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Team:Evry/auxin uptake
From 2012.igem.org
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<h2>The experiment is described below:</h2> | <h2>The experiment is described below:</h2> | ||
| - | <p> | + | <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> | ||
| - | + | <br> | |
<h3>Optimal wavelength for auxin detection</h3><br> | <h3>Optimal wavelength for auxin detection</h3><br> | ||
| - | 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> | + | <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. <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> | <h2>Result interpretation:</h2><br> | ||
| - | Chromatogram pic correspondent to NAA is present in | + | <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). | + | 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> | ||
<h2>Data</h2> | <h2>Data</h2> | ||
<br> | <br> | ||
| - | <h3>Auxin absorbance | + | <h3>Auxin absorbance spectrum</h3> |
| - | + | ||
<center><img src="https://static.igem.org/mediawiki/2012/a/a6/Auxin_absorbancy.jpg" alt="" width="500px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/a/a6/Auxin_absorbancy.jpg" alt="" width="500px" /></center> | ||
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<br> | <br> | ||
<br> | <br> | ||
| - | <i>IAA | + | <i>IAA 0µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/archive/d/da/20120918141429%21IAA_0uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/archive/d/da/20120918141429%21IAA_0uM.jpg" alt="" width="900px" /></center> | ||
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<br> | <br> | ||
| - | <i>IAA | + | <i>IAA 125µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/b/bf/IAA_125uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/b/bf/IAA_125uM.jpg" alt="" width="900px" /></center> | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <i>IAA | + | <i>IAA 250µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/2/2c/IAA_250uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/2/2c/IAA_250uM.jpg" alt="" width="900px" /></center> | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <i>IAA | + | <i>IAA 500µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/f/f4/IAA_500uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/f/f4/IAA_500uM.jpg" alt="" width="900px" /></center> | ||
<br> | <br> | ||
| Line 71: | Line 71: | ||
<br> | <br> | ||
| - | <i>NAA | + | <i>NAA 0µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/e/ea/NAA_0uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/e/ea/NAA_0uM.jpg" alt="" width="900px" /></center> | ||
| Line 77: | Line 77: | ||
<br> | <br> | ||
| - | <i>NAA | + | <i>NAA 125µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/8/84/NAA_125uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/8/84/NAA_125uM.jpg" alt="" width="900px" /></center> | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <i>NAA | + | <i>NAA 250µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/a/a3/NAA_250uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/a/a3/NAA_250uM.jpg" alt="" width="900px" /></center> | ||
<br> | <br> | ||
<br> | <br> | ||
| - | <i>NAA | + | <i>NAA 500µM: </i><br> |
<center><img src="https://static.igem.org/mediawiki/2012/c/c4/NAA_500uM.jpg" alt="" width="900px" /></center> | <center><img src="https://static.igem.org/mediawiki/2012/c/c4/NAA_500uM.jpg" alt="" width="900px" /></center> | ||
<br> | <br> | ||
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:
