Part:BBa_K4245131:Experience

Applications of BBa_K4245131

The middle sequence was incorporated into the padlock probe designs for RCA and then RCA protocols were run. The RCP was then tested with both the linear probes mechanism as well as the split Lettuce mechanism. Both mechanisms were also tested with the complement of the middle sequence. Both mechanisms were successful in binding the middle sequence and producing the desired output.

Experimental data with the use of hsa-miR-1-3p RCA Padlock Probe (BBa_K4245200), hsa-mir-133a-3p RCA Padlock Probe (BBa_K4245204), and hsa-mir-451a RCA Padlock Probe (BBa_K4245209) containing the middle sequence:

Rolling Circle Amplification (RCA) was successful with the RCA Padlock Probes mentioned above, which all contained the middle sequence. The products of RCA are long 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 rolling circle products (RCP) on an agarose gel, as a fluorescent band very close to the wells would indicate the presence of an extremely long DNA strand. The result was a really long DNA strand close to the well (see Fig. 1, 2, 3).

Figure 1. Image of gel ran with miRNA-1 padlock probe RCP product.

Figure 2. Image of gel ran with miRNA-133a padlock probe RCP product. RCP is visible in wells 7 and 8.

Figure 3. Image of gel ran with miRNA-451a padlock probe RCP product.

By analyzing the results on the gel, our team concluded that a very long strand of DNA, likely the RCP, was produced for all of the padlock probes used. The gels exhibited a fluorescent band of DNA very close to the well, which indicates that a long strand of DNA, greater than 1 kB was produced due to our reaction, as seen in Figure 1, Figure 2, and Figure 3. As a result, we can infer that the RCA reaction was successful with the padlock probes containing the middle sequence.

Experimental data with the use of the linear probes 6-carboxyl-fluorescein (FAM) Labeled DNA Probe (BBa_K4245130) and Black Hole Quencher 1 (BHQ1) DNA Probe (BBa_K4245132): Lambert iGEM collected data for basic parts BBa_K4245130 and BBa_K4245132 using the characterization protocol linked here.

There is a negative logarithmic correlation between the complement (of the middle sequence) concentrations ranging from 0.1-100 mM and the relative fluorescence units (RFU) (see Fig. 3). The 0 mM complement concentration produces less RFU than 0.1 mM, which does not align with the model. However, the large error bars at 0 mM suggests that there was some degree of significant error. Thus, this data point is insignificant and further trials should be performed to achieve more accurate results. Moreover, the data from 0.1-100 mM closely parallels the predictive ordinary differential equation (ODE) model (see Fig. 4) correlating miRNA to RFU (see Modeling).

The complement concentration in the characterization curve mimics RCP. Therefore, the overall data collected depicts an accurate relationship between the complement concentration and RFU.

Figure 4. Characterization curve for parts BBa_K4245130 and BBa_K4245132 showing a negative logarithmic relationship between RFU and complement concentrations ranging from 0.1-100 μM. Note: 0-0.1 μM shows a positive relationship, but large error bars at 0 μM suggest this was due to faulty pipetting.

Figure 5. Deterministic ODE Model Simulation of RFU output dependent on the concentration of RCP.

Experimental data with the use of the split Lettuce DNA fluorescent aptamer Lettuce left (BBa_K4245134) and Lettuce right (BBa_K4245135):

The Lettuce split aptamer has been tested with RCP produced through Rolling Circle Amplification (RCA), as well as a sequence complementary to the middle sequence.

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. 6). This suggests that the RCP produced through this middle sequence would allow for successful hybridization between the split Lettuce and RCP.

Figure 6. 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. 7)

Figure 7. 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.

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