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Team:Evry/Experimentals Parameters
From 2012.igem.org
Protocols to determinate the following experimentals parameters
Identify where the auxin concentrates
To determine where the auxin concentrates, you have to inject first the auxin in the heart of the tadpole, for it is directly associated into the bloodstream. Then, after waiting 20 minutes that auxin diffuses throughout the body, fixing the tadpole to permeabilize the tissue, then a revelation by fluorescent antibody anti-auxin, visualizes the areas where concentrated auxin.
How to determinate the Auxin diffusion from Skin to Water
To determine the diffusion coefficient of auxin from the skin to the environment (MMR), just in time to first soak for 20 minutes a tadpole skin volume, the volume has been previously measured in a solution MMR with auxin. Then transferring the tissue in a solution of MMR, and every 5 minutes taking a constant volume of media for each samples it is possible to determine the desired parameter. In fact, each samples shows the quantity of auxin diffused from the skin to the media, during a period. It is thus possible to plot a curve ( quantities fonction of time ), and determine the parameters about Auxin diffusion from Skin to Water.
How to determinate the Auxin diffusion from Blood to Muscle
To determine the diffusion coefficient of auxin, it will have injected directly into the heart auxin. Then let the tadpole in the MMR for 5 minutes ( recuperation time ). Then euthanize the tadpole and collect a volume of muscle. Determining the concentration of auxin in the piece of muscle removed. Repeat the experiment with at least 10 others tadpoles increasing the recuperation time of 5 minutes by experiments. Remark, you have to isolated always the same height of muscle. Thus, the values of auxin diffused from the blood to the muscle function of time will determine the desired parameters.
How to determinate the Auxin Half Life in the Skin
To determine the diffusion coefficient of auxin, it will have injected directly into the heart auxin. Then let the tadpole in the MMR for 5 minutes ( recuperation time ). Then euthanize the tadpole and collect a volume of muscle. Determining the concentration of auxin in the piece of muscle removed. Repeat the experiment with at least 10 others tadpoles increasing the recuperation time of 5 minutes by experiments. Remark, you have to isolated always the same height of muscle. Thus, the values of auxin diffused from the blood to the muscle function of time will determine the desired parameters.
How to determinate the Auxin Half Life in the Muscle
On the half-life time of auxin in tissues, we must first load the tissue of auxin by soaking in a large volume of solution MMR with auxin (volume 10,000 times greater than the volume of tissue) .
So we know how much auxin was present in the tissue when it was removed from the bath at high volume (because we have the volume of tissue, and the concentration in the tissue is the same as in the bath due to the strong difference between the volume of tissue and that of the bath).
Then place it in another volume of MMR with auxin concentration but the same volume of solution equal to the tissues.
Here we know how much auxin was present in the bath of small volume.
Wait 5 minutes, then remove the fabric and the environment.
Determine the auxin present in the tissue and the environment.
Then simply subtract this value from the degradation of auxin in the MMR only to know the amount of auxin degraded by tissue volume in 5 minutes
Repeat this experiment with at least 10 tadpoles, and by increasing the incubation time in the bath small volume of 5 minutes each time.
Finally just assign the value of auxin degradation over time to determine the parameters
