Part:BBa_K4245135:Experience

Lambert_GA 2022
The Lettuce split aptamer has been tested with the complement sequence of the middle sequence (BBa_K4245131) as well as with RCP produced through Rolling Circle Amplification (RCA).

We attempted to simulate the binding of the split Lettuce to the RCP by testing the split Lettuce with a sequence complementary to the middle sequence ordered from Integrated DNA Technologies. The complement, split Lettuce, and DFHBI-1T were mixed and heated at 70°C for 5 minutes, then cooled and held at 41°C for 1 hour. The fluorescence before and after were measured on the plate reader. From the results, we saw an increase in fluorescence in the presence of the complementary middle sequence and lettuce in the reaction as compared to the controls (see Fig. 1). This suggests that the RCP produced through this middle sequence would allow for successful hybridization between the split Lettuce and RCP.

Figure 1. Graph of split Lettuce reaction with middle sequence complement.

RCA was run with hsa-miR-1-3p RCA Padlock Probe BBa_K4245200. The products, split Lettuce, and DFHBI-1T were mixed and heated at 70°C for 5 minutes, then cooled and held at 41°C for 1 hour. The fluorescence before and after were measured on the plate reader. (see Fig. 2)

Figure 2. Graph of split Lettuce reaction with RCP. The values represent the change in fluorescence before and after the reaction with DFHBI-1T took place.

The increase in fluorescence of the RCP + Lettuce + dye was significantly greater than the controls, which suggests that the split Lettuce was successfully bound to the RCP and induced fluorescence in DFHBI-1T.

Lambert_GA 2023
Lettuce
Lettuce was tested as a reporter for rolling circle amplification (RCA) and was used for the successful characterization of hsa-miR-1-3p in the 2022 season (see Lambert iGEM Wiki RCA, 2022). Linear DNA probes are an off-state reporter, therefore they produce an indirect relationship between microRNA (miRNA) concentration and fluorescence output. Utilizing an on-state reporter such as Lettuce would result in a direct relationship, in which a higher concentration of fluorescence will correspond to a higher concentration of miRNA.

We characterized and quantified RCP through the Lettuce reporting mechanism (see Experiments: Lettuce with RCP). There is a positive logarithmic correlation between the miRNA concentrations and the relative fluorescence units (RFU) (see Fig. 3).

Figure 3. Characterization curve showing a positive logarithmic relationship between miR-1 concentrations and RFU from split Lettuce aptamer.

Comparison Lettuce resulted in significant SEM overlap between the lower miRNA concentrations, making accurate differentiation of miRNAs difficult. However, there was no indication that Lettuce outperformed linear DNA probes. Therefore, we continued to conduct further experiments with linear probes.

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