Farnesene construction</h3> </a>
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       <p align="justify">Methyl salicylate is a pheromone released by plants when they are attacked by aphids. It activates plant defence systems, as well as attract predators of the aphids, such as the ladybug or the green lacewing. In the lab we have focused on increasing the production of methyl salicylate of an existing brick, by increasing the production of its precursor, chorismate.</p>
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       <p align="justify">EBF is the most universal aphid alarm pheromone. It is released from the cornicles of the aphids to warn itself and others against upcoming danger, such as the natural predators of aphids. The aphids undergo a change in gene expression that motivates them to mobilise and leave the plant but because EBF is highly susceptible to oxidation, we our BanAphids to produce EBF regularly.</p>
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     <h3>Methyl Salicylate construction</h3> </a>
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       <p align="justify">We have modeled the production system of methyl salicylate by using the transcription, translation and protein degradation rate in order to calculate the mRNA and protein flux. We also brought the kinetics of methyl salicylate synthesis into account.</p>
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       <p align="justify">Methyl Salicylate is a phytohormone released by plants when they are under attack, for instance, upon aphid infestation. It activates plant defence systems, as well as attract aphid predators, such as the ladybug or the green lacewing. We have focussed on increasing the MeS production of an existing brick, by increasing the production of its precursor, chorismate.</p>
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       <p align="justify">We did a qPCR to check whether our genes of interest are properly transcribed. Also, the amount of mRNA we can measure with qPCR could serve as input data for our methyl salicylate model.</p>
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       <p align="justify">To check whether our genes of the MeS brick are properly transcribed, we performed a qPCR. On top of this, the amount of mRNA we can measure serves as input data for the <a href="https://2013.igem.org/Team:KU_Leuven/Project/Modelling/Cellular_Level">Methyl Salicylate model</a>.</p>
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Latest revision as of 21:40, 28 October 2013

iGem

Secret garden

Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!


  • A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
  • For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
  • We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?

Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!

tree ladybugcartoon

Aphid Background

Crashcourse in aphid biology

Honeydew System

You are here!

Sticker System

BanAphid population oscillates!

E. coligy

Validating the BanAphids in vivo

Parts

BioBrick 'm all!

Data Page

All our achievements on one small page!

We aim to achieve a sustainable way to reduce the damage caused by aphid pests, and offer an effective alternative for insecticides. Our modified E. coli, the BanAphid (meaning ‘to ban aphids’ as well as with ‘benefits’), would imitate the effect of insecticides by using the aphid’s own alarm pheromone, E-β-farnesene, (EBF) to repel them off the plant. On top of that we want to attract aphid predators such as the ladybug by using Methyl Salicylate(MeS), a phytohormone. This way we make sure the aphids are thoroughly removed from the plant. MeS also activates plant defense mechanisms and EBF also attracts predators.

We have established possible hurdles in introducing this system in the agricultural industry and took steps to tackle them.
First we have to make sure that the plant cell’s metabolism is not overburdened .
Secondly we have to take into account that aphids might habituate to constitutive expression of EBF (De Vos et al., 2010, Kunert et al., 2010).
Finally, we do not want to attract the aphid’s natural predators when they are not needed. These factors along with input from potential end users all played a role in the design of the Honeydew system.

Designing the Honeydew System

In this Honeydew System we opt to spray our BanAphids onto the plants. If aphids do happen to escape the EBF repellent signal, they activate the MeS BioBrick due to the presence of honeydew. This direct contact with honeydew is why we call this our Honeydew System. MeS acts as a second line of defence and attracts natural predators of the aphids such as ladybugs and green lacewings.

E-β-Farnesene construction

EBF is the most universal aphid alarm pheromone. It is released from the cornicles of the aphids to warn itself and others against upcoming danger, such as the natural predators of aphids. The aphids undergo a change in gene expression that motivates them to mobilise and leave the plant but because EBF is highly susceptible to oxidation, we our BanAphids to produce EBF regularly.

Methyl Salicylate construction

Methyl Salicylate is a phytohormone released by plants when they are under attack, for instance, upon aphid infestation. It activates plant defence systems, as well as attract aphid predators, such as the ladybug or the green lacewing. We have focussed on increasing the MeS production of an existing brick, by increasing the production of its precursor, chorismate.

Methyl Salicylate - qPCR

To check whether our genes of the MeS brick are properly transcribed, we performed a qPCR. On top of this, the amount of mRNA we can measure serves as input data for the Methyl Salicylate model.

Retrieved from "http://2013.igem.org/Team:KU_Leuven/Project/HoneydewSystem"