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From 2012.igem.org

Summary

 

We aimed to design and assemble tunable scaffolds. In order to reach this goal, we utilized the working mechanism of riboswitches. A designed theophylline aptamer was added on the basic RNA scaffold in order to achieve the controllability of it. Since this kind of tunable scaffolds contains an allosteric aptamer, we named them alloscaffolds. The series of alloscaffolds we designed are called ‘clover’. Three versions of clovers were designed, which have different interaction sites and different relative positions between theophylline aptamer and MS2 aptamer as well.

If scaffold library is a disperse scaffold bank, alloscaffold series serves as a successional scaffold bank which includes a variety of scaffolds with diverse efficiencies, realizing by theophylline.

As a preliminary experiment, we use theophylline of different concentrations to test two existing parts, which are riboswitches containing a theophylline aptamer.

Clover version 2 and 3 has been synthesized and characterized. They were respectively 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 tunable alloscaffolds are achieved. Compared with clover version 2, version 3 has a better and more stable control effect. Besides, to our surprise, the fluorescent could reach a higher intensity than the basic RNA scaffold, which means our clovers can bring a higher efficiency to pathways.

More work will be done in the future. Except alloscaffold, we are aiming to find new and better methods to design and assemble tunable RNA scaffolds. We believe that more RNA scaffolds with higher efficiency and better regulative effect will be designed and applied in the future.