Team:Evry/auxin uptake

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<h1>Auxin uptake experiment</h1>
<h1>Auxin uptake experiment</h1>
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<center><img src="https://static.igem.org/mediawiki/2012/6/62/MathFrog.png" width=200px></center>
 
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<h2>The experiment is described below:</h2>
<h2>The experiment is described below:</h2>
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<p>Eggs after <i>in vitro</i> fertilization were placed in medium with different concentration (125µM, 250µM, 500µM) of natural (IAA) or synthetic auxin (NAA) then incubated at 21 degrees Celsius for one day.<br>
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<p>Embryos after <i>in vitro</i> fertilization were placed in medium with different concentration (125µM, 250µM, 500µM) of natural (IAA) or synthetic auxin (NAA) then incubated at 21 degrees Celsius for one day.<br>
After one day the embryos were taken out from the medium and washed four times in MiliQ Water in order to remove all auxins residues on the embryo’s skin. Washed embryos were transferred to the Eppendorf tubes (10 embryos per tube), quenched immediately with 55µL of cold Acetonitrile/Methanol/Water (2:2:1 v/v/v) and homogenized with a mortar. Lysed and dispersed cells were incubated on ice for 20’ in order to release all metabolites from cytoplasm. The final step was samples centrifugation (14000rpm, 5min) and collection of supernatant.<br>
After one day the embryos were taken out from the medium and washed four times in MiliQ Water in order to remove all auxins residues on the embryo’s skin. Washed embryos were transferred to the Eppendorf tubes (10 embryos per tube), quenched immediately with 55µL of cold Acetonitrile/Methanol/Water (2:2:1 v/v/v) and homogenized with a mortar. Lysed and dispersed cells were incubated on ice for 20’ in order to release all metabolites from cytoplasm. The final step was samples centrifugation (14000rpm, 5min) and collection of supernatant.<br>
All metabolites (including auxins) were supposed to be present in the supernatant, which was confirmed by series of HPLC tests.
All metabolites (including auxins) were supposed to be present in the supernatant, which was confirmed by series of HPLC tests.
<br>
<br>
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Because auxin contains an aromatic ring we assumed that we should perform an HPLC coupled with UV detection. To determinate the optimal wavelength for auxin detection we did spectrophotometric measurement. <br>
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<br>
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Shown spectrophotometric result prove that the maximum absorption was obtained at λ=220 nm, so all UV detection will be performed in this wavelength. <br>
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<h3>Optimal wavelength for auxin detection</h3><br>
 +
<p>Because auxin contains an aromatic ring we assumed that we should perform an HPLC coupled with UV detection. To determinate the optimal wavelength for auxin detection we did spectrophotometric measurement. <br>
 +
Shown spectrophotometric result prove that the maximum absorption was obtained at λ=220 nm, so all UV detection will be performed in this wavelength.<p> <br>
 +
 
 +
<h2>Result interpretation:</h2><br>
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<p>Chromatogram pic correspondent to NAA is present in embryo’s extracts from three different concentration of NAA (but 0µM). <br>
 +
IAA pic was found only in the 500µM and 250µM samples. When auxin concentration in medium was smaller, it was hardly uptaken (IAA pic is in the baseline). <br>
 +
NAA is more easy to detect and hence it may be the auxin of choice for the experiments </p>
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<h2>Data</h2>
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<h2>Preparing the work of the future generations on iGEMers working on complex organism</h2>
 
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<p>When writing down our work, we have made an especial effort for our models and hypothesis to be very understandable, in order help the work of the future generation of iGEMers working on tadpoles, and on multicellular organisms in general. You can access general informations on the models by clicking on the ODE, PDE and AB simulations on the image below, and all the instructions are provided for you to run our simulations either on your own computer or directly in your web browser (!) using the full power of the Java Applets created using the Netlogo program.</p>
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<h3>Auxin absorbance spectrum</h3>
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<p>We hope our work will be useful for other to learn, enjoy and create new models for their projects in the future!</p>
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<center><img src="https://static.igem.org/mediawiki/2012/a/a6/Auxin_absorbancy.jpg" alt="" width="500px" /></center>
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<h2>Parameters estimation</h2>
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<h3>IAA (natural auxin) HPLC test results:</h3>
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<br>
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<i>IAA standard: </i><br>
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A great part of our modeling work has been to find or estimate the values of the parameters used in the model. For a better readability, we created a special pages regrouping them all: <a href="https://2012.igem.org/Team:Evry/parameters">here</a>.
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<center><img src="https://static.igem.org/mediawiki/2012/0/08/IAA_standard.jpg" alt="" width="900px" /></center>
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<i>IAA 0µM: </i><br>
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<h2>Overall map of models</h2>
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<center><img src="https://static.igem.org/mediawiki/2012/archive/d/da/20120918141429%21IAA_0uM.jpg" alt="" width="900px" /></center>
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<p>This schematic represents the different parts of the models we have created, as well as general information on the modeling methods used in these models. Clicks on the different elements of this image to access read the different models:</p>
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<i>IAA 125µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/b/bf/IAA_125uM.jpg" alt="" width="900px" /></center>
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<br>
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<i>IAA 250µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/2/2c/IAA_250uM.jpg" alt="" width="900px" /></center>
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<i>IAA 500µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/f/f4/IAA_500uM.jpg" alt="" width="900px" /></center>
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<img src="https://static.igem.org/mediawiki/2012/8/88/Schematic_modeling.png" width="840" height="555" border="0" class="mapper" usemap="#map" />
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<br>
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<h3>NAA (synthetic auxin) HPLC test results:</h3>
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<br>
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<i>NAA standard: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/c/c4/NAA_standard.jpg" alt="" width="900px" /></center>
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<br>
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<i>NAA 0µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/e/ea/NAA_0uM.jpg" alt="" width="900px" /></center>
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<br>
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<i>NAA 125µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/8/84/NAA_125uM.jpg" alt="" width="900px" /></center>
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<br>
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<br>
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<i>NAA 250µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/a/a3/NAA_250uM.jpg" alt="" width="900px" /></center>
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<br>
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<br>
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<i>NAA 500µM: </i><br>
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<center><img src="https://static.igem.org/mediawiki/2012/c/c4/NAA_500uM.jpg" alt="" width="900px" /></center>
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Latest revision as of 10:04, 26 September 2012

Auxin uptake experiment


In order to determine whether Xenopus embryos can uptake auxin, we did auxin extraction followed by detection by High Performance Liquid Chromatography (HPLC). This way we determined that the concentration below which auxin was undetectable was 125µM for NAA and 500µM for IAA.


The experiment is described below:

Embryos after in vitro fertilization were placed in medium with different concentration (125µM, 250µM, 500µM) of natural (IAA) or synthetic auxin (NAA) then incubated at 21 degrees Celsius for one day.
After one day the embryos were taken out from the medium and washed four times in MiliQ Water in order to remove all auxins residues on the embryo’s skin. Washed embryos were transferred to the Eppendorf tubes (10 embryos per tube), quenched immediately with 55µL of cold Acetonitrile/Methanol/Water (2:2:1 v/v/v) and homogenized with a mortar. Lysed and dispersed cells were incubated on ice for 20’ in order to release all metabolites from cytoplasm. The final step was samples centrifugation (14000rpm, 5min) and collection of supernatant.
All metabolites (including auxins) were supposed to be present in the supernatant, which was confirmed by series of HPLC tests.

Optimal wavelength for auxin detection


Because auxin contains an aromatic ring we assumed that we should perform an HPLC coupled with UV detection. To determinate the optimal wavelength for auxin detection we did spectrophotometric measurement.
Shown spectrophotometric result prove that the maximum absorption was obtained at λ=220 nm, so all UV detection will be performed in this wavelength.


Result interpretation:


Chromatogram pic correspondent to NAA is present in embryo’s extracts from three different concentration of NAA (but 0µM).
IAA pic was found only in the 500µM and 250µM samples. When auxin concentration in medium was smaller, it was hardly uptaken (IAA pic is in the baseline).
NAA is more easy to detect and hence it may be the auxin of choice for the experiments

Data


Auxin absorbance spectrum


IAA (natural auxin) HPLC test results:


IAA standard:


IAA 0µM:


IAA 125µM:


IAA 250µM:


IAA 500µM:



NAA (synthetic auxin) HPLC test results:


NAA standard:


NAA 0µM:


NAA 125µM:


NAA 250µM:


NAA 500µM:


Retrieved from "http://2012.igem.org/Team:Evry/auxin_uptake"