Part:BBa_K4245201:Experience

Rolling Circle Transcription (RCT) was run with this part but proved to be unsuccessful. The products of RCT are long RNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCT can be determined is by running the RCT products on an agarose gel, since a fluorescent band very close to the wells would indicate the presence of extremely long nucleic acid strands. However, the products of RCT did not appear on a 1% agarose gel (see Fig. 1).

Figure 1. Picture of RCT products (circled in green) run on a 1% agarose gel. There were no visible bands that indicate the production of long RNA strands.

The RCT products were also tested with DFHBI-1T, a fluorophore that fluoresces when reacting with the Broccoli RNA fluorescent aptamer. DFHBI-1T was added to the RCT products, and the fluorescence was read on the plate reader.

Figure 2. Graph of fluorescence output before and after the addition of DFHBI-1T to RCT products and controls.

However, when we added DFHBI-1T to the RCT products, there was no significant increase in fluorescence observed as compared to the controls (see Fig 2.).

From these results, Lambert iGEM determined that the process of RCT itself was not successful in our lab.

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