Team:Evry/AuxinTOX

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<h1> Auxin toxicity test in tadpoles </h1>  
<h1> Auxin toxicity test in tadpoles </h1>  
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<h2> The purpose of this experiement </h2>
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<h2> The purpose of this experiment </h2>
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<p>A recent study led by Japanese researches implemented a rapid protein depletion system using Auxin, a plant hormone. The Auxin-Inducible Depletion (AID) system allowed a rapid and reversible degradation of target proteins in response to Auxin (indole-3-acetic acid (IAA) and 1-naphthaleneacetic acid (NAA)). The originality of this project lies in the  fact that the AID system has been transplanted in non-plant cells as well as yeast and cell lines derived from chicken, mouse, hamster, monkey and human cells. Concerning cell treatment by the adding of Auxin in media, Kohei Nishimura et al. carried out experiences using 500µM of Auxin. Our project aimed to integrate the AID system in tadpoles for the control of protein synthesis in specific organ (link to exp). First we planned an Auxin toxicity test (IAA and NAA) in order to determine the suitable Auxin concentration for tadpoles. Experiments were conducted in [0, 125, 250, and 500] µM Auxin over 3 days and each morning the media were renewed.<b>(Preparation of different media for embryos development see experiments link). </b></p>
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<p>Before testing hormonal system in embryos (see <a href="https://2012.igem.org/Team:Evry/AIDSystem">here</a>), we conducted auxin toxicity test. Auxin concentrations used were 500, 250, 125 and 0 µM dissolved in MMR, with renewal of the medium each morning. (see medium preparation <a href=" https://2012.igem.org/Team:Evry/Protocols#Auxin_toxicity_test"> here </a>) </b></p>
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<h2>Results</h2>
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<h2>Result interpretation:</h2><br>
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<p>The survival rate of embryos in both 1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) were observed in triplicate during three days of experiment. <br>
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As a control sample, embryos were placed in MMR 0.1x medium without an auxin.<br/>
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The initial number of embryos for each medium condition was superior than 40.<br>
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The embryo's mortality rate was about:<br>
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<b>for NAA</b> 23% for day 1 (D1) and 45% for day 2 (D2) <br>
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<b>for IAA</b> 20% for D1 and 51% for D2 <br>
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<br>
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However embryos mortality depends not only on the auxin presence in media, but also on the rearing conditions, frequency of sorting dead and live embryos and media's changing. Usually the normal mortality ratio is no bigger than 30%. <br>
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We are aware that our experiment should be repeated to calculate the error deviation. But even without this data we confirmed our hypothesis that presence of <b>auxin in MMR medium is not toxic for embryos</b> and it doesn't cause any morphological abnormalities. </p>
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<img src="https://static.igem.org/mediawiki/2012/b/be/Image_NAA.png" alt="text to print if image not found" /> <br/>
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<h2>Data:</h2><br>
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<center><img src="https://static.igem.org/mediawiki/2012/6/6b/Naa_tox.jpg" alt="" width="500px" /></center> <br/>
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<p>Globally, the survival rate of embryos in 1-naphthaleneacetic acid (NAA) evolved in two times. In control the number of embryos remains constant between D0 and D1 and it decreases by half in D2. We observe no much change for 125 and 250µM in NAA. The number of surviving tadpoles in 500µM NAA decreases also between D0 and D2 following the same shape.<br/> </p>
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<br>
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<center><img src="https://static.igem.org/mediawiki/2012/8/8d/Iaa_toxicity1.jpg" alt="" width="500px" /></center>
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<img src="https://static.igem.org/mediawiki/2012/f/f0/Image_IAA.png" alt="text to print if image not found" />
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<br/>
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<i>Control sample- embryos in MMR medium</i><br>
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<br>
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<center><img src="https://static.igem.org/mediawiki/2012/8/8c/Naa_control.jpg" alt="" width="350px" /></center><br>
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<p>As in NAA case, the same trend is observed for the different media. The number of embryos is almost the same between D0 and D1 in control media and it decreases in D2. The survival rate is stable for 125µM, 250µM and 500µM IAA the first two days. Then it decreases by half in D2.</p>
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<i>Embryos in MMR medium with 250µM NAA</i><br>
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<br>
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<h2>Discussion</h2>
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<center><img src="https://static.igem.org/mediawiki/2012/3/3a/Tadpole_naa.jpg" alt="" width="350px" /></center><br>
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<p>The number of embryos (D0) and tadpoles (D1-D2) decrease globally in NAA and IAA for the different gradients. Compare to the control, NAA addition in the media doesn’t seem to affect tadpoles survival. The same observation for IAA addition since there isn’t correlation between tadpoles death and the gradient addition of IAA. Contrary to what it could be expected, for the last day, an important rate of tadpoles died in Auxin media as well as in control. It’s probably linked to the state of our plates. They weren’t changed and the dirty could have killed the tadpoles.</p>      
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<h2>Results</h2>
 
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<p>For the first time a plant hormone expression is implemented in a complex system. The auxin toxicity test (IAA and NAA) was made in order to determine the suitable auxin concentration for its use in tadpoles. According to the results we can’t assume an auxin toxicity in tadpoles.</p>
 
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Latest revision as of 02:19, 27 September 2012

Auxin toxicity test in tadpoles

The purpose of this experiment

Before testing hormonal system in embryos (see here), we conducted auxin toxicity test. Auxin concentrations used were 500, 250, 125 and 0 µM dissolved in MMR, with renewal of the medium each morning. (see medium preparation here )

Result interpretation:


The survival rate of embryos in both 1-naphthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) were observed in triplicate during three days of experiment.
As a control sample, embryos were placed in MMR 0.1x medium without an auxin.
The initial number of embryos for each medium condition was superior than 40.
The embryo's mortality rate was about:
for NAA 23% for day 1 (D1) and 45% for day 2 (D2)
for IAA 20% for D1 and 51% for D2

However embryos mortality depends not only on the auxin presence in media, but also on the rearing conditions, frequency of sorting dead and live embryos and media's changing. Usually the normal mortality ratio is no bigger than 30%.
We are aware that our experiment should be repeated to calculate the error deviation. But even without this data we confirmed our hypothesis that presence of auxin in MMR medium is not toxic for embryos and it doesn't cause any morphological abnormalities.

Data:





Control sample- embryos in MMR medium


Embryos in MMR medium with 250µM NAA


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