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From 2012.igem.org

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:

Eggs 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.
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.

Preparing the work of the future generations on iGEMers working on complex organism

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.

We hope our work will be useful for other to learn, enjoy and create new models for their projects in the future!

Parameters estimation

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: here.

Overall map of models

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: