Difference between revisions of "Part:BBa K738000"

Revision as of 19:41, 25 September 2012

RNA Scaffold generator

Scaffold D0 was constructed from a single RNA module d0, which folded into a duplex with PP7 and MS2 aptamer domains that bind PP7 and MS2 fusion proteins. Secondary and tertiary structure of RNA scaffold D0

GFP split into two halves (FA and FB), each of which is fused to MS2 or PP7. FA-MS2 and FB-PP7 then bind to their respective aptamers. FA and FB will give out fluorescence only when they are close enough. With help of RNA scaffold D0, FA and FB will be bring together by binding process between MS2 or PP7 and their respective aptamers. Fluorescence provides strong evidence that the RNA scaffold is functional. Function test of the RNA scaffold D0 has been done through split GFP assay. Pictures blow were taken with confocal microscope (olympus fluoview fv1000)

A Bacteria with FA-MS2 and FB-PP7, but without RNA scaffold D0. B Bacteria with FA-MS2, FB-PP7 and RNA scaffold D0 Fluorescence per OD were measured with plate reader (Synergy H1 Hybrid Reader, bioteck).

Bacteria with FA, FB and D0 is 1.63±0.05 fold brighter than bacteria with FA and FB but without D0.

Usage and Biology

In cells, multienzymatic pathways are often physically and spatially organized onto scaffolds or clusters or into microcompartments. Spatial organization helps substrates flow between interacting proteins, limits cross-talk between signaling pathways, and increases yields of sequential metabolic reactions. But multi-enzyme pathways in vivo widely used in chemical and medical industry are not naturally. So there is lacking in relative scaffolds used as spatial organization. BBa_K738000 is a discrete engineered RNA module for controling the spatial organization of bound proteins, which is able to make the production of the multi-enzyme pathways more efficient.

Sequence and Features