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From 2012.igem.org

Characterization of the degron system !

In between the two jamborees, we finished the construction of our TirI degron system and had the time to test it once in Xenupus. Unfortunately the results were negatives. They are presented in this page.

Preparation of the RNAs

Starting from the gene, cloned into pSC1C3 for TirI or pSC2+ for GFP-AID, we amplified them with a sp6 promoter on the front of the gene and PCR purified. The mRNAs were then prepared from the PCR product using the Sp6 phage RNA polymerase. The RNAs were then precipitated and poly-adenilated with a RNA poly-adelylase. The mRNA were then treated with a mix of capping proteins before being injected.

The RNAs of TirI and GPD-AID were then pooled in equimolar ratio before being injected for the GFP-AID TirI.

GFP-AID - Pep2A - TirI in pCS2+

The two genes GFP-AID and TirI were fused with a Pep2A peptide, to be transcribed as a single protein. The Pep2A sequence comes from the Thosea asigna virus 2A, creating a peptide bridge in between two proteins. When the ribosome transcribe this sequence protein, one of the amino-acids bound formation is not catalyzed, giving birth to two distinct protein. This is a common method to design polysistronic mRNA in eukaryotes. It is also especially relevant system to use here, because it gives a one to one protein ratio, that we wanted to achieve to make sure the GFP will be properly degraded in the presence of auxin.

Protocol

The embryos coming from the in-vitro fertilization were micro-injected from 1 cell to 10 cell stage, and left at 22°C overnight. On the morning, we sorted them and prepared different groups. After the first picture, we placed the tadpoles in a solution containing 5 mM of auxin IAA. Four hours later, we observed the tadpole again and tried to observe a decrease of the fluorescence signal. Unfortunately we didn't observe any noticeable change, and most of our tadpoles died during the 4 hours.

Results

In order to go further

Further controls that needs to be done. The first one is to check the correct expression of TirI doing immuno-fluorescence on the myc tag that is fused to the TirI. We should also try with a longer incubation time, but since all our tadpoles died during the process, this was not doable for this experiment. We are going to re-do this experiment after the jamborees with this additional controls for publication if it works.