Part:BBa_K4245133:Experience

Applications of BBa_K4245133

Lambert_GA 2022 The Lettuce DNA Aptamer has been tested with DFHBI-1T. The Method adopted by researchers at the Jaffrey Lab was used, and the fluorescence was read on the plate reader. (see Fig. 1)

Figure 1. Graph of fluorescence after DFHBI-1T was added.

The fluorescence of the reaction between the Lettuce aptamer and DFHBI-1T, as seen in Figure 1, appear to be significantly greater than the controls, which contained water, dye, and buffer, as used in the original research. This proved the functionality of the Lettuce DNA fluorescent aptamer, so Lambert iGEM continued with the design and use of the split Lettuce aptamer to better fit the reporting mechanism used in conjunction with Rolling Circle Amplification (RCA).

Lambert_GA 2023
This part was used as a control to test whether Exponential Rolling Circle Amplification (eRCA) was successful. The products of eRCA are short DNA strands composed of repeating complementary sequences of the used padlock probe. Therefore, one way in which the success of RCA can be determined is by running the exponential rolling circle products (eRCP) on an agarose gel. Since a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand, no/dim bands near the top of the well indicate that short DNA was produced(see fig. 1).

Figure 1. Image of gel ran with miRNA-1 RCP product; A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)

By analyzing the results on the gel, our team concluded that short strands of DNA were produced, likely the eRCP.
The eRCP was also tested with DFHBI-1T dye as the RCP would consist of Lettuce Aptamer sequences. The fluorescence was read on the plate reader (see fig. 2).

Figure 2. Graph of fluorescence after DFHBI-1T was added

As seen in Figure 2, the increase in fluorescence of the eRCP+dye was significantly greater than the controls (including the one with Lettuce+DFHBI), which suggests that Lettuce aptamers were produced. According to these results, eRCA was successful.

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