Difference between revisions of "Part:BBa K1777016"

Revision as of 08:40, 20 September 2015

Device to produce circRNA

The Fudan iGEM team 2015 designed the device with two inverted repeat sequence which can produce circular mir-21 sponge. We nicknamed it “Ouroboros”.The ouroboros or uroboros (/jʊərɵˈbɒrəs/; /ɔːˈrɒbɔrəs/, from the Greek οὐροβόρος ὄφις tail-devouring snake) is an ancient symbol depicting a serpent or dragon eating its own tail. Just like the ouroboros of myth, our Ouroboros cyclized itself by 'biting' its own. This is the plan A of our cyclizing device. We used the inverted repeat sequence, which is inserted into the flanking intron of the circularizing sequence.
We generate long inverted repeat sequence of 400bp and insert this sequence into the flanking intron of the circularizing exon. We choose the inserted sequence which have higher binding affinity to increase the cyclization efficiency. The flanking intron is β-globin intron1, which can improve the expression level of our device RNA，and provide long linker for the back-splicing mechanism.

Figure 1: Construct design. The Ouroboros contains inverted repeat sequence and 6 miR-21 binding sites.

The prototype of Ouroboros is to generate circRNA sponge to regulate oncomiRs, and this is based on the long half-life time of circRNA. In order to validate this assumption, we designed circRNA degradation experiment to measure circRNA half-life time.In this experiment, we use our device to cyclize the RNA and simulate the prototype in which the Ouroboros are used as research device.
The circularizing exon have 6 mir-21 binding site, which is reported to be the most efficient number for miRNA sponges. The circularizing exon is constructed by overlap PCR, and the binding site is designed to have the highest binding affinity.
In order to build a tool box, we are synthesize the inverted sequence and the circularizing sequence separately, and finally combine these two parts together. Other than mir-21 sponge, we also designed mir-17 sponge circularizing exon. Our toolbox will finally able to regulate different kinds of miRNAs, and facilitate the reseach and health care issues related to miRNA.
To support our testing experiment, we designed a reporter Luciferase device. We inserted two mir-21 binding site to the 3’-UTR of the Luciferase, and it can report the mir-21 concentration by the intense of fluorescent.

Sequence and Features