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From 2012.igem.org

Summary

 

On the fundament that RNA scaffold in vivo has been achieved, we aimed to design and assemble controllable scaffolds. A designed theophylline aptamer was added on the original scaffold D0 in order to produce an interaction with MS2 aptamer in the absence of theophylline, thus disturbing the bind of MS2 aptamer and corresponding protein. However in the presence of theophylline, the interaction would disappear with the change of theophylline aptamer. We called these controllable scaffolds 'clovers'. Three versions of clover were designed, which have different interaction sites and different relative positions between the theophylline and MS2 aptamers.

As a preliminary experiment, we use theophylline of different concentrations to test two existing parts, which consist of the gene of a theophylline aptamer and a fluorescent protein.

Clover version two has been synthesized; it was co-transformated into E.coli with Fa-MS2 and Fb-PP7 fusion proteins. As a result, in a certain range of theophylline concentration, the fluorescent intensity has a positive correlation with it, which means controllable scaffold is achieved. Besides, to our surprise, the fluorescent could reach a higher intensity than the original scaffold D0, which means our clover, makes lovers closer!

More future work will be done in the next period. We are going to synthesize clover version one and version three, which seem to have a better control effect. A comparison will be made to find the best design principle of controllable RNA scaffold. We believe that more RNA scaffolds with higher efficiency and better regulative effect will be designed and applied.