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From 2012.igem.org

Auxin toxicity test in tadpoles

The purpose of this experiement

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.(Preparation of different media for embryos development see experiments link).

Results

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.

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.

Discussion

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.

Results

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.