Team:KU Leuven/Project

From 2013.igem.org

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   <h3>The Project</h3>
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   <h3 class="bg-green"><i>E. coligy</i>: Plants with BanAphids</h3>
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One of man’s basic needs is feeding him/herself. Finding or producing food has always been the primary priority of mankind, of all animals in fact. So as the world’s population expands to over 7 billion but the world’s resources such as land remain limited, the World is in a desperate struggle to increase productivity of the lands already in use and encourage <a href="http://www.youtube.com/watch?v=2nFQOkzEjxQ">innovative agriculture.</a> Weather, disease and pests such as aphids, can reduce crop yields by up to 75% and since weather is difficult to control most farmers try to control the damage caused by disease and pests. Many farmers then turn to the use of pesticides and insecticides, and society is now questioning whether their ‘fresh’ food isn’t in fact ‘poison’ due to the amount of pesticides that can be detected on their food. <br/>
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<b>Our goal is to create a sustainable system to reduce the damage that aphids cause to our agricultural industry and reduce the use of insecticides.</b> <br/><br/>
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The current solution usually requires huge amounts of insecticides, damaging ecosystems in several ways. First, the indiscriminate weakening of insects means that beneficial insects (pollinators, ...) are also affected. Second, insecticides affect vertebrates, including humans, damaging our health. Third, insecticide residues (catabolites) accumulate in each food-chain. The highest organism, be it humans or for example predatory birds, will end up with potentially high levels of residues and their negative effects.<br/>
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Our goal is to reduce the damage that aphids cause to our agricultural industry in a sustainable way. The current solution is almost always using huge amounts of insecticides, which damage the ecosystem in several ways. First of all, the indiscriminately weakening of insects means that beneficial insects for agriculture are also affected. Insecticides affect vertebrates, including humans, as they have been shown to be damaging for our health <I>(Bjørling-Poulsen et al.)</I>. Besides that, residues or catabolites of insecticides accumulate in higher organisms that eat the insects, which in turn end up in our food chain.<br/>
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We aim to offer an <b>effective and sustainable alternative for insecticides.</b> Our modified <I>E. coli</I>, the <B>BanAphids</B> (meaning ‘to ban aphids’ as well as provide plants with ‘benefits’), would produce the aphid’s own alarm pheromone, <b>E-β-farnesene (EBF)</b>, to repel them off the plant. On top of that we want to attract aphid predators such as the ladybug via the production of <b>Methyl Salicylate (MeS)</b>, a phytohormone, by our BanAphids. This way we ensure aphids are thoroughly removed from the plant.
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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 (‘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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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 <I>(De Vos et al, 2010, Kunert, 2010)</I>. Finally, we do not want to attract the aphid’s natural predators when they are not needed. <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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<img src="https://static.igem.org/mediawiki/2013/8/86/TetRconstruct.png" width=500>
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    <p>Figure 1ǀ Tet repressor under low glucose promoter. </p>
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    <p>Figure 2ǀ AroG, LacI construct.</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=800>
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    <p>Figure 3ǀ Chorismate pathway</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> Figure 3ǀ MIT BioBrick 2006 for salicylic acid to MeS conversion</p>
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<img src="https://static.igem.org/mediawiki/2013/5/54/MeSconstruct.png" width=400>
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    <p>Figure 4ǀ MeS construct to convert salicylic acid into MeS</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>Figure 5ǀ EBF construct</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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Another way to avoid habituation of the aphids is to choose for an oscillating production of EBF, which requires a colony-wide synchronized oscillating system. We also developed this oscillating system so that the BanAphids could alternate between MeS and EBF production without the use of a direct signal, the honeydew. This way, the BanAphids would not need to be in direct contact with the aphids but could be kept separate from the plant. Our second method is therefore to keep the BanAphids in a container so that they are isolated from the environment but so that MeS and EBF can still pass. We turned to the sticker model of the Groningen 2012 iGEM team to use as a container. The sticker is composed of a plastic film, whose pores are too small for bacteria to pass but through which volatiles can still pass. The Groningen 2012 iGEM team has already investigated the biosafety of this sticker.<br/>
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<img src="https://static.igem.org/mediawiki/2013/1/1f/Stickermodel.jpg" width=300>
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     <p>Figure 6ǀ Sticker model</p>
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    <p align = "justify"> We devised <b>two different systems</b> that might use these molecules. We did this because of response we received from both private end users and the general public.
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<br/><br/>In the first system, our <b><a href="https://2013.igem.org/Team:KU_Leuven/Project/HoneydewSystem"> honeydew system</a></b>, we spray our BanAphids directly onto the plants. In this system our BanAphids will <b>only produce MeS when aphids are present</b> because they are sensitive to the high concentrations of glucose found in the honeydew produced by the aphids. This interaction minimises the burden on the plant’s metabolism, attracts predators only when needed and avoids aphid habituation.</p>
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  <img src="https://static.igem.org/mediawiki/2013/0/07/Tekeningproject.jpg" alt="banaphids">
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    <p><b>Honeydew System</b><br/> Shows our BanAphids producing EBF to get rid of aphids and MeS to attract ladybugs.</p>
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      <h3>Honeydew system</h3>
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We devised a second system with input from the private end user: the <b><a href="https://2013.igem.org/Team:KU_Leuven/Project/StickerSystem">sticker system</a></b>.
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Our second method starts from the principle that the <b>BanAphids</b> may not come in direct contact with the plants because of safety and ethical concerns. For this we turned to the <a href="https://2012.igem.org/Team:Groningen/Sticker">sticker model of the Groningen 2012 iGEM team</a> to use as a container. The sticker is composed of a plastic film, whose pores are too small for the BanAphids to pass, but large enough so that the volatile pheromones it produces still pass. The Groningen 2012 iGEM team has already investigated the biosafety of this sticker. However this means the BanAphids lose direct contact with the honeydew and the Honeydew System fails. To overcome this and permit this containment, we designed our <b><a href="https://2013.igem.org/Team:KU_Leuven/Project/Oscillator/Description">own oscillating system</a></b> so that there is an <b>alternating MeS and EBF production</b>.<br/><br/>
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Finally we want to define the impact of both these systems on the ecosystem. In collaboration with the relevant industries we performed various plant and insect experiments for the <b><i><a href="https://2013.igem.org/Team:KU_Leuven/Project/Ecological/wetlab">E. coligy</a></b></i> part of our project. This allows us to demonstrate the effect of our systems on the ecosystem.<br/></p>
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    <img src="https://static.igem.org/mediawiki/2013/5/5d/Stickermodelverkleind.png">
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     <p><b>Sticker system</b><br/> How the sticker could be placed in a rose bush to repel aphids and attract ladybugs.</p>  
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     <h3>Background</h3>
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     <h3>Aphid Background</h3>
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     <p>Crashcourse in aphid biology.</p>
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     <p>Crashcourse in aphid biology</p>
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     <p>Our BanAphids react to honeydew</p>
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     <p>Helps predators find the aphid.</p>
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     <p>BanAphid population oscillates!</p>
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     <h3><i>E. coligy</i></h3>
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     <p>Warning: engineers only.</p>
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     <p>Validating the BanAphids in vivo</p>
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     <p>BioBrick 'm all!</p>
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     <p>BioBrick 'm all!</p>
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     <p>All our achievements on one small page!</p>
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Latest revision as of 01:21, 29 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

Our goal is to create a sustainable system to reduce the damage that aphids cause to our agricultural industry and reduce the use of insecticides.

The current solution usually requires huge amounts of insecticides, damaging ecosystems in several ways. First, the indiscriminate weakening of insects means that beneficial insects (pollinators, ...) are also affected. Second, insecticides affect vertebrates, including humans, damaging our health. Third, insecticide residues (catabolites) accumulate in each food-chain. The highest organism, be it humans or for example predatory birds, will end up with potentially high levels of residues and their negative effects.
We aim to offer an effective and sustainable alternative for insecticides. Our modified E. coli, the BanAphids (meaning ‘to ban aphids’ as well as provide plants with ‘benefits’), would produce 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 via the production of Methyl Salicylate (MeS), a phytohormone, by our BanAphids. This way we ensure aphids are thoroughly removed from the plant.

We devised two different systems that might use these molecules. We did this because of response we received from both private end users and the general public.

In the first system, our honeydew system, we spray our BanAphids directly onto the plants. In this system our BanAphids will only produce MeS when aphids are present because they are sensitive to the high concentrations of glucose found in the honeydew produced by the aphids. This interaction minimises the burden on the plant’s metabolism, attracts predators only when needed and avoids aphid habituation.

banaphids

Honeydew System
Shows our BanAphids producing EBF to get rid of aphids and MeS to attract ladybugs.

Honeydew system


We devised a second system with input from the private end user: the sticker system. Our second method starts from the principle that the BanAphids may not come in direct contact with the plants because of safety and ethical concerns. For this we turned to the sticker model of the Groningen 2012 iGEM team to use as a container. The sticker is composed of a plastic film, whose pores are too small for the BanAphids to pass, but large enough so that the volatile pheromones it produces still pass. The Groningen 2012 iGEM team has already investigated the biosafety of this sticker. However this means the BanAphids lose direct contact with the honeydew and the Honeydew System fails. To overcome this and permit this containment, we designed our own oscillating system so that there is an alternating MeS and EBF production.

Finally we want to define the impact of both these systems on the ecosystem. In collaboration with the relevant industries we performed various plant and insect experiments for the E. coligy part of our project. This allows us to demonstrate the effect of our systems on the ecosystem.

Sticker system
How the sticker could be placed in a rose bush to repel aphids and attract ladybugs.

Aphid Background

Crashcourse in aphid biology

Honeydew System

Our BanAphids react to honeydew

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!